ZA200708939B - Lactobacillus acidophilus nucleic acid sequences encoding carbohydrate utilization-related proteins and uses therefor - Google Patents
Lactobacillus acidophilus nucleic acid sequences encoding carbohydrate utilization-related proteins and uses therefor Download PDFInfo
- Publication number
- ZA200708939B ZA200708939B ZA200708939A ZA200708939A ZA200708939B ZA 200708939 B ZA200708939 B ZA 200708939B ZA 200708939 A ZA200708939 A ZA 200708939A ZA 200708939 A ZA200708939 A ZA 200708939A ZA 200708939 B ZA200708939 B ZA 200708939B
- Authority
- ZA
- South Africa
- Prior art keywords
- protein
- polypeptide
- sequence
- nucleic acid
- seq
- Prior art date
Links
- 150000007523 nucleic acids Chemical group 0.000 title claims description 170
- 230000025938 carbohydrate utilization Effects 0.000 title claims description 160
- 108090000623 proteins and genes Proteins 0.000 title description 426
- 102000004169 proteins and genes Human genes 0.000 title description 353
- 240000001046 Lactobacillus acidophilus Species 0.000 title description 51
- 235000013956 Lactobacillus acidophilus Nutrition 0.000 title description 50
- 229940039695 lactobacillus acidophilus Drugs 0.000 title description 50
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 204
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 197
- 229920001184 polypeptide Polymers 0.000 claims description 192
- 102000039446 nucleic acids Human genes 0.000 claims description 157
- 108020004707 nucleic acids Proteins 0.000 claims description 157
- 125000003729 nucleotide group Chemical group 0.000 claims description 135
- 239000002773 nucleotide Substances 0.000 claims description 133
- 238000000034 method Methods 0.000 claims description 120
- 150000001720 carbohydrates Chemical class 0.000 claims description 84
- 230000000694 effects Effects 0.000 claims description 66
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 65
- 235000013305 food Nutrition 0.000 claims description 34
- 239000013598 vector Substances 0.000 claims description 31
- 238000004519 manufacturing process Methods 0.000 claims description 25
- 244000005700 microbiome Species 0.000 claims description 25
- 102000040430 polynucleotide Human genes 0.000 claims description 24
- 108091033319 polynucleotide Proteins 0.000 claims description 24
- 239000002157 polynucleotide Substances 0.000 claims description 24
- 230000000295 complement effect Effects 0.000 claims description 20
- 239000003814 drug Substances 0.000 claims description 19
- 229940079593 drug Drugs 0.000 claims description 19
- 238000012545 processing Methods 0.000 claims description 15
- 102000040811 transporter activity Human genes 0.000 claims description 15
- 108091092194 transporter activity Proteins 0.000 claims description 15
- 210000001035 gastrointestinal tract Anatomy 0.000 claims description 12
- 239000000796 flavoring agent Substances 0.000 claims description 11
- 235000019634 flavors Nutrition 0.000 claims description 11
- 238000003860 storage Methods 0.000 claims description 9
- 238000012258 culturing Methods 0.000 claims description 2
- 235000018102 proteins Nutrition 0.000 description 349
- 108010078791 Carrier Proteins Proteins 0.000 description 171
- 235000001014 amino acid Nutrition 0.000 description 155
- 229940024606 amino acid Drugs 0.000 description 153
- 150000001413 amino acids Chemical class 0.000 description 153
- 102000004190 Enzymes Human genes 0.000 description 133
- 108090000790 Enzymes Proteins 0.000 description 133
- 229940088598 enzyme Drugs 0.000 description 133
- 241000196324 Embryophyta Species 0.000 description 93
- 210000004027 cell Anatomy 0.000 description 91
- 230000014509 gene expression Effects 0.000 description 82
- 235000014633 carbohydrates Nutrition 0.000 description 78
- 239000000523 sample Substances 0.000 description 77
- 235000000346 sugar Nutrition 0.000 description 63
- 239000012634 fragment Substances 0.000 description 61
- 108020004414 DNA Proteins 0.000 description 59
- 108091000080 Phosphotransferase Proteins 0.000 description 53
- 102000020233 phosphotransferase Human genes 0.000 description 53
- 230000032258 transport Effects 0.000 description 50
- 241000894006 Bacteria Species 0.000 description 48
- 230000001580 bacterial effect Effects 0.000 description 48
- 230000027455 binding Effects 0.000 description 43
- 108091028043 Nucleic acid sequence Proteins 0.000 description 42
- 108010006533 ATP-Binding Cassette Transporters Proteins 0.000 description 41
- 102000005416 ATP-Binding Cassette Transporters Human genes 0.000 description 41
- 238000009396 hybridization Methods 0.000 description 37
- 239000002253 acid Substances 0.000 description 33
- 108020004999 messenger RNA Proteins 0.000 description 31
- 230000000692 anti-sense effect Effects 0.000 description 30
- 102000037865 fusion proteins Human genes 0.000 description 30
- 108020001507 fusion proteins Proteins 0.000 description 30
- 230000001105 regulatory effect Effects 0.000 description 30
- 230000006870 function Effects 0.000 description 27
- 102000003939 Membrane transport proteins Human genes 0.000 description 26
- 108090000301 Membrane transport proteins Proteins 0.000 description 26
- 239000000047 product Substances 0.000 description 26
- GUBGYTABKSRVRQ-CUHNMECISA-N D-Cellobiose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-CUHNMECISA-N 0.000 description 25
- 238000002864 sequence alignment Methods 0.000 description 25
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 24
- 102000014914 Carrier Proteins Human genes 0.000 description 22
- 241000588724 Escherichia coli Species 0.000 description 22
- 230000015572 biosynthetic process Effects 0.000 description 22
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 22
- 239000000758 substrate Substances 0.000 description 22
- 230000002103 transcriptional effect Effects 0.000 description 22
- 239000013615 primer Substances 0.000 description 21
- 238000004422 calculation algorithm Methods 0.000 description 20
- 239000013604 expression vector Substances 0.000 description 20
- 230000009261 transgenic effect Effects 0.000 description 20
- 238000003752 polymerase chain reaction Methods 0.000 description 19
- 230000035897 transcription Effects 0.000 description 19
- 238000013518 transcription Methods 0.000 description 19
- 108091026890 Coding region Proteins 0.000 description 18
- 150000007513 acids Chemical group 0.000 description 18
- 108091008324 binding proteins Proteins 0.000 description 17
- 239000012528 membrane Substances 0.000 description 16
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 15
- 238000003776 cleavage reaction Methods 0.000 description 15
- 230000007017 scission Effects 0.000 description 15
- 230000009466 transformation Effects 0.000 description 15
- 241000193998 Streptococcus pneumoniae Species 0.000 description 14
- 238000006243 chemical reaction Methods 0.000 description 14
- 229940031000 streptococcus pneumoniae Drugs 0.000 description 14
- 230000014616 translation Effects 0.000 description 14
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 description 13
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 13
- 108091034117 Oligonucleotide Proteins 0.000 description 13
- 108010076504 Protein Sorting Signals Proteins 0.000 description 13
- 241000193996 Streptococcus pyogenes Species 0.000 description 13
- 210000004899 c-terminal region Anatomy 0.000 description 13
- 239000008101 lactose Substances 0.000 description 13
- 238000013519 translation Methods 0.000 description 13
- 150000008495 β-glucosides Chemical class 0.000 description 13
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 12
- 125000000539 amino acid group Chemical group 0.000 description 12
- 238000003556 assay Methods 0.000 description 12
- 108010005774 beta-Galactosidase Proteins 0.000 description 12
- 238000001514 detection method Methods 0.000 description 12
- -1 infant formulae Substances 0.000 description 12
- 239000004310 lactic acid Substances 0.000 description 12
- 235000014655 lactic acid Nutrition 0.000 description 12
- 230000026731 phosphorylation Effects 0.000 description 12
- 238000006366 phosphorylation reaction Methods 0.000 description 12
- 241000186660 Lactobacillus Species 0.000 description 11
- 239000002299 complementary DNA Substances 0.000 description 11
- 230000004151 fermentation Effects 0.000 description 11
- 238000000855 fermentation Methods 0.000 description 11
- 239000000203 mixture Substances 0.000 description 11
- DTBNBXWJWCWCIK-UHFFFAOYSA-K phosphonatoenolpyruvate Chemical compound [O-]C(=O)C(=C)OP([O-])([O-])=O DTBNBXWJWCWCIK-UHFFFAOYSA-K 0.000 description 11
- 238000006467 substitution reaction Methods 0.000 description 11
- 241000193401 Clostridium acetobutylicum Species 0.000 description 10
- HMFHBZSHGGEWLO-SOOFDHNKSA-N D-ribofuranose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@@H]1O HMFHBZSHGGEWLO-SOOFDHNKSA-N 0.000 description 10
- 229930091371 Fructose Natural products 0.000 description 10
- 239000005715 Fructose Substances 0.000 description 10
- PYMYPHUHKUWMLA-LMVFSUKVSA-N Ribose Natural products OC[C@@H](O)[C@@H](O)[C@@H](O)C=O PYMYPHUHKUWMLA-LMVFSUKVSA-N 0.000 description 10
- HMFHBZSHGGEWLO-UHFFFAOYSA-N alpha-D-Furanose-Ribose Natural products OCC1OC(O)C(O)C1O HMFHBZSHGGEWLO-UHFFFAOYSA-N 0.000 description 10
- 238000004458 analytical method Methods 0.000 description 10
- 230000003197 catalytic effect Effects 0.000 description 10
- 150000001875 compounds Chemical class 0.000 description 10
- 238000000338 in vitro Methods 0.000 description 10
- 230000004060 metabolic process Effects 0.000 description 10
- 239000006041 probiotic Substances 0.000 description 10
- 230000000529 probiotic effect Effects 0.000 description 10
- 235000018291 probiotics Nutrition 0.000 description 10
- 238000003786 synthesis reaction Methods 0.000 description 10
- 239000003053 toxin Substances 0.000 description 10
- 231100000765 toxin Toxicity 0.000 description 10
- 108700012359 toxins Proteins 0.000 description 10
- 238000012546 transfer Methods 0.000 description 10
- 108010062877 Bacteriocins Proteins 0.000 description 9
- 102100026189 Beta-galactosidase Human genes 0.000 description 9
- WQZGKKKJIJFFOK-QTVWNMPRSA-N D-mannopyranose Chemical compound OC[C@H]1OC(O)[C@@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-QTVWNMPRSA-N 0.000 description 9
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 description 9
- 102000004157 Hydrolases Human genes 0.000 description 9
- 108090000604 Hydrolases Proteins 0.000 description 9
- 230000004071 biological effect Effects 0.000 description 9
- 230000004927 fusion Effects 0.000 description 9
- 230000002068 genetic effect Effects 0.000 description 9
- 230000001965 increasing effect Effects 0.000 description 9
- 230000037361 pathway Effects 0.000 description 9
- 108010055837 phosphocarrier protein HPr Proteins 0.000 description 9
- 150000008163 sugars Chemical class 0.000 description 9
- 108700034291 EC 2.7.1.69 Proteins 0.000 description 8
- ZHNUHDYFZUAESO-UHFFFAOYSA-N Formamide Chemical compound NC=O ZHNUHDYFZUAESO-UHFFFAOYSA-N 0.000 description 8
- 241000194041 Lactococcus lactis subsp. lactis Species 0.000 description 8
- 241000186805 Listeria innocua Species 0.000 description 8
- 241000186779 Listeria monocytogenes Species 0.000 description 8
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 8
- 235000014969 Streptococcus diacetilactis Nutrition 0.000 description 8
- 229930006000 Sucrose Natural products 0.000 description 8
- 240000008042 Zea mays Species 0.000 description 8
- WQZGKKKJIJFFOK-PHYPRBDBSA-N alpha-D-galactose Chemical compound OC[C@H]1O[C@H](O)[C@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-PHYPRBDBSA-N 0.000 description 8
- 230000001419 dependent effect Effects 0.000 description 8
- 229930182830 galactose Natural products 0.000 description 8
- 108091006104 gene-regulatory proteins Proteins 0.000 description 8
- 102000034356 gene-regulatory proteins Human genes 0.000 description 8
- 230000034659 glycolysis Effects 0.000 description 8
- 235000014304 histidine Nutrition 0.000 description 8
- 229940115931 listeria monocytogenes Drugs 0.000 description 8
- 230000035772 mutation Effects 0.000 description 8
- 229930029653 phosphoenolpyruvate Natural products 0.000 description 8
- 230000009049 secondary transport Effects 0.000 description 8
- 239000000126 substance Substances 0.000 description 8
- 239000005720 sucrose Substances 0.000 description 8
- 230000005945 translocation Effects 0.000 description 8
- 241000006382 Bacillus halodurans Species 0.000 description 7
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 7
- 102000053602 DNA Human genes 0.000 description 7
- 102100031132 Glucose-6-phosphate isomerase Human genes 0.000 description 7
- 108010070600 Glucose-6-phosphate isomerase Proteins 0.000 description 7
- 108010015895 Glycerone kinase Proteins 0.000 description 7
- 229910019142 PO4 Inorganic materials 0.000 description 7
- 108091081024 Start codon Proteins 0.000 description 7
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 7
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 7
- 230000000845 anti-microbial effect Effects 0.000 description 7
- 108010051210 beta-Fructofuranosidase Proteins 0.000 description 7
- 229910052799 carbon Inorganic materials 0.000 description 7
- 125000001547 cellobiose group Chemical group 0.000 description 7
- 239000003795 chemical substances by application Substances 0.000 description 7
- 239000003623 enhancer Substances 0.000 description 7
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 7
- 125000000487 histidyl group Chemical group [H]N([H])C(C(=O)O*)C([H])([H])C1=C([H])N([H])C([H])=N1 0.000 description 7
- 230000001939 inductive effect Effects 0.000 description 7
- 238000003780 insertion Methods 0.000 description 7
- 230000037431 insertion Effects 0.000 description 7
- 229940039696 lactobacillus Drugs 0.000 description 7
- 239000000463 material Substances 0.000 description 7
- 230000004048 modification Effects 0.000 description 7
- 238000012986 modification Methods 0.000 description 7
- 241000894007 species Species 0.000 description 7
- 239000007858 starting material Substances 0.000 description 7
- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 description 6
- 108090000994 Catalytic RNA Proteins 0.000 description 6
- 102000053642 Catalytic RNA Human genes 0.000 description 6
- 102000004163 DNA-directed RNA polymerases Human genes 0.000 description 6
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 6
- 108010070675 Glutathione transferase Proteins 0.000 description 6
- 102000005720 Glutathione transferase Human genes 0.000 description 6
- HNDVDQJCIGZPNO-YFKPBYRVSA-N L-histidine Chemical compound OC(=O)[C@@H](N)CC1=CN=CN1 HNDVDQJCIGZPNO-YFKPBYRVSA-N 0.000 description 6
- 101710175625 Maltose/maltodextrin-binding periplasmic protein Proteins 0.000 description 6
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 description 6
- 235000016383 Zea mays subsp huehuetenangensis Nutrition 0.000 description 6
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 description 6
- 230000000875 corresponding effect Effects 0.000 description 6
- 235000013365 dairy product Nutrition 0.000 description 6
- 238000012217 deletion Methods 0.000 description 6
- 230000037430 deletion Effects 0.000 description 6
- 238000001727 in vivo Methods 0.000 description 6
- 235000009973 maize Nutrition 0.000 description 6
- 238000010369 molecular cloning Methods 0.000 description 6
- 238000002703 mutagenesis Methods 0.000 description 6
- 231100000350 mutagenesis Toxicity 0.000 description 6
- 239000010452 phosphate Substances 0.000 description 6
- 108091092562 ribozyme Proteins 0.000 description 6
- 238000012216 screening Methods 0.000 description 6
- 210000001519 tissue Anatomy 0.000 description 6
- 230000003827 upregulation Effects 0.000 description 6
- 102000021527 ATP binding proteins Human genes 0.000 description 5
- 108091011108 ATP binding proteins Proteins 0.000 description 5
- 244000063299 Bacillus subtilis Species 0.000 description 5
- 235000014469 Bacillus subtilis Nutrition 0.000 description 5
- 108010077805 Bacterial Proteins Proteins 0.000 description 5
- 108010029692 Bisphosphoglycerate mutase Proteins 0.000 description 5
- 241000193468 Clostridium perfringens Species 0.000 description 5
- 229920002670 Fructan Polymers 0.000 description 5
- 102000002464 Galactosidases Human genes 0.000 description 5
- 108010093031 Galactosidases Proteins 0.000 description 5
- 102000051366 Glycosyltransferases Human genes 0.000 description 5
- 108700023372 Glycosyltransferases Proteins 0.000 description 5
- 108700023483 L-lactate dehydrogenases Proteins 0.000 description 5
- 241001465754 Metazoa Species 0.000 description 5
- 108020004711 Nucleic Acid Probes Proteins 0.000 description 5
- 108091005461 Nucleic proteins Proteins 0.000 description 5
- 108091005804 Peptidases Proteins 0.000 description 5
- 102000035195 Peptidases Human genes 0.000 description 5
- 108010069341 Phosphofructokinases Proteins 0.000 description 5
- 102000001105 Phosphofructokinases Human genes 0.000 description 5
- LCTONWCANYUPML-UHFFFAOYSA-M Pyruvate Chemical compound CC(=O)C([O-])=O LCTONWCANYUPML-UHFFFAOYSA-M 0.000 description 5
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 description 5
- 241000194019 Streptococcus mutans Species 0.000 description 5
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 5
- 238000007792 addition Methods 0.000 description 5
- 108090000637 alpha-Amylases Proteins 0.000 description 5
- 238000003491 array Methods 0.000 description 5
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 5
- 230000015556 catabolic process Effects 0.000 description 5
- 238000010367 cloning Methods 0.000 description 5
- 230000001086 cytosolic effect Effects 0.000 description 5
- 239000008103 glucose Substances 0.000 description 5
- 150000004676 glycans Chemical class 0.000 description 5
- 230000012010 growth Effects 0.000 description 5
- 230000007062 hydrolysis Effects 0.000 description 5
- 238000006460 hydrolysis reaction Methods 0.000 description 5
- 230000000977 initiatory effect Effects 0.000 description 5
- 238000002372 labelling Methods 0.000 description 5
- 239000011159 matrix material Substances 0.000 description 5
- 230000001404 mediated effect Effects 0.000 description 5
- 235000013336 milk Nutrition 0.000 description 5
- 239000008267 milk Substances 0.000 description 5
- 210000004080 milk Anatomy 0.000 description 5
- 239000002853 nucleic acid probe Substances 0.000 description 5
- 239000013612 plasmid Substances 0.000 description 5
- 229920001282 polysaccharide Polymers 0.000 description 5
- 239000005017 polysaccharide Substances 0.000 description 5
- 230000009046 primary transport Effects 0.000 description 5
- 239000013589 supplement Substances 0.000 description 5
- 235000013618 yogurt Nutrition 0.000 description 5
- 244000283070 Abies balsamea Species 0.000 description 4
- 235000007173 Abies balsamea Nutrition 0.000 description 4
- 241000589155 Agrobacterium tumefaciens Species 0.000 description 4
- 102000005369 Aldehyde Dehydrogenase Human genes 0.000 description 4
- 108020002663 Aldehyde Dehydrogenase Proteins 0.000 description 4
- 241000193830 Bacillus <bacterium> Species 0.000 description 4
- 108091033380 Coding strand Proteins 0.000 description 4
- 108020004705 Codon Proteins 0.000 description 4
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 4
- 108090000626 DNA-directed RNA polymerases Proteins 0.000 description 4
- 108010068561 Fructose-Bisphosphate Aldolase Proteins 0.000 description 4
- 102000001390 Fructose-Bisphosphate Aldolase Human genes 0.000 description 4
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 4
- 102000005744 Glycoside Hydrolases Human genes 0.000 description 4
- 108010031186 Glycoside Hydrolases Proteins 0.000 description 4
- 101001059454 Homo sapiens Serine/threonine-protein kinase MARK2 Proteins 0.000 description 4
- 108010028688 Isoamylase Proteins 0.000 description 4
- 102000003855 L-lactate dehydrogenase Human genes 0.000 description 4
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 4
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 4
- 229920002097 Lichenin Polymers 0.000 description 4
- 102000015841 Major facilitator superfamily Human genes 0.000 description 4
- 108050004064 Major facilitator superfamily Proteins 0.000 description 4
- 229930195725 Mannitol Natural products 0.000 description 4
- 235000002637 Nicotiana tabacum Nutrition 0.000 description 4
- 244000061176 Nicotiana tabacum Species 0.000 description 4
- 108700026244 Open Reading Frames Proteins 0.000 description 4
- 108010092494 Periplasmic binding proteins Proteins 0.000 description 4
- 102000012288 Phosphopyruvate Hydratase Human genes 0.000 description 4
- 108010022181 Phosphopyruvate Hydratase Proteins 0.000 description 4
- 108020005115 Pyruvate Kinase Proteins 0.000 description 4
- 102000013009 Pyruvate Kinase Human genes 0.000 description 4
- 102100028904 Serine/threonine-protein kinase MARK2 Human genes 0.000 description 4
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 4
- 238000009825 accumulation Methods 0.000 description 4
- 239000000556 agonist Substances 0.000 description 4
- 239000005557 antagonist Substances 0.000 description 4
- 239000004599 antimicrobial Substances 0.000 description 4
- 238000013459 approach Methods 0.000 description 4
- 230000009286 beneficial effect Effects 0.000 description 4
- RNBGYGVWRKECFJ-ARQDHWQXSA-N beta-D-fructofuranose 1,6-bisphosphate Chemical compound O[C@H]1[C@H](O)[C@@](O)(COP(O)(O)=O)O[C@@H]1COP(O)(O)=O RNBGYGVWRKECFJ-ARQDHWQXSA-N 0.000 description 4
- 230000033228 biological regulation Effects 0.000 description 4
- 230000001851 biosynthetic effect Effects 0.000 description 4
- 210000000170 cell membrane Anatomy 0.000 description 4
- 238000006731 degradation reaction Methods 0.000 description 4
- GNGACRATGGDKBX-UHFFFAOYSA-N dihydroxyacetone phosphate Chemical compound OCC(=O)COP(O)(O)=O GNGACRATGGDKBX-UHFFFAOYSA-N 0.000 description 4
- 102000006602 glyceraldehyde-3-phosphate dehydrogenase Human genes 0.000 description 4
- 108020004445 glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 description 4
- 125000003147 glycosyl group Chemical group 0.000 description 4
- 235000011073 invertase Nutrition 0.000 description 4
- 150000002500 ions Chemical class 0.000 description 4
- 238000002955 isolation Methods 0.000 description 4
- 239000000594 mannitol Substances 0.000 description 4
- 235000010355 mannitol Nutrition 0.000 description 4
- 239000003550 marker Substances 0.000 description 4
- 230000007246 mechanism Effects 0.000 description 4
- 229930182817 methionine Natural products 0.000 description 4
- 238000002493 microarray Methods 0.000 description 4
- 230000000813 microbial effect Effects 0.000 description 4
- 230000002018 overexpression Effects 0.000 description 4
- 239000002243 precursor Substances 0.000 description 4
- 238000002360 preparation method Methods 0.000 description 4
- 239000002987 primer (paints) Substances 0.000 description 4
- 101150045242 ptsH gene Proteins 0.000 description 4
- 238000003259 recombinant expression Methods 0.000 description 4
- 230000002441 reversible effect Effects 0.000 description 4
- 150000003839 salts Chemical class 0.000 description 4
- 239000011780 sodium chloride Substances 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- 241001515965 unidentified phage Species 0.000 description 4
- 238000011144 upstream manufacturing Methods 0.000 description 4
- 238000005406 washing Methods 0.000 description 4
- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 description 3
- ZFTFOHBYVDOAMH-XNOIKFDKSA-N (2r,3s,4s,5r)-5-[[(2r,3s,4s,5r)-5-[[(2r,3s,4s,5r)-3,4-dihydroxy-2,5-bis(hydroxymethyl)oxolan-2-yl]oxymethyl]-3,4-dihydroxy-2-(hydroxymethyl)oxolan-2-yl]oxymethyl]-2-(hydroxymethyl)oxolane-2,3,4-triol Chemical class O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)OC[C@@H]1[C@@H](O)[C@H](O)[C@](CO)(OC[C@@H]2[C@H]([C@H](O)[C@@](O)(CO)O2)O)O1 ZFTFOHBYVDOAMH-XNOIKFDKSA-N 0.000 description 3
- LXJXRIRHZLFYRP-VKHMYHEASA-L (R)-2-Hydroxy-3-(phosphonooxy)-propanal Natural products O=C[C@H](O)COP([O-])([O-])=O LXJXRIRHZLFYRP-VKHMYHEASA-L 0.000 description 3
- OSJPPGNTCRNQQC-UHFFFAOYSA-N 3-phosphoglyceric acid Chemical compound OC(=O)C(O)COP(O)(O)=O OSJPPGNTCRNQQC-UHFFFAOYSA-N 0.000 description 3
- 108700014167 ABC-type maltose transporter activity proteins Proteins 0.000 description 3
- 244000241257 Cucumis melo Species 0.000 description 3
- XPYBSIWDXQFNMH-UHFFFAOYSA-N D-fructose 1,6-bisphosphate Natural products OP(=O)(O)OCC(O)C(O)C(O)C(=O)COP(O)(O)=O XPYBSIWDXQFNMH-UHFFFAOYSA-N 0.000 description 3
- LXJXRIRHZLFYRP-VKHMYHEASA-N D-glyceraldehyde 3-phosphate Chemical compound O=C[C@H](O)COP(O)(O)=O LXJXRIRHZLFYRP-VKHMYHEASA-N 0.000 description 3
- 229920002444 Exopolysaccharide Polymers 0.000 description 3
- 102100026859 FAD-AMP lyase (cyclizing) Human genes 0.000 description 3
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 3
- 239000004471 Glycine Substances 0.000 description 3
- 229920002527 Glycogen Polymers 0.000 description 3
- 102000052862 Glycoside hydrolase family 31 Human genes 0.000 description 3
- 108700035759 Glycoside hydrolase family 31 Proteins 0.000 description 3
- 241000282412 Homo Species 0.000 description 3
- 241000282414 Homo sapiens Species 0.000 description 3
- 206010020649 Hyperkeratosis Diseases 0.000 description 3
- 102000004195 Isomerases Human genes 0.000 description 3
- 108090000769 Isomerases Proteins 0.000 description 3
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 description 3
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 description 3
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 3
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 description 3
- 244000199866 Lactobacillus casei Species 0.000 description 3
- 235000013958 Lactobacillus casei Nutrition 0.000 description 3
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 description 3
- 241000186781 Listeria Species 0.000 description 3
- 108050001696 Major intrinsic proteins Proteins 0.000 description 3
- 102000011364 Major intrinsic proteins Human genes 0.000 description 3
- 108010026217 Malate Dehydrogenase Proteins 0.000 description 3
- 102000013460 Malate Dehydrogenase Human genes 0.000 description 3
- 241000124008 Mammalia Species 0.000 description 3
- 108091092724 Noncoding DNA Proteins 0.000 description 3
- 101710163270 Nuclease Proteins 0.000 description 3
- 102000011755 Phosphoglycerate Kinase Human genes 0.000 description 3
- 102000004160 Phosphoric Monoester Hydrolases Human genes 0.000 description 3
- 108090000608 Phosphoric Monoester Hydrolases Proteins 0.000 description 3
- MUPFEKGTMRGPLJ-RMMQSMQOSA-N Raffinose Natural products O(C[C@H]1[C@@H](O)[C@H](O)[C@@H](O)[C@@H](O[C@@]2(CO)[C@H](O)[C@@H](O)[C@@H](CO)O2)O1)[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 MUPFEKGTMRGPLJ-RMMQSMQOSA-N 0.000 description 3
- 240000006394 Sorghum bicolor Species 0.000 description 3
- 241000194017 Streptococcus Species 0.000 description 3
- 101710180600 Sucrose operon repressor Proteins 0.000 description 3
- 108050007025 Sugar transport proteins Proteins 0.000 description 3
- 102000017952 Sugar transport proteins Human genes 0.000 description 3
- 102000003673 Symporters Human genes 0.000 description 3
- 108090000088 Symporters Proteins 0.000 description 3
- 101001099217 Thermotoga maritima (strain ATCC 43589 / DSM 3109 / JCM 10099 / NBRC 100826 / MSB8) Triosephosphate isomerase Proteins 0.000 description 3
- 108090000190 Thrombin Proteins 0.000 description 3
- 241000723792 Tobacco etch virus Species 0.000 description 3
- 108700019146 Transgenes Proteins 0.000 description 3
- MUPFEKGTMRGPLJ-UHFFFAOYSA-N UNPD196149 Natural products OC1C(O)C(CO)OC1(CO)OC1C(O)C(O)C(O)C(COC2C(C(O)C(O)C(CO)O2)O)O1 MUPFEKGTMRGPLJ-UHFFFAOYSA-N 0.000 description 3
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Natural products CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 description 3
- 241000700605 Viruses Species 0.000 description 3
- 108091022872 aldose 1-epimerase Proteins 0.000 description 3
- 102000020006 aldose 1-epimerase Human genes 0.000 description 3
- 108010030291 alpha-Galactosidase Proteins 0.000 description 3
- 230000004075 alteration Effects 0.000 description 3
- 230000000890 antigenic effect Effects 0.000 description 3
- 102000005936 beta-Galactosidase Human genes 0.000 description 3
- 101150029503 bglG gene Proteins 0.000 description 3
- 239000013060 biological fluid Substances 0.000 description 3
- 108091006374 cAMP receptor proteins Proteins 0.000 description 3
- 230000001925 catabolic effect Effects 0.000 description 3
- 230000008878 coupling Effects 0.000 description 3
- 238000010168 coupling process Methods 0.000 description 3
- 238000005859 coupling reaction Methods 0.000 description 3
- 210000000805 cytoplasm Anatomy 0.000 description 3
- 235000011180 diphosphates Nutrition 0.000 description 3
- 230000003828 downregulation Effects 0.000 description 3
- 238000001952 enzyme assay Methods 0.000 description 3
- RNBGYGVWRKECFJ-UHFFFAOYSA-N fructose-1,6-phosphate Natural products OC1C(O)C(O)(COP(O)(O)=O)OC1COP(O)(O)=O RNBGYGVWRKECFJ-UHFFFAOYSA-N 0.000 description 3
- 230000005714 functional activity Effects 0.000 description 3
- FBPFZTCFMRRESA-GUCUJZIJSA-N galactitol Chemical compound OC[C@H](O)[C@@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-GUCUJZIJSA-N 0.000 description 3
- 235000013922 glutamic acid Nutrition 0.000 description 3
- 239000004220 glutamic acid Substances 0.000 description 3
- 229940096919 glycogen Drugs 0.000 description 3
- 108700014210 glycosyltransferase activity proteins Proteins 0.000 description 3
- 239000001963 growth medium Substances 0.000 description 3
- 230000036541 health Effects 0.000 description 3
- 230000001976 improved effect Effects 0.000 description 3
- 238000006317 isomerization reaction Methods 0.000 description 3
- 229940017800 lactobacillus casei Drugs 0.000 description 3
- 239000003446 ligand Substances 0.000 description 3
- 125000000311 mannosyl group Chemical group C1([C@@H](O)[C@@H](O)[C@H](O)[C@H](O1)CO)* 0.000 description 3
- 230000036961 partial effect Effects 0.000 description 3
- 108010071189 phosphoenolpyruvate-glucose phosphotransferase Proteins 0.000 description 3
- KCRZDTROFIOPBP-UHFFFAOYSA-N phosphono 2,3-dihydroxypropanoate Chemical compound OCC(O)C(=O)OP(O)(O)=O KCRZDTROFIOPBP-UHFFFAOYSA-N 0.000 description 3
- 235000019833 protease Nutrition 0.000 description 3
- 230000004952 protein activity Effects 0.000 description 3
- 238000003498 protein array Methods 0.000 description 3
- MUPFEKGTMRGPLJ-ZQSKZDJDSA-N raffinose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO[C@@H]2[C@@H]([C@@H](O)[C@@H](O)[C@@H](CO)O2)O)O1 MUPFEKGTMRGPLJ-ZQSKZDJDSA-N 0.000 description 3
- 230000002829 reductive effect Effects 0.000 description 3
- 229920005989 resin Polymers 0.000 description 3
- 239000011347 resin Substances 0.000 description 3
- 238000012552 review Methods 0.000 description 3
- 238000007423 screening assay Methods 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 230000004083 survival effect Effects 0.000 description 3
- 229960003495 thiamine Drugs 0.000 description 3
- 229960004072 thrombin Drugs 0.000 description 3
- 230000005030 transcription termination Effects 0.000 description 3
- 230000001131 transforming effect Effects 0.000 description 3
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 3
- 239000004474 valine Substances 0.000 description 3
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 2
- 108090000344 1,4-alpha-Glucan Branching Enzyme Proteins 0.000 description 2
- 102000003925 1,4-alpha-Glucan Branching Enzyme Human genes 0.000 description 2
- XOHUEYCVLUUEJJ-UHFFFAOYSA-N 2,3-Bisphosphoglyceric acid Chemical compound OP(=O)(O)OC(C(=O)O)COP(O)(O)=O XOHUEYCVLUUEJJ-UHFFFAOYSA-N 0.000 description 2
- 108010082310 2-hydroxyacid dehydrogenase Proteins 0.000 description 2
- MWMOPIVLTLEUJO-UHFFFAOYSA-N 2-oxopropanoic acid;phosphoric acid Chemical compound OP(O)(O)=O.CC(=O)C(O)=O MWMOPIVLTLEUJO-UHFFFAOYSA-N 0.000 description 2
- GXIURPTVHJPJLF-UWTATZPHSA-N 2-phosphoglycerate Natural products OC[C@H](C(O)=O)OP(O)(O)=O GXIURPTVHJPJLF-UWTATZPHSA-N 0.000 description 2
- GXIURPTVHJPJLF-UHFFFAOYSA-N 2-phosphoglyceric acid Chemical compound OCC(C(O)=O)OP(O)(O)=O GXIURPTVHJPJLF-UHFFFAOYSA-N 0.000 description 2
- 102000009878 3-Hydroxysteroid Dehydrogenases Human genes 0.000 description 2
- HSJKGGMUJITCBW-UHFFFAOYSA-N 3-hydroxybutanal Chemical compound CC(O)CC=O HSJKGGMUJITCBW-UHFFFAOYSA-N 0.000 description 2
- 108010034869 6-phospho-beta-glucosidase Proteins 0.000 description 2
- 229920000936 Agarose Polymers 0.000 description 2
- 102100026277 Alpha-galactosidase A Human genes 0.000 description 2
- 108010039224 Amidophosphoribosyltransferase Proteins 0.000 description 2
- 244000144725 Amygdalus communis Species 0.000 description 2
- 235000011437 Amygdalus communis Nutrition 0.000 description 2
- 102000003669 Antiporters Human genes 0.000 description 2
- 108090000084 Antiporters Proteins 0.000 description 2
- 241000219194 Arabidopsis Species 0.000 description 2
- 244000105624 Arachis hypogaea Species 0.000 description 2
- 101100174784 Bacillus subtilis (strain 168) ganR gene Proteins 0.000 description 2
- 101100128225 Bacillus subtilis (strain 168) licT gene Proteins 0.000 description 2
- 108020004513 Bacterial RNA Proteins 0.000 description 2
- 241000335053 Beta vulgaris Species 0.000 description 2
- 102100036200 Bisphosphoglycerate mutase Human genes 0.000 description 2
- 241001674345 Callitropsis nootkatensis Species 0.000 description 2
- 235000009467 Carica papaya Nutrition 0.000 description 2
- 240000006432 Carica papaya Species 0.000 description 2
- 235000003255 Carthamus tinctorius Nutrition 0.000 description 2
- 244000020518 Carthamus tinctorius Species 0.000 description 2
- 241000207199 Citrus Species 0.000 description 2
- 241000193461 Clostridium longisporum Species 0.000 description 2
- 235000013162 Cocos nucifera Nutrition 0.000 description 2
- 244000060011 Cocos nucifera Species 0.000 description 2
- 241000723377 Coffea Species 0.000 description 2
- 241000218631 Coniferophyta Species 0.000 description 2
- 108091035707 Consensus sequence Proteins 0.000 description 2
- 235000009847 Cucumis melo var cantalupensis Nutrition 0.000 description 2
- 240000008067 Cucumis sativus Species 0.000 description 2
- 108090000695 Cytokines Proteins 0.000 description 2
- 102000004127 Cytokines Human genes 0.000 description 2
- GSXOAOHZAIYLCY-UHFFFAOYSA-N D-F6P Natural products OCC(=O)C(O)C(O)C(O)COP(O)(O)=O GSXOAOHZAIYLCY-UHFFFAOYSA-N 0.000 description 2
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 description 2
- FBPFZTCFMRRESA-JGWLITMVSA-N D-glucitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-JGWLITMVSA-N 0.000 description 2
- 108010066133 D-octopine dehydrogenase Proteins 0.000 description 2
- 239000003298 DNA probe Substances 0.000 description 2
- 230000004568 DNA-binding Effects 0.000 description 2
- 108020005199 Dehydrogenases Proteins 0.000 description 2
- 229920001353 Dextrin Polymers 0.000 description 2
- 239000004375 Dextrin Substances 0.000 description 2
- QSJXEFYPDANLFS-UHFFFAOYSA-N Diacetyl Chemical group CC(=O)C(C)=O QSJXEFYPDANLFS-UHFFFAOYSA-N 0.000 description 2
- 238000002965 ELISA Methods 0.000 description 2
- 244000078127 Eleusine coracana Species 0.000 description 2
- ULGZDMOVFRHVEP-RWJQBGPGSA-N Erythromycin Chemical compound O([C@@H]1[C@@H](C)C(=O)O[C@@H]([C@@]([C@H](O)[C@@H](C)C(=O)[C@H](C)C[C@@](C)(O)[C@H](O[C@H]2[C@@H]([C@H](C[C@@H](C)O2)N(C)C)O)[C@H]1C)(C)O)CC)[C@H]1C[C@@](C)(OC)[C@@H](O)[C@H](C)O1 ULGZDMOVFRHVEP-RWJQBGPGSA-N 0.000 description 2
- 241000701959 Escherichia virus Lambda Species 0.000 description 2
- 108010074860 Factor Xa Proteins 0.000 description 2
- 241000192125 Firmicutes Species 0.000 description 2
- 101001076781 Fructilactobacillus sanfranciscensis (strain ATCC 27651 / DSM 20451 / JCM 5668 / CCUG 30143 / KCTC 3205 / NCIMB 702811 / NRRL B-3934 / L-12) Ribose-5-phosphate isomerase A Proteins 0.000 description 2
- 108700039691 Genetic Promoter Regions Proteins 0.000 description 2
- 229920001503 Glucan Polymers 0.000 description 2
- 235000010469 Glycine max Nutrition 0.000 description 2
- 244000068988 Glycine max Species 0.000 description 2
- 244000299507 Gossypium hirsutum Species 0.000 description 2
- NYHBQMYGNKIUIF-UUOKFMHZSA-N Guanosine Chemical compound C1=NC=2C(=O)NC(N)=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O NYHBQMYGNKIUIF-UUOKFMHZSA-N 0.000 description 2
- 244000020551 Helianthus annuus Species 0.000 description 2
- 235000003222 Helianthus annuus Nutrition 0.000 description 2
- 102000005548 Hexokinase Human genes 0.000 description 2
- 108700040460 Hexokinases Proteins 0.000 description 2
- 235000005206 Hibiscus Nutrition 0.000 description 2
- 235000007185 Hibiscus lunariifolius Nutrition 0.000 description 2
- 244000284380 Hibiscus rosa sinensis Species 0.000 description 2
- 244000267823 Hydrangea macrophylla Species 0.000 description 2
- 235000014486 Hydrangea macrophylla Nutrition 0.000 description 2
- 108010050904 Interferons Proteins 0.000 description 2
- 102000014150 Interferons Human genes 0.000 description 2
- 108091092195 Intron Proteins 0.000 description 2
- 108010044467 Isoenzymes Proteins 0.000 description 2
- LKDRXBCSQODPBY-AMVSKUEXSA-N L-(-)-Sorbose Chemical compound OCC1(O)OC[C@H](O)[C@@H](O)[C@@H]1O LKDRXBCSQODPBY-AMVSKUEXSA-N 0.000 description 2
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 2
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 2
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 2
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 description 2
- AYFVYJQAPQTCCC-GBXIJSLDSA-N L-threonine Chemical compound C[C@@H](O)[C@H](N)C(O)=O AYFVYJQAPQTCCC-GBXIJSLDSA-N 0.000 description 2
- 108010054278 Lac Repressors Proteins 0.000 description 2
- JVTAAEKCZFNVCJ-UHFFFAOYSA-M Lactate Chemical compound CC(O)C([O-])=O JVTAAEKCZFNVCJ-UHFFFAOYSA-M 0.000 description 2
- 241000209510 Liliopsida Species 0.000 description 2
- 235000007688 Lycopersicon esculentum Nutrition 0.000 description 2
- 241000710118 Maize chlorotic mottle virus Species 0.000 description 2
- 241000723994 Maize dwarf mosaic virus Species 0.000 description 2
- 235000014826 Mangifera indica Nutrition 0.000 description 2
- 240000007228 Mangifera indica Species 0.000 description 2
- 240000003183 Manihot esculenta Species 0.000 description 2
- 108090000428 Mannitol-1-phosphate 5-dehydrogenases Proteins 0.000 description 2
- 101710161955 Mannitol-specific phosphotransferase enzyme IIA component Proteins 0.000 description 2
- 240000004658 Medicago sativa Species 0.000 description 2
- 108010052285 Membrane Proteins Proteins 0.000 description 2
- 102000014842 Multidrug resistance proteins Human genes 0.000 description 2
- 108050005144 Multidrug resistance proteins Proteins 0.000 description 2
- 108700035572 NAD-dependent epimerase/dehydratases Proteins 0.000 description 2
- 102000054449 NAD-dependent epimerase/dehydratases Human genes 0.000 description 2
- 241000234479 Narcissus Species 0.000 description 2
- 206010028980 Neoplasm Diseases 0.000 description 2
- 108010025020 Nerve Growth Factor Proteins 0.000 description 2
- 101710141454 Nucleoprotein Proteins 0.000 description 2
- 240000007817 Olea europaea Species 0.000 description 2
- 108010026867 Oligo-1,6-Glucosidase Proteins 0.000 description 2
- 240000007594 Oryza sativa Species 0.000 description 2
- 235000007164 Oryza sativa Nutrition 0.000 description 2
- 235000007199 Panicum miliaceum Nutrition 0.000 description 2
- 235000007195 Pennisetum typhoides Nutrition 0.000 description 2
- 102000007079 Peptide Fragments Human genes 0.000 description 2
- 108091093037 Peptide nucleic acid Proteins 0.000 description 2
- 244000025272 Persea americana Species 0.000 description 2
- 235000008673 Persea americana Nutrition 0.000 description 2
- 240000007377 Petunia x hybrida Species 0.000 description 2
- 235000010617 Phaseolus lunatus Nutrition 0.000 description 2
- 102000011025 Phosphoglycerate Mutase Human genes 0.000 description 2
- 235000008331 Pinus X rigitaeda Nutrition 0.000 description 2
- 235000011613 Pinus brutia Nutrition 0.000 description 2
- 241000018646 Pinus brutia Species 0.000 description 2
- 241000218606 Pinus contorta Species 0.000 description 2
- 235000013267 Pinus ponderosa Nutrition 0.000 description 2
- 235000008577 Pinus radiata Nutrition 0.000 description 2
- 241000218621 Pinus radiata Species 0.000 description 2
- 235000008566 Pinus taeda Nutrition 0.000 description 2
- 241000218679 Pinus taeda Species 0.000 description 2
- 239000004365 Protease Substances 0.000 description 2
- 108010009341 Protein Serine-Threonine Kinases Proteins 0.000 description 2
- 102000009516 Protein Serine-Threonine Kinases Human genes 0.000 description 2
- 240000001416 Pseudotsuga menziesii Species 0.000 description 2
- 108020004511 Recombinant DNA Proteins 0.000 description 2
- 108010034634 Repressor Proteins Proteins 0.000 description 2
- 241000208422 Rhododendron Species 0.000 description 2
- 102000046755 Ribokinases Human genes 0.000 description 2
- 102000006382 Ribonucleases Human genes 0.000 description 2
- 108010083644 Ribonucleases Proteins 0.000 description 2
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 2
- 235000007238 Secale cereale Nutrition 0.000 description 2
- 244000082988 Secale cereale Species 0.000 description 2
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 2
- 240000005498 Setaria italica Species 0.000 description 2
- 240000003768 Solanum lycopersicum Species 0.000 description 2
- 235000011684 Sorghum saccharatum Nutrition 0.000 description 2
- 241000191973 Staphylococcus xylosus Species 0.000 description 2
- 108020005038 Terminator Codon Proteins 0.000 description 2
- 239000004098 Tetracycline Substances 0.000 description 2
- 244000269722 Thea sinensis Species 0.000 description 2
- 244000299461 Theobroma cacao Species 0.000 description 2
- 235000009470 Theobroma cacao Nutrition 0.000 description 2
- JZRWCGZRTZMZEH-UHFFFAOYSA-N Thiamine Natural products CC1=C(CCO)SC=[N+]1CC1=CN=C(C)N=C1N JZRWCGZRTZMZEH-UHFFFAOYSA-N 0.000 description 2
- 108700038108 Thiamine pyrophosphate enzyme Proteins 0.000 description 2
- 102000052482 Thiamine pyrophosphate enzyme Human genes 0.000 description 2
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 description 2
- 239000004473 Threonine Substances 0.000 description 2
- 241000218638 Thuja plicata Species 0.000 description 2
- 241000723873 Tobacco mosaic virus Species 0.000 description 2
- 235000021307 Triticum Nutrition 0.000 description 2
- 241000209140 Triticum Species 0.000 description 2
- 108010058532 UTP-hexose-1-phosphate uridylyltransferase Proteins 0.000 description 2
- 102000006321 UTP-hexose-1-phosphate uridylyltransferase Human genes 0.000 description 2
- 102000037089 Uniporters Human genes 0.000 description 2
- 108091006293 Uniporters Proteins 0.000 description 2
- 238000002441 X-ray diffraction Methods 0.000 description 2
- 230000004913 activation Effects 0.000 description 2
- 239000012190 activator Substances 0.000 description 2
- 108091006088 activator proteins Proteins 0.000 description 2
- 230000009056 active transport Effects 0.000 description 2
- 235000004279 alanine Nutrition 0.000 description 2
- 150000001323 aldoses Chemical class 0.000 description 2
- 102000004139 alpha-Amylases Human genes 0.000 description 2
- 229940024171 alpha-amylase Drugs 0.000 description 2
- 229960000723 ampicillin Drugs 0.000 description 2
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 2
- 238000000137 annealing Methods 0.000 description 2
- 239000003242 anti bacterial agent Substances 0.000 description 2
- 229940088710 antibiotic agent Drugs 0.000 description 2
- 239000000074 antisense oligonucleotide Substances 0.000 description 2
- 238000012230 antisense oligonucleotides Methods 0.000 description 2
- 235000003704 aspartic acid Nutrition 0.000 description 2
- 210000003578 bacterial chromosome Anatomy 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- BGWGXPAPYGQALX-ARQDHWQXSA-N beta-D-fructofuranose 6-phosphate Chemical compound OC[C@@]1(O)O[C@H](COP(O)(O)=O)[C@@H](O)[C@@H]1O BGWGXPAPYGQALX-ARQDHWQXSA-N 0.000 description 2
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 description 2
- 244000022203 blackseeded proso millet Species 0.000 description 2
- 230000000903 blocking effect Effects 0.000 description 2
- 239000011575 calcium Substances 0.000 description 2
- 150000001768 cations Chemical class 0.000 description 2
- 230000001413 cellular effect Effects 0.000 description 2
- 238000012512 characterization method Methods 0.000 description 2
- 235000013351 cheese Nutrition 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000004587 chromatography analysis Methods 0.000 description 2
- 230000002759 chromosomal effect Effects 0.000 description 2
- 235000020971 citrus fruits Nutrition 0.000 description 2
- ASARMUCNOOHMLO-WLORSUFZSA-L cobalt(2+);[(2r,3s,4r,5s)-5-(5,6-dimethylbenzimidazol-1-yl)-4-hydroxy-2-(hydroxymethyl)oxolan-3-yl] [(2s)-1-[3-[(1r,2r,3r,4z,7s,9z,12s,13s,14z,17s,18s,19r)-2,13,18-tris(2-amino-2-oxoethyl)-7,12,17-tris(3-amino-3-oxopropyl)-3,5,8,8,13,15,18,19-octamethyl-2 Chemical compound [Co+2].[N-]([C@@H]1[C@H](CC(N)=O)[C@@]2(C)CCC(=O)NC[C@H](C)OP([O-])(=O)O[C@H]3[C@H]([C@H](O[C@@H]3CO)N3C4=CC(C)=C(C)C=C4N=C3)O)\C2=C(C)/C([C@H](C\2(C)C)CCC(N)=O)=N/C/2=C\C([C@H]([C@@]/2(CC(N)=O)C)CCC(N)=O)=N\C\2=C(C)/C2=N[C@]1(C)[C@@](C)(CC(N)=O)[C@@H]2CCC(N)=O ASARMUCNOOHMLO-WLORSUFZSA-L 0.000 description 2
- 238000002742 combinatorial mutagenesis Methods 0.000 description 2
- 239000003184 complementary RNA Substances 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 2
- 235000018417 cysteine Nutrition 0.000 description 2
- 125000000151 cysteine group Chemical group N[C@@H](CS)C(=O)* 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 235000019425 dextrin Nutrition 0.000 description 2
- 235000005911 diet Nutrition 0.000 description 2
- 235000015872 dietary supplement Nutrition 0.000 description 2
- 230000004069 differentiation Effects 0.000 description 2
- 239000000539 dimer Substances 0.000 description 2
- XPPKVPWEQAFLFU-UHFFFAOYSA-J diphosphate(4-) Chemical compound [O-]P([O-])(=O)OP([O-])([O-])=O XPPKVPWEQAFLFU-UHFFFAOYSA-J 0.000 description 2
- 230000006806 disease prevention Effects 0.000 description 2
- 239000012636 effector Substances 0.000 description 2
- 238000004520 electroporation Methods 0.000 description 2
- 210000002257 embryonic structure Anatomy 0.000 description 2
- 230000002708 enhancing effect Effects 0.000 description 2
- 230000006353 environmental stress Effects 0.000 description 2
- 241001233957 eudicotyledons Species 0.000 description 2
- 210000003527 eukaryotic cell Anatomy 0.000 description 2
- 230000004720 fertilization Effects 0.000 description 2
- 230000037406 food intake Effects 0.000 description 2
- 235000003599 food sweetener Nutrition 0.000 description 2
- 230000004110 gluconeogenesis Effects 0.000 description 2
- 230000004153 glucose metabolism Effects 0.000 description 2
- 229930182478 glucoside Natural products 0.000 description 2
- 150000008131 glucosides Chemical class 0.000 description 2
- 230000002414 glycolytic effect Effects 0.000 description 2
- 239000004009 herbicide Substances 0.000 description 2
- 229910052739 hydrogen Inorganic materials 0.000 description 2
- 239000001257 hydrogen Substances 0.000 description 2
- 230000002209 hydrophobic effect Effects 0.000 description 2
- 230000002163 immunogen Effects 0.000 description 2
- 230000008676 import Effects 0.000 description 2
- 230000006698 induction Effects 0.000 description 2
- 238000007689 inspection Methods 0.000 description 2
- 230000003993 interaction Effects 0.000 description 2
- 229940079322 interferon Drugs 0.000 description 2
- 239000000543 intermediate Substances 0.000 description 2
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 description 2
- 229960000310 isoleucine Drugs 0.000 description 2
- BJHIKXHVCXFQLS-PQLUHFTBSA-N keto-D-tagatose Chemical compound OC[C@@H](O)[C@H](O)[C@H](O)C(=O)CO BJHIKXHVCXFQLS-PQLUHFTBSA-N 0.000 description 2
- 101150043267 lacR gene Proteins 0.000 description 2
- 108020001756 ligand binding domains Proteins 0.000 description 2
- 230000000670 limiting effect Effects 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 230000033001 locomotion Effects 0.000 description 2
- 229910001425 magnesium ion Inorganic materials 0.000 description 2
- 238000012423 maintenance Methods 0.000 description 2
- 230000035800 maturation Effects 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 230000002503 metabolic effect Effects 0.000 description 2
- 239000003068 molecular probe Substances 0.000 description 2
- 230000036457 multidrug resistance Effects 0.000 description 2
- 210000004897 n-terminal region Anatomy 0.000 description 2
- 230000007935 neutral effect Effects 0.000 description 2
- 238000003499 nucleic acid array Methods 0.000 description 2
- 235000016709 nutrition Nutrition 0.000 description 2
- 229920001542 oligosaccharide Polymers 0.000 description 2
- 150000002482 oligosaccharides Chemical class 0.000 description 2
- 230000003647 oxidation Effects 0.000 description 2
- 238000007254 oxidation reaction Methods 0.000 description 2
- 101150100557 pfkB gene Proteins 0.000 description 2
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 description 2
- 235000021317 phosphate Nutrition 0.000 description 2
- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 description 2
- LFGREXWGYUGZLY-UHFFFAOYSA-N phosphoryl Chemical group [P]=O LFGREXWGYUGZLY-UHFFFAOYSA-N 0.000 description 2
- 230000000865 phosphorylative effect Effects 0.000 description 2
- 230000008488 polyadenylation Effects 0.000 description 2
- 230000002265 prevention Effects 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 210000001236 prokaryotic cell Anatomy 0.000 description 2
- 235000019419 proteases Nutrition 0.000 description 2
- 235000004252 protein component Nutrition 0.000 description 2
- 210000001938 protoplast Anatomy 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 108020003175 receptors Proteins 0.000 description 2
- 230000014493 regulation of gene expression Effects 0.000 description 2
- 238000009877 rendering Methods 0.000 description 2
- 230000008439 repair process Effects 0.000 description 2
- 230000004044 response Effects 0.000 description 2
- 230000028327 secretion Effects 0.000 description 2
- 230000001568 sexual effect Effects 0.000 description 2
- 229910001415 sodium ion Inorganic materials 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 239000000600 sorbitol Substances 0.000 description 2
- 238000012289 standard assay Methods 0.000 description 2
- 239000003765 sweetening agent Substances 0.000 description 2
- 230000002194 synthesizing effect Effects 0.000 description 2
- 229960002180 tetracycline Drugs 0.000 description 2
- 229930101283 tetracycline Natural products 0.000 description 2
- 235000019364 tetracycline Nutrition 0.000 description 2
- 150000003522 tetracyclines Chemical class 0.000 description 2
- 235000019157 thiamine Nutrition 0.000 description 2
- 239000011721 thiamine Substances 0.000 description 2
- KYMBYSLLVAOCFI-UHFFFAOYSA-N thiamine Chemical compound CC1=C(CCO)SCN1CC1=CN=C(C)N=C1N KYMBYSLLVAOCFI-UHFFFAOYSA-N 0.000 description 2
- 231100000167 toxic agent Toxicity 0.000 description 2
- 230000005026 transcription initiation Effects 0.000 description 2
- 108091008023 transcriptional regulators Proteins 0.000 description 2
- 108091006107 transcriptional repressors Proteins 0.000 description 2
- 238000001890 transfection Methods 0.000 description 2
- 238000011426 transformation method Methods 0.000 description 2
- 230000001052 transient effect Effects 0.000 description 2
- 230000007704 transition Effects 0.000 description 2
- 238000011282 treatment Methods 0.000 description 2
- 125000000026 trimethylsilyl group Chemical group [H]C([H])([H])[Si]([*])(C([H])([H])[H])C([H])([H])[H] 0.000 description 2
- 235000013311 vegetables Nutrition 0.000 description 2
- XDIYNQZUNSSENW-RPQBYJBYSA-N (2s,3s,4r,5r)-2,3,4,5,6-pentahydroxyhexanal;(2r,3s,4r,5r)-2,3,4,5,6-pentahydroxyhexanal Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C=O.OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)C=O XDIYNQZUNSSENW-RPQBYJBYSA-N 0.000 description 1
- OKZYCXHTTZZYSK-ZCFIWIBFSA-N (R)-5-phosphomevalonic acid Chemical compound OC(=O)C[C@@](O)(C)CCOP(O)(O)=O OKZYCXHTTZZYSK-ZCFIWIBFSA-N 0.000 description 1
- KJTLQQUUPVSXIM-ZCFIWIBFSA-M (R)-mevalonate Chemical compound OCC[C@](O)(C)CC([O-])=O KJTLQQUUPVSXIM-ZCFIWIBFSA-M 0.000 description 1
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 1
- UKAUYVFTDYCKQA-UHFFFAOYSA-N -2-Amino-4-hydroxybutanoic acid Natural products OC(=O)C(N)CCO UKAUYVFTDYCKQA-UHFFFAOYSA-N 0.000 description 1
- 108091064702 1 family Proteins 0.000 description 1
- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 description 1
- 108020004465 16S ribosomal RNA Proteins 0.000 description 1
- CHUGKEQJSLOLHL-UHFFFAOYSA-N 2,2-Bis(bromomethyl)propane-1,3-diol Chemical group OCC(CO)(CBr)CBr CHUGKEQJSLOLHL-UHFFFAOYSA-N 0.000 description 1
- AYRXSINWFIIFAE-UHFFFAOYSA-N 2,3,4,5-tetrahydroxy-6-[3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxyhexanal Chemical compound OCC1OC(OCC(O)C(O)C(O)C(O)C=O)C(O)C(O)C1O AYRXSINWFIIFAE-UHFFFAOYSA-N 0.000 description 1
- XOHUEYCVLUUEJJ-UHFFFAOYSA-I 2,3-Diphosphoglycerate Chemical compound [O-]P(=O)([O-])OC(C(=O)[O-])COP([O-])([O-])=O XOHUEYCVLUUEJJ-UHFFFAOYSA-I 0.000 description 1
- ADTZYXYDFDENOA-UHFFFAOYSA-N 2-(carboxymethylamino)acetic acid;nickel Chemical compound [Ni].OC(=O)CNCC(O)=O ADTZYXYDFDENOA-UHFFFAOYSA-N 0.000 description 1
- IFNXFIJXYVEYLF-UHFFFAOYSA-N 2-Propylglutaric acid Chemical compound CCCC(C(O)=O)CCC(O)=O IFNXFIJXYVEYLF-UHFFFAOYSA-N 0.000 description 1
- FMYBFLOWKQRBST-UHFFFAOYSA-N 2-[bis(carboxymethyl)amino]acetic acid;nickel Chemical compound [Ni].OC(=O)CN(CC(O)=O)CC(O)=O FMYBFLOWKQRBST-UHFFFAOYSA-N 0.000 description 1
- ASJSAQIRZKANQN-CRCLSJGQSA-N 2-deoxy-D-ribose Chemical compound OC[C@@H](O)[C@@H](O)CC=O ASJSAQIRZKANQN-CRCLSJGQSA-N 0.000 description 1
- 108091071337 20 family Proteins 0.000 description 1
- OSJPPGNTCRNQQC-UWTATZPHSA-N 3-phospho-D-glyceric acid Chemical compound OC(=O)[C@H](O)COP(O)(O)=O OSJPPGNTCRNQQC-UWTATZPHSA-N 0.000 description 1
- LJQLQCAXBUHEAZ-UWTATZPHSA-N 3-phospho-D-glyceroyl dihydrogen phosphate Chemical compound OP(=O)(O)OC[C@@H](O)C(=O)OP(O)(O)=O LJQLQCAXBUHEAZ-UWTATZPHSA-N 0.000 description 1
- AUNGANRZJHBGPY-MBNYWOFBSA-N 7,8-dimethyl-10-[(2R,3R,4S)-2,3,4,5-tetrahydroxypentyl]benzo[g]pteridine-2,4-dione Chemical compound OC[C@H](O)[C@H](O)[C@H](O)CN1C=2C=C(C)C(C)=CC=2N=C2C1=NC(=O)NC2=O AUNGANRZJHBGPY-MBNYWOFBSA-N 0.000 description 1
- 101710191093 A' protein Proteins 0.000 description 1
- 108050001144 ABC transporter, permeases Proteins 0.000 description 1
- 108700014205 ABC-type glycerol-3-phosphate transporter activity proteins Proteins 0.000 description 1
- 108700014181 ABC-type protein transporter activity proteins Proteins 0.000 description 1
- 108091006112 ATPases Proteins 0.000 description 1
- 235000004507 Abies alba Nutrition 0.000 description 1
- 235000014081 Abies amabilis Nutrition 0.000 description 1
- 244000101408 Abies amabilis Species 0.000 description 1
- 244000178606 Abies grandis Species 0.000 description 1
- 235000017894 Abies grandis Nutrition 0.000 description 1
- 235000004710 Abies lasiocarpa Nutrition 0.000 description 1
- 240000005020 Acaciella glauca Species 0.000 description 1
- 108010000700 Acetolactate synthase Proteins 0.000 description 1
- 101100295756 Acinetobacter baumannii (strain ATCC 19606 / DSM 30007 / JCM 6841 / CCUG 19606 / CIP 70.34 / NBRC 109757 / NCIMB 12457 / NCTC 12156 / 81) omp38 gene Proteins 0.000 description 1
- 101710159080 Aconitate hydratase A Proteins 0.000 description 1
- 101710159078 Aconitate hydratase B Proteins 0.000 description 1
- 102100033647 Activity-regulated cytoskeleton-associated protein Human genes 0.000 description 1
- 102000057290 Adenosine Triphosphatases Human genes 0.000 description 1
- 102100032157 Adenylate cyclase type 10 Human genes 0.000 description 1
- 241000193798 Aerococcus Species 0.000 description 1
- 241000589158 Agrobacterium Species 0.000 description 1
- 101100125297 Agrobacterium vitis (strain S4 / ATCC BAA-846) iaaH gene Proteins 0.000 description 1
- 108010031025 Alanine Dehydrogenase Proteins 0.000 description 1
- 108090000072 Aldehyde-Lyases Proteins 0.000 description 1
- 102000003677 Aldehyde-Lyases Human genes 0.000 description 1
- 241000724328 Alfalfa mosaic virus Species 0.000 description 1
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 1
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 1
- 108700028369 Alleles Proteins 0.000 description 1
- 241000186033 Alloiococcus Species 0.000 description 1
- 241001212612 Allora Species 0.000 description 1
- 108050005273 Amino acid transporters Proteins 0.000 description 1
- 102000034263 Amino acid transporters Human genes 0.000 description 1
- 229920000945 Amylopectin Polymers 0.000 description 1
- 229920000856 Amylose Polymers 0.000 description 1
- 235000001271 Anacardium Nutrition 0.000 description 1
- 241000693997 Anacardium Species 0.000 description 1
- 244000226021 Anacardium occidentale Species 0.000 description 1
- 244000099147 Ananas comosus Species 0.000 description 1
- 235000007119 Ananas comosus Nutrition 0.000 description 1
- 108020005544 Antisense RNA Proteins 0.000 description 1
- 102000010637 Aquaporins Human genes 0.000 description 1
- 108010063290 Aquaporins Proteins 0.000 description 1
- 235000010777 Arachis hypogaea Nutrition 0.000 description 1
- 239000004475 Arginine Substances 0.000 description 1
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 description 1
- 235000007319 Avena orientalis Nutrition 0.000 description 1
- 244000075850 Avena orientalis Species 0.000 description 1
- 101100354312 Bacillus subtilis (strain 168) licC gene Proteins 0.000 description 1
- 101100299636 Bacillus subtilis (strain 168) treP gene Proteins 0.000 description 1
- 101100053714 Bacillus subtilis (strain 168) ywbA gene Proteins 0.000 description 1
- 108700003860 Bacterial Genes Proteins 0.000 description 1
- 108050007854 Bacterial extracellular solute-binding proteins Proteins 0.000 description 1
- 235000016068 Berberis vulgaris Nutrition 0.000 description 1
- 235000021533 Beta vulgaris Nutrition 0.000 description 1
- 102100023995 Beta-nerve growth factor Human genes 0.000 description 1
- 108090001003 Beta-phosphoglucomutases Proteins 0.000 description 1
- 101001015517 Betula pendula Germin-like protein 1 Proteins 0.000 description 1
- 241000589969 Borreliella burgdorferi Species 0.000 description 1
- 235000011331 Brassica Nutrition 0.000 description 1
- 241000219198 Brassica Species 0.000 description 1
- 240000002791 Brassica napus Species 0.000 description 1
- 240000008100 Brassica rapa Species 0.000 description 1
- 241000220243 Brassica sp. Species 0.000 description 1
- 235000004936 Bromus mango Nutrition 0.000 description 1
- 241000589562 Brucella Species 0.000 description 1
- 125000001433 C-terminal amino-acid group Chemical group 0.000 description 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- 244000045232 Canavalia ensiformis Species 0.000 description 1
- 101710132601 Capsid protein Proteins 0.000 description 1
- 241000206594 Carnobacterium Species 0.000 description 1
- 241000218645 Cedrus Species 0.000 description 1
- 101710134867 Chalcone synthase 3 Proteins 0.000 description 1
- 108091006146 Channels Proteins 0.000 description 1
- 108010035563 Chloramphenicol O-acetyltransferase Proteins 0.000 description 1
- 235000007516 Chrysanthemum Nutrition 0.000 description 1
- 244000189548 Chrysanthemum x morifolium Species 0.000 description 1
- 101710094648 Coat protein Proteins 0.000 description 1
- 102000029816 Collagenase Human genes 0.000 description 1
- 108060005980 Collagenase Proteins 0.000 description 1
- 108020004394 Complementary RNA Proteins 0.000 description 1
- 229920000742 Cotton Polymers 0.000 description 1
- 208000010859 Creutzfeldt-Jakob disease Diseases 0.000 description 1
- MIKUYHXYGGJMLM-GIMIYPNGSA-N Crotonoside Natural products C1=NC2=C(N)NC(=O)N=C2N1[C@H]1O[C@@H](CO)[C@H](O)[C@@H]1O MIKUYHXYGGJMLM-GIMIYPNGSA-N 0.000 description 1
- 241000219112 Cucumis Species 0.000 description 1
- 235000010071 Cucumis prophetarum Nutrition 0.000 description 1
- 235000010799 Cucumis sativus var sativus Nutrition 0.000 description 1
- NBSCHQHZLSJFNQ-GASJEMHNSA-N D-Glucose 6-phosphate Chemical compound OC1O[C@H](COP(O)(O)=O)[C@@H](O)[C@H](O)[C@H]1O NBSCHQHZLSJFNQ-GASJEMHNSA-N 0.000 description 1
- NYHBQMYGNKIUIF-UHFFFAOYSA-N D-guanosine Natural products C1=2NC(N)=NC(=O)C=2N=CN1C1OC(CO)C(O)C1O NYHBQMYGNKIUIF-UHFFFAOYSA-N 0.000 description 1
- KJTLQQUUPVSXIM-UHFFFAOYSA-N DL-mevalonic acid Natural products OCCC(O)(C)CC(O)=O KJTLQQUUPVSXIM-UHFFFAOYSA-N 0.000 description 1
- 108020003215 DNA Probes Proteins 0.000 description 1
- 238000000018 DNA microarray Methods 0.000 description 1
- 239000003155 DNA primer Substances 0.000 description 1
- 244000000626 Daucus carota Species 0.000 description 1
- 235000002767 Daucus carota Nutrition 0.000 description 1
- 101710088194 Dehydrogenase Proteins 0.000 description 1
- 229920002307 Dextran Polymers 0.000 description 1
- 235000009355 Dianthus caryophyllus Nutrition 0.000 description 1
- 240000006497 Dianthus caryophyllus Species 0.000 description 1
- 206010012735 Diarrhoea Diseases 0.000 description 1
- 241000588700 Dickeya chrysanthemi Species 0.000 description 1
- 108090000204 Dipeptidase 1 Proteins 0.000 description 1
- 241001147751 Dolosigranulum Species 0.000 description 1
- 235000014466 Douglas bleu Nutrition 0.000 description 1
- 101100019554 Drosophila melanogaster Adk2 gene Proteins 0.000 description 1
- 235000007349 Eleusine coracana Nutrition 0.000 description 1
- 235000013499 Eleusine coracana subsp coracana Nutrition 0.000 description 1
- 241000710188 Encephalomyocarditis virus Species 0.000 description 1
- 102000009476 Enolase, C-terminal TIM barrel domains Human genes 0.000 description 1
- 108050000394 Enolase, C-terminal TIM barrel domains Proteins 0.000 description 1
- 241000194033 Enterococcus Species 0.000 description 1
- 241000194031 Enterococcus faecium Species 0.000 description 1
- 108010013369 Enteropeptidase Proteins 0.000 description 1
- 102100029727 Enteropeptidase Human genes 0.000 description 1
- 101710204837 Envelope small membrane protein Proteins 0.000 description 1
- YQYJSBFKSSDGFO-UHFFFAOYSA-N Epihygromycin Natural products OC1C(O)C(C(=O)C)OC1OC(C(=C1)O)=CC=C1C=C(C)C(=O)NC1C(O)C(O)C2OCOC2C1O YQYJSBFKSSDGFO-UHFFFAOYSA-N 0.000 description 1
- 101100410352 Escherichia coli (strain K12) chbC gene Proteins 0.000 description 1
- 101100129092 Escherichia coli hic gene Proteins 0.000 description 1
- 101100004352 Escherichia coli lacZ gene Proteins 0.000 description 1
- 101000686777 Escherichia phage T7 T7 RNA polymerase Proteins 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 241000206602 Eukaryota Species 0.000 description 1
- 241000221079 Euphorbia <genus> Species 0.000 description 1
- 240000002395 Euphorbia pulcherrima Species 0.000 description 1
- 108010054218 Factor VIII Proteins 0.000 description 1
- 102000001690 Factor VIII Human genes 0.000 description 1
- 241000218218 Ficus <angiosperm> Species 0.000 description 1
- 102000003793 Fructokinases Human genes 0.000 description 1
- 108090000156 Fructokinases Proteins 0.000 description 1
- 101710086299 Fructose-2,6-bisphosphatase Proteins 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 241000605956 Fusobacterium mortiferum Species 0.000 description 1
- 102000030782 GTP binding Human genes 0.000 description 1
- 108091000058 GTP-Binding Proteins 0.000 description 1
- 108060003306 Galactosyltransferase Proteins 0.000 description 1
- 102000030902 Galactosyltransferase Human genes 0.000 description 1
- 108700028146 Genetic Enhancer Elements Proteins 0.000 description 1
- 241000193385 Geobacillus stearothermophilus Species 0.000 description 1
- VFRROHXSMXFLSN-UHFFFAOYSA-N Glc6P Natural products OP(=O)(O)OCC(O)C(O)C(O)C(O)C=O VFRROHXSMXFLSN-UHFFFAOYSA-N 0.000 description 1
- 108010056771 Glucosidases Proteins 0.000 description 1
- 102000004366 Glucosidases Human genes 0.000 description 1
- 108010060309 Glucuronidase Proteins 0.000 description 1
- 102000053187 Glucuronidase Human genes 0.000 description 1
- 102000007390 Glycogen Phosphorylase Human genes 0.000 description 1
- 108010046163 Glycogen Phosphorylase Proteins 0.000 description 1
- 108010001483 Glycogen Synthase Proteins 0.000 description 1
- 102000001426 Glycoside hydrolase family 1 Human genes 0.000 description 1
- 108050009637 Glycoside hydrolase family 1 Proteins 0.000 description 1
- 239000005562 Glyphosate Substances 0.000 description 1
- 101150087426 Gnal gene Proteins 0.000 description 1
- 102100021181 Golgi phosphoprotein 3 Human genes 0.000 description 1
- 240000000047 Gossypium barbadense Species 0.000 description 1
- 235000009429 Gossypium barbadense Nutrition 0.000 description 1
- 235000009432 Gossypium hirsutum Nutrition 0.000 description 1
- 101100508941 Halobacterium salinarum (strain ATCC 700922 / JCM 11081 / NRC-1) ppa gene Proteins 0.000 description 1
- 108010093488 His-His-His-His-His-His Proteins 0.000 description 1
- 101001017818 Homo sapiens ATP-dependent translocase ABCB1 Proteins 0.000 description 1
- 101000775498 Homo sapiens Adenylate cyclase type 10 Proteins 0.000 description 1
- 101000899240 Homo sapiens Endoplasmic reticulum chaperone BiP Proteins 0.000 description 1
- 240000005979 Hordeum vulgare Species 0.000 description 1
- 235000007340 Hordeum vulgare Nutrition 0.000 description 1
- AVXURJPOCDRRFD-UHFFFAOYSA-N Hydroxylamine Chemical compound ON AVXURJPOCDRRFD-UHFFFAOYSA-N 0.000 description 1
- 108060003951 Immunoglobulin Proteins 0.000 description 1
- 108020005350 Initiator Codon Proteins 0.000 description 1
- 206010022678 Intestinal infections Diseases 0.000 description 1
- 108010042889 Inulosucrase Proteins 0.000 description 1
- 244000017020 Ipomoea batatas Species 0.000 description 1
- 235000002678 Ipomoea batatas Nutrition 0.000 description 1
- 102400000471 Isomaltase Human genes 0.000 description 1
- ONIBWKKTOPOVIA-BYPYZUCNSA-N L-Proline Chemical compound OC(=O)[C@@H]1CCCN1 ONIBWKKTOPOVIA-BYPYZUCNSA-N 0.000 description 1
- ODKSFYDXXFIFQN-BYPYZUCNSA-P L-argininium(2+) Chemical compound NC(=[NH2+])NCCC[C@H]([NH3+])C(O)=O ODKSFYDXXFIFQN-BYPYZUCNSA-P 0.000 description 1
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 description 1
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 1
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 1
- UKAUYVFTDYCKQA-VKHMYHEASA-N L-homoserine Chemical compound OC(=O)[C@@H](N)CCO UKAUYVFTDYCKQA-VKHMYHEASA-N 0.000 description 1
- JVTAAEKCZFNVCJ-REOHCLBHSA-N L-lactic acid Chemical compound C[C@H](O)C(O)=O JVTAAEKCZFNVCJ-REOHCLBHSA-N 0.000 description 1
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 description 1
- 244000116699 Lactobacillus acidophilus NCFM Species 0.000 description 1
- 235000009195 Lactobacillus acidophilus NCFM Nutrition 0.000 description 1
- 244000199885 Lactobacillus bulgaricus Species 0.000 description 1
- 235000013960 Lactobacillus bulgaricus Nutrition 0.000 description 1
- 241001134659 Lactobacillus curvatus Species 0.000 description 1
- 241001147746 Lactobacillus delbrueckii subsp. lactis Species 0.000 description 1
- 241000218588 Lactobacillus rhamnosus Species 0.000 description 1
- 241000194036 Lactococcus Species 0.000 description 1
- 241000194035 Lactococcus lactis Species 0.000 description 1
- 201000010538 Lactose Intolerance Diseases 0.000 description 1
- 241000208822 Lactuca Species 0.000 description 1
- 235000003228 Lactuca sativa Nutrition 0.000 description 1
- 240000008415 Lactuca sativa Species 0.000 description 1
- 101710192606 Latent membrane protein 2 Proteins 0.000 description 1
- 241000219729 Lathyrus Species 0.000 description 1
- 241000192132 Leuconostoc Species 0.000 description 1
- 241000192129 Leuconostoc lactis Species 0.000 description 1
- 108060001084 Luciferase Proteins 0.000 description 1
- 239000005089 Luciferase Substances 0.000 description 1
- 102000004317 Lyases Human genes 0.000 description 1
- 108090000856 Lyases Proteins 0.000 description 1
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 1
- 239000004472 Lysine Substances 0.000 description 1
- 101710145006 Lysis protein Proteins 0.000 description 1
- 241000208467 Macadamia Species 0.000 description 1
- 235000018330 Macadamia integrifolia Nutrition 0.000 description 1
- 240000007575 Macadamia integrifolia Species 0.000 description 1
- FYYHWMGAXLPEAU-UHFFFAOYSA-N Magnesium Chemical compound [Mg] FYYHWMGAXLPEAU-UHFFFAOYSA-N 0.000 description 1
- JLVVSXFLKOJNIY-UHFFFAOYSA-N Magnesium ion Chemical compound [Mg+2] JLVVSXFLKOJNIY-UHFFFAOYSA-N 0.000 description 1
- 101710125418 Major capsid protein Proteins 0.000 description 1
- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 description 1
- 108010057899 Maltose phosphorylase Proteins 0.000 description 1
- 244000070406 Malus silvestris Species 0.000 description 1
- 240000002129 Malva sylvestris Species 0.000 description 1
- 235000006770 Malva sylvestris Nutrition 0.000 description 1
- 235000004456 Manihot esculenta Nutrition 0.000 description 1
- 235000016735 Manihot esculenta subsp esculenta Nutrition 0.000 description 1
- 101800000577 Maturation protease Proteins 0.000 description 1
- 235000010624 Medicago sativa Nutrition 0.000 description 1
- 235000017587 Medicago sativa ssp. sativa Nutrition 0.000 description 1
- 241001468189 Melissococcus Species 0.000 description 1
- 108010090306 Member 2 Subfamily G ATP Binding Cassette Transporter Proteins 0.000 description 1
- 102000018697 Membrane Proteins Human genes 0.000 description 1
- SVSKFMJQWMZCRD-MCDZGGTQSA-L MgADP Chemical compound [Mg+2].C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP([O-])(=O)OP(O)([O-])=O)[C@@H](O)[C@H]1O SVSKFMJQWMZCRD-MCDZGGTQSA-L 0.000 description 1
- ZOKXTWBITQBERF-UHFFFAOYSA-N Molybdenum Chemical compound [Mo] ZOKXTWBITQBERF-UHFFFAOYSA-N 0.000 description 1
- 101710155748 Multiple sugar-binding protein Proteins 0.000 description 1
- 101100523877 Mus musculus Rbks gene Proteins 0.000 description 1
- 101100095600 Mus musculus Serinc1 gene Proteins 0.000 description 1
- 241000234295 Musa Species 0.000 description 1
- 240000005561 Musa balbisiana Species 0.000 description 1
- 235000018290 Musa x paradisiaca Nutrition 0.000 description 1
- 108010021466 Mutant Proteins Proteins 0.000 description 1
- 102000008300 Mutant Proteins Human genes 0.000 description 1
- OVRNDRQMDRJTHS-CBQIKETKSA-N N-Acetyl-D-Galactosamine Chemical compound CC(=O)N[C@H]1[C@@H](O)O[C@H](CO)[C@H](O)[C@@H]1O OVRNDRQMDRJTHS-CBQIKETKSA-N 0.000 description 1
- MBLBDJOUHNCFQT-UHFFFAOYSA-N N-acetyl-D-galactosamine Natural products CC(=O)NC(C=O)C(O)C(O)C(O)CO MBLBDJOUHNCFQT-UHFFFAOYSA-N 0.000 description 1
- 108010007843 NADH oxidase Proteins 0.000 description 1
- 238000005481 NMR spectroscopy Methods 0.000 description 1
- 229930193140 Neomycin Natural products 0.000 description 1
- 102000007072 Nerve Growth Factors Human genes 0.000 description 1
- 241000192673 Nostoc sp. Species 0.000 description 1
- MUBMVGCGOYJTSS-FMTOCKGGSA-N OC[C@@H](O)[C@H](O)[C@H](O)[C@@H](O)C=O.O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@H]1[C@H](O)[C@@H](O)C(O)O[C@@H]1CO Chemical compound OC[C@@H](O)[C@H](O)[C@H](O)[C@@H](O)C=O.O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@H]1[C@H](O)[C@@H](O)C(O)O[C@@H]1CO MUBMVGCGOYJTSS-FMTOCKGGSA-N 0.000 description 1
- 241000202223 Oenococcus Species 0.000 description 1
- 235000002725 Olea europaea Nutrition 0.000 description 1
- 108020005187 Oligonucleotide Probes Proteins 0.000 description 1
- 101710116435 Outer membrane protein Proteins 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 241000192001 Pediococcus Species 0.000 description 1
- 241000191996 Pediococcus pentosaceus Species 0.000 description 1
- 108010087702 Penicillinase Proteins 0.000 description 1
- 244000038248 Pennisetum spicatum Species 0.000 description 1
- 244000115721 Pennisetum typhoides Species 0.000 description 1
- 108010033276 Peptide Fragments Proteins 0.000 description 1
- 244000100170 Phaseolus lunatus Species 0.000 description 1
- 235000010627 Phaseolus vulgaris Nutrition 0.000 description 1
- 244000046052 Phaseolus vulgaris Species 0.000 description 1
- 241000286209 Phasianidae Species 0.000 description 1
- 108010022684 Phosphofructokinase-1 Proteins 0.000 description 1
- 102000012435 Phosphofructokinase-1 Human genes 0.000 description 1
- 102000045595 Phosphoprotein Phosphatases Human genes 0.000 description 1
- 108700019535 Phosphoprotein Phosphatases Proteins 0.000 description 1
- 101710155159 Phosphotransferase enzyme IIA component Proteins 0.000 description 1
- 108050006122 Phosphotransferase system, EIIC Proteins 0.000 description 1
- 240000000020 Picea glauca Species 0.000 description 1
- 235000008127 Picea glauca Nutrition 0.000 description 1
- 241000218595 Picea sitchensis Species 0.000 description 1
- 241000709664 Picornaviridae Species 0.000 description 1
- 235000005205 Pinus Nutrition 0.000 description 1
- 241000218602 Pinus <genus> Species 0.000 description 1
- 235000008593 Pinus contorta Nutrition 0.000 description 1
- 244000019397 Pinus jeffreyi Species 0.000 description 1
- 241000555277 Pinus ponderosa Species 0.000 description 1
- 235000013269 Pinus ponderosa var ponderosa Nutrition 0.000 description 1
- 235000013268 Pinus ponderosa var scopulorum Nutrition 0.000 description 1
- 235000010582 Pisum sativum Nutrition 0.000 description 1
- 240000004713 Pisum sativum Species 0.000 description 1
- 241000710078 Potyvirus Species 0.000 description 1
- 101710083689 Probable capsid protein Proteins 0.000 description 1
- 101710184309 Probable sucrose-6-phosphate hydrolase Proteins 0.000 description 1
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 description 1
- 102000001253 Protein Kinase Human genes 0.000 description 1
- 101100084022 Pseudomonas aeruginosa (strain ATCC 15692 / DSM 22644 / CIP 104116 / JCM 14847 / LMG 12228 / 1C / PRS 101 / PAO1) lapA gene Proteins 0.000 description 1
- 235000008572 Pseudotsuga menziesii Nutrition 0.000 description 1
- 235000005386 Pseudotsuga menziesii var menziesii Nutrition 0.000 description 1
- 241000508269 Psidium Species 0.000 description 1
- 240000001679 Psidium guajava Species 0.000 description 1
- 235000013929 Psidium pyriferum Nutrition 0.000 description 1
- CZPWVGJYEJSRLH-UHFFFAOYSA-N Pyrimidine Chemical compound C1=CN=CN=C1 CZPWVGJYEJSRLH-UHFFFAOYSA-N 0.000 description 1
- 101150071963 RBSK gene Proteins 0.000 description 1
- 108091034057 RNA (poly(A)) Proteins 0.000 description 1
- 102000008991 RNA Recognition Motif Proteins Human genes 0.000 description 1
- 108010049094 RNA Recognition Motif Proteins Proteins 0.000 description 1
- 230000006819 RNA synthesis Effects 0.000 description 1
- 102000044126 RNA-Binding Proteins Human genes 0.000 description 1
- 230000004570 RNA-binding Effects 0.000 description 1
- 101710105008 RNA-binding protein Proteins 0.000 description 1
- 108090001066 Racemases and epimerases Proteins 0.000 description 1
- 102000004879 Racemases and epimerases Human genes 0.000 description 1
- 102000009661 Repressor Proteins Human genes 0.000 description 1
- 235000011449 Rosa Nutrition 0.000 description 1
- 235000004789 Rosa xanthina Nutrition 0.000 description 1
- 241000109329 Rosa xanthina Species 0.000 description 1
- 101000744001 Ruminococcus gnavus (strain ATCC 29149 / VPI C7-9) 3beta-hydroxysteroid dehydrogenase Proteins 0.000 description 1
- 241000209051 Saccharum Species 0.000 description 1
- 240000000111 Saccharum officinarum Species 0.000 description 1
- 235000007201 Saccharum officinarum Nutrition 0.000 description 1
- 241000293871 Salmonella enterica subsp. enterica serovar Typhi Species 0.000 description 1
- 241000293869 Salmonella enterica subsp. enterica serovar Typhimurium Species 0.000 description 1
- 240000000513 Santalum album Species 0.000 description 1
- ZVHPYMRQVUPXNX-UHFFFAOYSA-N Saurin Natural products OCC1C2CC(O)C(=C)C3C(C2OC1=O)C(=C)CC3=O ZVHPYMRQVUPXNX-UHFFFAOYSA-N 0.000 description 1
- 238000012300 Sequence Analysis Methods 0.000 description 1
- 241001116459 Sequoia Species 0.000 description 1
- 101710187074 Serine proteinase inhibitor Proteins 0.000 description 1
- 235000008515 Setaria glauca Nutrition 0.000 description 1
- 235000007226 Setaria italica Nutrition 0.000 description 1
- 241000207763 Solanum Species 0.000 description 1
- 235000002634 Solanum Nutrition 0.000 description 1
- 235000002595 Solanum tuberosum Nutrition 0.000 description 1
- 244000061456 Solanum tuberosum Species 0.000 description 1
- 235000007230 Sorghum bicolor Nutrition 0.000 description 1
- 244000062793 Sorghum vulgare Species 0.000 description 1
- 238000002105 Southern blotting Methods 0.000 description 1
- 241000251131 Sphyrna Species 0.000 description 1
- 235000009184 Spondias indica Nutrition 0.000 description 1
- UQZIYBXSHAGNOE-USOSMYMVSA-N Stachyose Natural products O(C[C@H]1[C@@H](O)[C@H](O)[C@H](O)[C@@H](O[C@@]2(CO)[C@H](O)[C@@H](O)[C@@H](CO)O2)O1)[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@H](CO[C@@H]2[C@@H](O)[C@@H](O)[C@@H](O)[C@H](CO)O2)O1 UQZIYBXSHAGNOE-USOSMYMVSA-N 0.000 description 1
- 241000201788 Staphylococcus aureus subsp. aureus Species 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- 108010090804 Streptavidin Proteins 0.000 description 1
- 241000193985 Streptococcus agalactiae Species 0.000 description 1
- 235000014897 Streptococcus lactis Nutrition 0.000 description 1
- 101100292254 Streptococcus mutans serotype c (strain ATCC 700610 / UA159) msmK gene Proteins 0.000 description 1
- 241000187432 Streptomyces coelicolor Species 0.000 description 1
- 102400000472 Sucrase Human genes 0.000 description 1
- 102100027918 Sucrase-isomaltase, intestinal Human genes 0.000 description 1
- 101710112652 Sucrose-6-phosphate hydrolase Proteins 0.000 description 1
- 101710181960 Sucrose:sucrose 1-fructosyltransferase Proteins 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 description 1
- 102000019197 Superoxide Dismutase Human genes 0.000 description 1
- 108010012715 Superoxide dismutase Proteins 0.000 description 1
- 241000192581 Synechocystis sp. Species 0.000 description 1
- 108700005078 Synthetic Genes Proteins 0.000 description 1
- 108010076818 TEV protease Proteins 0.000 description 1
- 101710109576 Terminal protein Proteins 0.000 description 1
- 241000500334 Tetragenococcus Species 0.000 description 1
- 235000006468 Thea sinensis Nutrition 0.000 description 1
- 241000204666 Thermotoga maritima Species 0.000 description 1
- RYYWUUFWQRZTIU-UHFFFAOYSA-N Thiophosphoric acid Chemical group OP(O)(S)=O RYYWUUFWQRZTIU-UHFFFAOYSA-N 0.000 description 1
- 108700009124 Transcription Initiation Site Proteins 0.000 description 1
- 102000004357 Transferases Human genes 0.000 description 1
- 108090000992 Transferases Proteins 0.000 description 1
- 241001635318 Trichococcus Species 0.000 description 1
- 241000722923 Tulipa Species 0.000 description 1
- 241000722921 Tulipa gesneriana Species 0.000 description 1
- HSCJRCZFDFQWRP-JZMIEXBBSA-N UDP-alpha-D-glucose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1OP(O)(=O)OP(O)(=O)OC[C@@H]1[C@@H](O)[C@@H](O)[C@H](N2C(NC(=O)C=C2)=O)O1 HSCJRCZFDFQWRP-JZMIEXBBSA-N 0.000 description 1
- 108090000848 Ubiquitin Proteins 0.000 description 1
- 102000044159 Ubiquitin Human genes 0.000 description 1
- HSCJRCZFDFQWRP-UHFFFAOYSA-N Uridindiphosphoglukose Natural products OC1C(O)C(O)C(CO)OC1OP(O)(=O)OP(O)(=O)OCC1C(O)C(O)C(N2C(NC(=O)C=C2)=O)O1 HSCJRCZFDFQWRP-UHFFFAOYSA-N 0.000 description 1
- 241000207194 Vagococcus Species 0.000 description 1
- ZVNYJIZDIRKMBF-UHFFFAOYSA-N Vesnarinone Chemical compound C1=C(OC)C(OC)=CC=C1C(=O)N1CCN(C=2C=C3CCC(=O)NC3=CC=2)CC1 ZVNYJIZDIRKMBF-UHFFFAOYSA-N 0.000 description 1
- 208000036142 Viral infection Diseases 0.000 description 1
- 229930003451 Vitamin B1 Natural products 0.000 description 1
- 241000202221 Weissella Species 0.000 description 1
- 241000204366 Xylella Species 0.000 description 1
- 235000007244 Zea mays Nutrition 0.000 description 1
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 1
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 description 1
- PTFCDOFLOPIGGS-UHFFFAOYSA-N Zinc dication Chemical compound [Zn+2] PTFCDOFLOPIGGS-UHFFFAOYSA-N 0.000 description 1
- 241000588902 Zymomonas mobilis Species 0.000 description 1
- XJLXINKUBYWONI-DQQFMEOOSA-N [[(2r,3r,4r,5r)-5-(6-aminopurin-9-yl)-3-hydroxy-4-phosphonooxyoxolan-2-yl]methoxy-hydroxyphosphoryl] [(2s,3r,4s,5s)-5-(3-carbamoylpyridin-1-ium-1-yl)-3,4-dihydroxyoxolan-2-yl]methyl phosphate Chemical compound NC(=O)C1=CC=C[N+]([C@@H]2[C@H]([C@@H](O)[C@H](COP([O-])(=O)OP(O)(=O)OC[C@@H]3[C@H]([C@@H](OP(O)(O)=O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 XJLXINKUBYWONI-DQQFMEOOSA-N 0.000 description 1
- 230000036579 abiotic stress Effects 0.000 description 1
- 230000001133 acceleration Effects 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 125000000641 acridinyl group Chemical group C1(=CC=CC2=NC3=CC=CC=C3C=C12)* 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 238000001261 affinity purification Methods 0.000 description 1
- 150000001299 aldehydes Chemical class 0.000 description 1
- 125000001931 aliphatic group Chemical group 0.000 description 1
- 230000003281 allosteric effect Effects 0.000 description 1
- 235000020224 almond Nutrition 0.000 description 1
- HXXFSFRBOHSIMQ-FPRJBGLDSA-N alpha-D-galactose 1-phosphate Chemical compound OC[C@H]1O[C@H](OP(O)(O)=O)[C@H](O)[C@@H](O)[C@H]1O HXXFSFRBOHSIMQ-FPRJBGLDSA-N 0.000 description 1
- 102000005840 alpha-Galactosidase Human genes 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 230000006538 anaerobic glycolysis Effects 0.000 description 1
- 239000004410 anthocyanin Substances 0.000 description 1
- 235000010208 anthocyanin Nutrition 0.000 description 1
- 229930002877 anthocyanin Natural products 0.000 description 1
- 150000004636 anthocyanins Chemical class 0.000 description 1
- 239000002246 antineoplastic agent Substances 0.000 description 1
- 229940041181 antineoplastic drug Drugs 0.000 description 1
- 101150023525 araH gene Proteins 0.000 description 1
- PYMYPHUHKUWMLA-WDCZJNDASA-N arabinose Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)C=O PYMYPHUHKUWMLA-WDCZJNDASA-N 0.000 description 1
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 description 1
- 101150042295 arfA gene Proteins 0.000 description 1
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 1
- 150000003934 aromatic aldehydes Chemical class 0.000 description 1
- 125000003118 aryl group Chemical group 0.000 description 1
- 235000009582 asparagine Nutrition 0.000 description 1
- 229960001230 asparagine Drugs 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000010165 autogamy Effects 0.000 description 1
- 230000029586 bacterial cell surface binding Effects 0.000 description 1
- 230000010310 bacterial transformation Effects 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- 238000005452 bending Methods 0.000 description 1
- DZBUGLKDJFMEHC-UHFFFAOYSA-N benzoquinolinylidene Chemical group C1=CC=CC2=CC3=CC=CC=C3N=C21 DZBUGLKDJFMEHC-UHFFFAOYSA-N 0.000 description 1
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 description 1
- 102000006995 beta-Glucosidase Human genes 0.000 description 1
- 108010047754 beta-Glucosidase Proteins 0.000 description 1
- 102000006635 beta-lactamase Human genes 0.000 description 1
- DLRVVLDZNNYCBX-ZZFZYMBESA-N beta-melibiose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1OC[C@@H]1[C@@H](O)[C@H](O)[C@@H](O)[C@H](O)O1 DLRVVLDZNNYCBX-ZZFZYMBESA-N 0.000 description 1
- 101150086929 bglF gene Proteins 0.000 description 1
- 230000001588 bifunctional effect Effects 0.000 description 1
- 238000004166 bioassay Methods 0.000 description 1
- 238000010364 biochemical engineering Methods 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 230000031018 biological processes and functions Effects 0.000 description 1
- 230000006696 biosynthetic metabolic pathway Effects 0.000 description 1
- 229960002685 biotin Drugs 0.000 description 1
- 235000020958 biotin Nutrition 0.000 description 1
- 239000011616 biotin Substances 0.000 description 1
- 125000005340 bisphosphate group Chemical group 0.000 description 1
- 150000005693 branched-chain amino acids Chemical class 0.000 description 1
- 210000000481 breast Anatomy 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000006172 buffering agent Substances 0.000 description 1
- 235000014121 butter Nutrition 0.000 description 1
- 235000015155 buttermilk Nutrition 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
- 239000001506 calcium phosphate Substances 0.000 description 1
- 229910000389 calcium phosphate Inorganic materials 0.000 description 1
- 235000011010 calcium phosphates Nutrition 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 125000004432 carbon atom Chemical group C* 0.000 description 1
- 230000009015 carbon catabolite repression of transcription Effects 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 235000020226 cashew nut Nutrition 0.000 description 1
- 239000003054 catalyst Substances 0.000 description 1
- 101150080131 celB gene Proteins 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 230000032823 cell division Effects 0.000 description 1
- 230000009087 cell motility Effects 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- 230000005754 cellular signaling Effects 0.000 description 1
- 230000004700 cellular uptake Effects 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 235000010980 cellulose Nutrition 0.000 description 1
- 235000013339 cereals Nutrition 0.000 description 1
- 229920001429 chelating resin Polymers 0.000 description 1
- 239000012707 chemical precursor Substances 0.000 description 1
- 230000035605 chemotaxis Effects 0.000 description 1
- 229960005091 chloramphenicol Drugs 0.000 description 1
- WIIZWVCIJKGZOK-RKDXNWHRSA-N chloramphenicol Chemical compound ClC(Cl)C(=O)N[C@H](CO)[C@H](O)C1=CC=C([N+]([O-])=O)C=C1 WIIZWVCIJKGZOK-RKDXNWHRSA-N 0.000 description 1
- RPKLZQLYODPWTM-KBMWBBLPSA-N cholanoic acid Chemical compound C1CC2CCCC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@@H](CCC(O)=O)C)[C@@]1(C)CC2 RPKLZQLYODPWTM-KBMWBBLPSA-N 0.000 description 1
- 238000000975 co-precipitation Methods 0.000 description 1
- 229910017052 cobalt Inorganic materials 0.000 description 1
- 239000010941 cobalt Substances 0.000 description 1
- GUTLYIVDDKVIGB-UHFFFAOYSA-N cobalt atom Chemical compound [Co] GUTLYIVDDKVIGB-UHFFFAOYSA-N 0.000 description 1
- 229960002424 collagenase Drugs 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 238000009833 condensation Methods 0.000 description 1
- 230000005494 condensation Effects 0.000 description 1
- 230000021615 conjugation Effects 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 239000013068 control sample Substances 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 235000005822 corn Nutrition 0.000 description 1
- 235000015140 cultured milk Nutrition 0.000 description 1
- 235000014048 cultured milk product Nutrition 0.000 description 1
- ATDGTVJJHBUTRL-UHFFFAOYSA-N cyanogen bromide Chemical compound BrC#N ATDGTVJJHBUTRL-UHFFFAOYSA-N 0.000 description 1
- OPTASPLRGRRNAP-UHFFFAOYSA-N cytosine Chemical class NC=1C=CNC(=O)N=1 OPTASPLRGRRNAP-UHFFFAOYSA-N 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 230000018044 dehydration Effects 0.000 description 1
- 238000006297 dehydration reaction Methods 0.000 description 1
- 101150106284 deoR gene Proteins 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 230000015155 detection of stimulus involved in sensory perception Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 230000037213 diet Effects 0.000 description 1
- 230000000378 dietary effect Effects 0.000 description 1
- 210000002249 digestive system Anatomy 0.000 description 1
- RXKJFZQQPQGTFL-UHFFFAOYSA-N dihydroxyacetone Chemical compound OCC(=O)CO RXKJFZQQPQGTFL-UHFFFAOYSA-N 0.000 description 1
- 238000006471 dimerization reaction Methods 0.000 description 1
- 150000002016 disaccharides Chemical class 0.000 description 1
- 238000010494 dissociation reaction Methods 0.000 description 1
- 230000005593 dissociations Effects 0.000 description 1
- 238000012377 drug delivery Methods 0.000 description 1
- 244000013123 dwarf bean Species 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 230000008030 elimination Effects 0.000 description 1
- 238000003379 elimination reaction Methods 0.000 description 1
- 239000002158 endotoxin Substances 0.000 description 1
- 230000037149 energy metabolism Effects 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 230000009088 enzymatic function Effects 0.000 description 1
- 238000009585 enzyme analysis Methods 0.000 description 1
- 101150043985 epsE gene Proteins 0.000 description 1
- 229960003276 erythromycin Drugs 0.000 description 1
- 230000029142 excretion Effects 0.000 description 1
- 230000001747 exhibiting effect Effects 0.000 description 1
- 108010055265 exo-1,6-alpha-glucosidase Proteins 0.000 description 1
- 238000001125 extrusion Methods 0.000 description 1
- 210000003746 feather Anatomy 0.000 description 1
- 210000003608 fece Anatomy 0.000 description 1
- 235000021107 fermented food Nutrition 0.000 description 1
- 239000012467 final product Substances 0.000 description 1
- 108010060641 flavanone synthetase Proteins 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 229940014144 folate Drugs 0.000 description 1
- OVBPIULPVIDEAO-LBPRGKRZSA-N folic acid Chemical compound C=1N=C2NC(N)=NC(=O)C2=NC=1CNC1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 OVBPIULPVIDEAO-LBPRGKRZSA-N 0.000 description 1
- 235000019152 folic acid Nutrition 0.000 description 1
- 239000011724 folic acid Substances 0.000 description 1
- BJHIKXHVCXFQLS-UYFOZJQFSA-N fructose group Chemical group OCC(=O)[C@@H](O)[C@H](O)[C@H](O)CO BJHIKXHVCXFQLS-UYFOZJQFSA-N 0.000 description 1
- 230000002538 fungal effect Effects 0.000 description 1
- 230000005021 gait Effects 0.000 description 1
- 230000002496 gastric effect Effects 0.000 description 1
- 101150045100 gatY gene Proteins 0.000 description 1
- 230000030279 gene silencing Effects 0.000 description 1
- 238000012226 gene silencing method Methods 0.000 description 1
- 101150013858 glgC gene Proteins 0.000 description 1
- 101150072146 glgD gene Proteins 0.000 description 1
- 230000004190 glucose uptake Effects 0.000 description 1
- 229940045189 glucose-6-phosphate Drugs 0.000 description 1
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 1
- 229930182470 glycoside Natural products 0.000 description 1
- 150000002338 glycosides Chemical class 0.000 description 1
- 108091072197 glycosyl hydrolase 42 family Proteins 0.000 description 1
- 125000003630 glycyl group Chemical group [H]N([H])C([H])([H])C(*)=O 0.000 description 1
- XDDAORKBJWWYJS-UHFFFAOYSA-N glyphosate Chemical compound OC(=O)CNCP(O)(O)=O XDDAORKBJWWYJS-UHFFFAOYSA-N 0.000 description 1
- 229940097068 glyphosate Drugs 0.000 description 1
- 101150094642 gntR gene Proteins 0.000 description 1
- 235000021331 green beans Nutrition 0.000 description 1
- 229940029575 guanosine Drugs 0.000 description 1
- 230000005802 health problem Effects 0.000 description 1
- 108010067006 heat stable toxin (E coli) Proteins 0.000 description 1
- 230000002363 herbicidal effect Effects 0.000 description 1
- 150000002402 hexoses Chemical class 0.000 description 1
- 125000004435 hydrogen atom Chemical class [H]* 0.000 description 1
- 230000003301 hydrolyzing effect Effects 0.000 description 1
- 235000015243 ice cream Nutrition 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 238000010166 immunofluorescence Methods 0.000 description 1
- 102000018358 immunoglobulin Human genes 0.000 description 1
- 238000001114 immunoprecipitation Methods 0.000 description 1
- 238000007901 in situ hybridization Methods 0.000 description 1
- 238000011065 in-situ storage Methods 0.000 description 1
- 238000010348 incorporation Methods 0.000 description 1
- 239000000411 inducer Substances 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 230000002452 interceptive effect Effects 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- 108010090785 inulinase Proteins 0.000 description 1
- 239000001573 invertase Substances 0.000 description 1
- 229960000318 kanamycin Drugs 0.000 description 1
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 1
- 229930027917 kanamycin Natural products 0.000 description 1
- 229930182823 kanamycin A Natural products 0.000 description 1
- 101150109450 kbaY gene Proteins 0.000 description 1
- 238000011005 laboratory method Methods 0.000 description 1
- 101150018339 lacS gene Proteins 0.000 description 1
- 229940004208 lactobacillus bulgaricus Drugs 0.000 description 1
- 108010060845 lactose permease Proteins 0.000 description 1
- 108010005131 levanase Proteins 0.000 description 1
- 101150047523 lexA gene Proteins 0.000 description 1
- 150000002632 lipids Chemical group 0.000 description 1
- 238000001638 lipofection Methods 0.000 description 1
- 229920006008 lipopolysaccharide Polymers 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- 235000014684 lodgepole pine Nutrition 0.000 description 1
- 230000002132 lysosomal effect Effects 0.000 description 1
- 229910052749 magnesium Inorganic materials 0.000 description 1
- 239000011777 magnesium Substances 0.000 description 1
- 229940049920 malate Drugs 0.000 description 1
- BJEPYKJPYRNKOW-UHFFFAOYSA-N malic acid Chemical compound OC(=O)C(O)CC(O)=O BJEPYKJPYRNKOW-UHFFFAOYSA-N 0.000 description 1
- 230000021121 meiosis Effects 0.000 description 1
- 238000002844 melting Methods 0.000 description 1
- 230000008018 melting Effects 0.000 description 1
- 230000000442 meristematic effect Effects 0.000 description 1
- 238000012269 metabolic engineering Methods 0.000 description 1
- 230000037353 metabolic pathway Effects 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 229910021645 metal ion Inorganic materials 0.000 description 1
- MYWUZJCMWCOHBA-VIFPVBQESA-N methamphetamine Chemical compound CN[C@@H](C)CC1=CC=CC=C1 MYWUZJCMWCOHBA-VIFPVBQESA-N 0.000 description 1
- 244000005706 microflora Species 0.000 description 1
- 238000000520 microinjection Methods 0.000 description 1
- 230000003228 microsomal effect Effects 0.000 description 1
- 235000019713 millet Nutrition 0.000 description 1
- 230000002438 mitochondrial effect Effects 0.000 description 1
- 238000001823 molecular biology technique Methods 0.000 description 1
- 229910052750 molybdenum Inorganic materials 0.000 description 1
- 239000011733 molybdenum Substances 0.000 description 1
- 239000000178 monomer Substances 0.000 description 1
- 231100000219 mutagenic Toxicity 0.000 description 1
- 230000003505 mutagenic effect Effects 0.000 description 1
- 210000003365 myofibril Anatomy 0.000 description 1
- 229960004927 neomycin Drugs 0.000 description 1
- 229940053128 nerve growth factor Drugs 0.000 description 1
- 210000002569 neuron Anatomy 0.000 description 1
- 239000003900 neurotrophic factor Substances 0.000 description 1
- 238000006386 neutralization reaction Methods 0.000 description 1
- 108010058731 nopaline synthase Proteins 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 235000020939 nutritional additive Nutrition 0.000 description 1
- 239000002751 oligonucleotide probe Substances 0.000 description 1
- 238000002515 oligonucleotide synthesis Methods 0.000 description 1
- 101150087557 omcB gene Proteins 0.000 description 1
- 101150115693 ompA gene Proteins 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 150000002894 organic compounds Chemical class 0.000 description 1
- 101150095512 otsB gene Proteins 0.000 description 1
- 238000012261 overproduction Methods 0.000 description 1
- KHPXUQMNIQBQEV-UHFFFAOYSA-N oxaloacetic acid Chemical compound OC(=O)CC(=O)C(O)=O KHPXUQMNIQBQEV-UHFFFAOYSA-N 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 230000020477 pH reduction Effects 0.000 description 1
- 235000002252 panizo Nutrition 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 235000020232 peanut Nutrition 0.000 description 1
- 229950009506 penicillinase Drugs 0.000 description 1
- 238000010647 peptide synthesis reaction Methods 0.000 description 1
- 230000002093 peripheral effect Effects 0.000 description 1
- 210000001322 periplasm Anatomy 0.000 description 1
- 238000002823 phage display Methods 0.000 description 1
- 230000002974 pharmacogenomic effect Effects 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- HTYIXCKSEQQCJO-UHFFFAOYSA-N phenaglycodol Chemical compound CC(C)(O)C(C)(O)C1=CC=C(Cl)C=C1 HTYIXCKSEQQCJO-UHFFFAOYSA-N 0.000 description 1
- 101150009573 phoA gene Proteins 0.000 description 1
- RGCLLPNLLBQHPF-HJWRWDBZSA-N phosphamidon Chemical compound CCN(CC)C(=O)C(\Cl)=C(/C)OP(=O)(OC)OC RGCLLPNLLBQHPF-HJWRWDBZSA-N 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 108010093591 phosphoenolpyruvate-sucrose phosphotransferase Proteins 0.000 description 1
- FDIKHVQUPVCJFA-UHFFFAOYSA-N phosphohistidine Chemical compound OP(=O)(O)NC(C(=O)O)CC1=CN=CN1 FDIKHVQUPVCJFA-UHFFFAOYSA-N 0.000 description 1
- 108010006451 phosphomethylpyrimidine kinase Proteins 0.000 description 1
- 150000003013 phosphoric acid derivatives Chemical class 0.000 description 1
- 238000003566 phosphorylation assay Methods 0.000 description 1
- 230000000704 physical effect Effects 0.000 description 1
- 238000003976 plant breeding Methods 0.000 description 1
- 244000000003 plant pathogen Species 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 229920005862 polyol Polymers 0.000 description 1
- 150000003077 polyols Chemical class 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- 230000002335 preservative effect Effects 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 230000004845 protein aggregation Effects 0.000 description 1
- 108060006633 protein kinase Proteins 0.000 description 1
- 230000002797 proteolythic effect Effects 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
- 230000002285 radioactive effect Effects 0.000 description 1
- 101150042846 rbsA gene Proteins 0.000 description 1
- 230000009257 reactivity Effects 0.000 description 1
- 238000010188 recombinant method Methods 0.000 description 1
- 230000006798 recombination Effects 0.000 description 1
- 238000005215 recombination Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 235000003499 redwood Nutrition 0.000 description 1
- 230000001172 regenerating effect Effects 0.000 description 1
- 230000008929 regeneration Effects 0.000 description 1
- 238000011069 regeneration method Methods 0.000 description 1
- 230000022532 regulation of transcription, DNA-dependent Effects 0.000 description 1
- 230000008844 regulatory mechanism Effects 0.000 description 1
- 230000010076 replication Effects 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 235000009566 rice Nutrition 0.000 description 1
- 229940098714 salmonella enterica subsp. enterica serovar typhi Drugs 0.000 description 1
- 238000010187 selection method Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 239000003001 serine protease inhibitor Substances 0.000 description 1
- 235000000673 shore pine Nutrition 0.000 description 1
- 238000002741 site-directed mutagenesis Methods 0.000 description 1
- 239000001509 sodium citrate Substances 0.000 description 1
- 239000007790 solid phase Substances 0.000 description 1
- 235000021262 sour milk Nutrition 0.000 description 1
- 230000002269 spontaneous effect Effects 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- UQZIYBXSHAGNOE-XNSRJBNMSA-N stachyose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO[C@@H]2[C@@H]([C@@H](O)[C@@H](O)[C@@H](CO[C@@H]3[C@@H]([C@@H](O)[C@@H](O)[C@@H](CO)O3)O)O2)O)O1 UQZIYBXSHAGNOE-XNSRJBNMSA-N 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 108010085346 steroid delta-isomerase Proteins 0.000 description 1
- 239000003270 steroid hormone Substances 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 210000002784 stomach Anatomy 0.000 description 1
- KDYFGRWQOYBRFD-UHFFFAOYSA-N succinic acid Chemical compound OC(=O)CCC(O)=O KDYFGRWQOYBRFD-UHFFFAOYSA-N 0.000 description 1
- 125000000185 sucrose group Chemical group 0.000 description 1
- 150000005846 sugar alcohols Chemical class 0.000 description 1
- 239000000829 suppository Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 238000004114 suspension culture Methods 0.000 description 1
- 230000009897 systematic effect Effects 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 238000001214 thermospray mass spectrometry Methods 0.000 description 1
- DPJRMOMPQZCRJU-UHFFFAOYSA-M thiamine hydrochloride Chemical compound Cl.[Cl-].CC1=C(CCO)SC=[N+]1CC1=CN=C(C)N=C1N DPJRMOMPQZCRJU-UHFFFAOYSA-M 0.000 description 1
- 229960002363 thiamine pyrophosphate Drugs 0.000 description 1
- 235000008170 thiamine pyrophosphate Nutrition 0.000 description 1
- 239000011678 thiamine pyrophosphate Substances 0.000 description 1
- YXVCLPJQTZXJLH-UHFFFAOYSA-N thiamine(1+) diphosphate chloride Chemical compound [Cl-].CC1=C(CCOP(O)(=O)OP(O)(O)=O)SC=[N+]1CC1=CN=C(C)N=C1N YXVCLPJQTZXJLH-UHFFFAOYSA-N 0.000 description 1
- 108010058651 thioglucosidase Proteins 0.000 description 1
- 101150046289 tms2 gene Proteins 0.000 description 1
- 230000000699 topical effect Effects 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 239000003440 toxic substance Substances 0.000 description 1
- 238000011491 transcranial magnetic stimulation Methods 0.000 description 1
- 238000010361 transduction Methods 0.000 description 1
- 230000026683 transduction Effects 0.000 description 1
- 230000014621 translational initiation Effects 0.000 description 1
- 108091005703 transmembrane proteins Proteins 0.000 description 1
- 102000035160 transmembrane proteins Human genes 0.000 description 1
- 230000017105 transposition Effects 0.000 description 1
- 101150055398 treB gene Proteins 0.000 description 1
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 1
- 230000004102 tricarboxylic acid cycle Effects 0.000 description 1
- HRXKRNGNAMMEHJ-UHFFFAOYSA-K trisodium citrate Chemical compound [Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O HRXKRNGNAMMEHJ-UHFFFAOYSA-K 0.000 description 1
- 229940038773 trisodium citrate Drugs 0.000 description 1
- 101150035767 trp gene Proteins 0.000 description 1
- 108010087967 type I signal peptidase Proteins 0.000 description 1
- 210000001215 vagina Anatomy 0.000 description 1
- 235000015112 vegetable and seed oil Nutrition 0.000 description 1
- 230000035899 viability Effects 0.000 description 1
- 108700026220 vif Genes Proteins 0.000 description 1
- 230000009385 viral infection Effects 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
- 235000019156 vitamin B Nutrition 0.000 description 1
- 239000011720 vitamin B Substances 0.000 description 1
- 235000010374 vitamin B1 Nutrition 0.000 description 1
- 239000011691 vitamin B1 Substances 0.000 description 1
- 239000011534 wash buffer Substances 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 230000007279 water homeostasis Effects 0.000 description 1
- 238000001262 western blot Methods 0.000 description 1
- 229910052725 zinc Inorganic materials 0.000 description 1
- 239000011701 zinc Substances 0.000 description 1
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Peptides Or Proteins (AREA)
Description
LACTOBACILLUS ACIDOPHILUS NUCLEIC ACID SEQUENCES ENCODING :
CARBOHYDRATE UTILIZATION-RELATED PROTEINS AND USES
THEREFOR E TU -
CROSS REFERENCE TO RELATED APPLICATIONS \\ BE. i This application claims priority to U.S. application serial number - TT filed March 7, 2005, entitled "LACTOBA CILLUS ACIDOPHILUS NUCLEIC ACID - © SEQUENCES ENCODING CARBOHYDRATE UTILIZATION-RELATED . ’
PROTEINS AND USES THEREFOR," listing inventors Todd R. Klaenhammer, Eric
Altermann, Rodolphe Barrangou, W. Michael Russell and Tri Duong and identified as
Attorney Docket No. 505 1-693, which claims the benefit of U.S. Provisional : : Application Serial No. 60/551,121, filed March 8, 2004, the contents of each of which are herein incorporated by reference in their entireties.
: oo This invention relates to polynucleotides isolated from lactic acid bacteria, namely Lactobacillus acidophilus, and polypeptides encoded by them, as well as _ methods for using the polypeptides and organisms expressing them.
Lactobacillus acidophilus is a Gram-positive, rod-shaped, non-spore forming, : E oo homofermentative bacterium that is a normal inhabitant of the gastrointestinal and genitourinary tracts. Since its original isolation by Moro (1900) from infant feces, the : cw - “acid loving” organism has been found in the intestinal tract of humans, breast-fed infants, and persons consuming high milk, lactose, or dextrin diets. Historically, oo
Lactobacillus acidophilus is the Lactobacillus species most often implicated as an intestinal probiotic capable of eliciting beneficial effects on the microflora of the gastrointestinal tract (Klaenhammer and Russell (2000) “Species of the Lactobacillus : acidophilus complex,” Encyclopedia of Food Microbiology, 2:1151—1157. Robinson ) 30 etal, eds. (Academic Press, San Diego, California). Lactobacillus acidophilus can ] ferment hexoses, including lactose and more complex oligosaccharides, to produce lactic acid and lower the pH of the environment where the organism is cultured. ~
Acidified environments (e.g., food, vagina, and regions within the gastrointestinal tract) can interfere with the growth of undesirable bacteria, pathogens, and yeasts. The organism is well known for its acid tolerance, survival in cultured dairy products, and viability during passage through the stomach and gastrointestinal tract. Lactobacilli i and other commensal bacteria, some of which are considered probiotic bacteria that : “favor life,” have been studied extensively for their effects on human health, oo particularly in the prevention or treatment of enteric infections, diarrheal disease, 3 prevention of cancer, and stimulation of the immune system. Lactobacilli have also : : been studied for their influence on dairy. product flavor, and functional and textural - characteristics. Genetic characterization of other Lactobacillus species (eg. L : johnsonii and L. rhamnosus) has been described (see e.g., U.S. Patent No. 6,476,200;
U.S. Patent No. 6,544,772; U.S. Patent Publication Nos. 20020159976, 2003013882 oo & 20040009490; PCT Publication No. WO 2004/031389; PCT Publication No. : 2003/084989; PCT Publication No. WO 2004/020467). g
Bacterial growth requires specific transport systeins to.import nutrients from the external environment. Lactic acid bacteria transport molecules into and out of the cell via three systems: primary transport, secondary transport, and group :
Co - translocation. In primary transport, chemical (primarily ATP), electrical, or solar energy 1s used to drive transport. ATP-binding cassette (ABC) transporters are the most abundant class of primary transport systems in lactic acid bacteria. In this 20. system, ATP hydrolysis is linked with substrate translocation across the membrane for ’ _ both the import of sugars and compatible solutes and the export of products such as drugs or toxins that are undesirable to the cell, or cellular components that function outside of the cell, such as cell wall polysaccharides. In general, ABC transporters are relatively specific for their substrates, but some are multispecific, such as the | } multidrug transporters. So B
Secondary transport systems use electrochemical gradients to provide the energy for sugar translocation. They comprise symporters, which cotransport two or : more solutes, uniporters, which transport one molecule, and antiporters, which :
Co countertransport two or more solutes. Symporters generally couple the uphill oo 30 movement of the substrate to the downhill movement of a proton (or ion), antiporters use the ion gradient for excretion of a product, and uniporters do not use a coupling . Ce .ton (Poolman (2002) Antonie van Leeuwenhoek 82:147-164). - Group translocation involves the phosphoenolpyruvate (PEP)-dependent ) Co phosphotransferase system (PTS), which couples the uptake of a carbohydrate or alditol with its phosphorylation (Poolman (2002), supra). The phosphate group originates from the conversion of PEP into pyruvate, and the subsequent phosphorylation involves the energy coupling proteins, Enzyme I and HPr, as well as ] substrate-specific phosphoryl transfer proteins IIA, IIB and IC. :
Multidrug transporters may be separated into two major classes, secondary a - multidrug transporters and ABC transporters. Secondary multidrug transporters may : be further divided into distinct families, including the major facilitator superfamily i (MFS), the small multidrug resistance family (SMR), the resistance-nodulation-cell . division family (RND), and the multidrug and toxic compound extrusion family (MATE) (Putman ef al. (2000) Microbiol. Mol Biol. Reviews 64:672-693). “Secondary multidrug transporters use the electrochemical gradients, as described herein, to extrude drugs from the cell. ABC-type multidrug transporters use energy from ATP hydrolysis to pump drugs out of the cell (Putman ef al. (2000), supra).
Bacteria are able to metabolize various carbohydrates by utilizing transport proteins and enzymes with different carbohydrate specificities, in addition to employing diverse regulatory mechanisms, such as catabolite repression. The ‘isolation and characterization of these proteins allows for the development of essential : probiotic products with numerous applications, including those that benefit human and/or animal health, and those concerned with food production and safety. The proteins can also be used in developing transgenic plants with altered growth or , survival capabilities.
Compositions and methods for modifying microorganisms and plants are provided. Compositions of the invention include isolated nucleic acids from
Lactobacillus acidophilus encoding carbohydrate utilization-related proteins, including proteins of the phosphotransferase system (PTS), ABC transporters, and other proteins involved in transport, degradation, and/or synthesis of sugars in
Lactobacillus acidophilus. Compositions also include isolated nucleic acids from . 30 Lactobacillus acidophilus that encode multidrug transporters. Specifically, the present invention provides isolated nucleic acid molecules comprising, consisting essentially of and/or consisting of the nucleotide sequence as set forth in odd numbered SEQID ~ : : NOS:1-363, singly and/or in any combination, and isolated nucleic acid molecules
: encoding the amino acid sequence as set forth found in even numbered SEQ ID
NOS:2-364, singly and/or in any combination. Also provided are isolated and/or recombinant polypeptides comprising, consisting essentially of and/or consisting of an : ] amino acid sequence encoded by a nucleic acid molecule described herein and/or as : set forth in even numbered SEQ ID NOS:2-364, singly and/or in any combination. ’ oo Variant nucleic acids and polypeptides sufficiently identical to the nucleotide oo sequences and amino acid sequences set forth in the Sequence Listing are | | " encompassed by the present invention. Additionally, fragments and sufficiently : identical fragments of the nucleotide sequences and amino acid sequences are : encompassed. Nucleotide sequences that are complementary to a nucleic acid sequence of the invention, or that hybridize to a nucleotide sequence of the invention, are also encompassed. ~~ Compositions further include vectors and prokaryotic, eukaryotic and plant cells for recombinant expression of the nucleic acids described herein, as well as transgenic microbial and plant populations comprising the vectors. Also included in the invention are methods for the recombinant production of the polypeptides of the oo invention, and methods for their use. Further included are methods and kits for detecting the presence of a nucleic acid and/or polypeptide sequence of the invention in a sample, and antibodies that bind to a polypeptide of the invention. Biologically pure cultures of bacteria comprising a nucleotide or amino acid sequence of the ' present invention are encompassed. Food containing these cultures are encompassed, including milk, yogurt, curd, cheese, fermented milks, ice creams, fermented cereal based products, milk based powders, infant formulae, tablets, liquid bacterial suspensions, dried oral supplement, and liquid oral supplements. ~The carbohydrate utilization-related and multidrug transporter molecules of ; the present invention are useful for the selection and production of recombinant ~ bacteria, particularly the production of bacteria with improved fermentative abilities.
Such bacteria include, but are not limited to, bacteria that have a modified ability to synthesize, transport, accumulate, and/or utilize various carbohydrates, bacteria with . 30 altered flavors or textures, bacteria that produce altered carbohydrates, and bacteria better able to survive stressful conditions, such as those encountered in food : processing and/or in the gastrointestinal tract of an animal. The multidrug transporter molecules of the present invention include those that allow bacteria to better survive _ contact with antimicrobial polypeptides, such as bacteriocins or other toxins. These carbohydrate utilization-related and multidrug transporter molecules are also useful for modifying plant species. Transgenic plants comprising one or more sequences of the present invention may be beneficial economically in‘'that they are more resistance ne 2 to environmental stresses, including, but not limited to, plant pathogens, high salt “ concentration, or dehydration. They may also be better able to withstand food processing and storage conditions. . : : The present invention provides an isolated nucleic acid selected from the i oo group consisting of a nucleic acid comprising, consisting of and/or consisting ) . essentially of a nucleotide sequence as set forth in SEQ ID NOS:1, 3,5, 7,9, 11, 13, 15,17,19,21,23,25,27,29, 31, 33, 35, 37, 39,41, 43, 45, 47,49, 51, 53, 55, 57, 59, 61,63, 65,67,69,71,73,75,77,79, 81, 83, 85, 87, 89, 91, 93,95, 97, 99, 101, 103, 105,107,109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167,169, 171, 173,175,177, 179, 181, 183, 182, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207,209,211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235,237, 239, 241, 243,245,247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, a 275,277,279, 281, 283, 285, 287, 289, 291, 293, 295, 297, 299, 301, 303,305, 307, 309,311,313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361 and/or 363 in any combination, including multiples of the same sequence, and/or a complement thereof, a nucleic acid comprising, consisting of and/or consisting essentially of a nucleotide sequence having at least 90% sequence identity to a nucleotide sequence as set forth in SEQ ID
NOS:1,3,5,7,9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, dg 47,49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, ) 93,95,97,99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123,125, 127, : 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157,159, 161, : 163,165, 167, 169, 171, 173,175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225,227, 229, i 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267,269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 293, 295, 297, ] 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361 and/or 363 in any combination, including multiples of the same sequence, and/or a complement ) . thereof, a nucleic acid comprising, consisting of and/or consisting essentially of a -
fragment of a nucleotide sequence as set forth in SEQ IDNOS:1, 3,5,7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47,49, 51,53, 55, 57, 59, 61, 63, 65, 67,69, 71,73,75, 77,79, 81, 83, 85, 87, 89, 91, 93, 95, 97,99, 101, 103, 105, 107,109, 111, 113, 115, 117, 119, 121, 123, 125, 127,129, 131, 133, 135,137,139, ~~ ° :
S 141, 143, 145, 147, 149,°151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175,177,179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, oo 209, 211, 213, 215, 217, 219, 221, 223, 225,227, 229, 231, 233, 235, 237, 239, 241, . 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277,279, 281, 283, 285, 287, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311,313, 315,317, 319, 321,323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361 and/or 363 in any combination, including - : multiples of the same sequence, and/or a complement thereof, a nucleic acid that encodes a polypeptide comprising an amino acid sequence as set forth in SEQ ID :
NO:2, 4,6, 8, 10, 12, i4, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, ’ 48,50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, : 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192,194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232,234,236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312,314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362 and/or 364 i in any combination, including multiples of the same sequence, and/or encoded by a nucleic acid molecule described herein, a nucleic acid comprising a nucleotide : sequence encoding a polypeptide having at least 90% amino acid sequence identity to the amino acid sequence as set forth in SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22,24, 26,28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, : 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110,112,114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, ] 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164,166, 168, 170, 172,174, 176, . 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212,214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, ; 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, :
280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314,316,318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, : 348, 350,352, 354, 356, 358, 360, 362 and/or 364 in any combination, including oo multiples of the same sequence, and/or encoded by a nucleic acid molecule described ] 5 herein, and a nucleic acid that hybridizes under stringent conditions to any of the above. - So - Compositions further include vectors comprising the nucleic acids described i herein, vectors further comprising a nucleic acid encoding a heterologous polypeptide, : "and cells, including bacterial, plant and eukaryotic cells, containing said vectors. Also included in the invention are methods for the recombinant production of the polypeptides of the invention, and methods for their use. Further included are methods ) and kits for detecting the presence of a nucleic acid or polypeptide sequence of the ; invention in a sample, and antibodies that bind to a polypeptide of the invention. :
The present invention further provides an isolated polypeptide selected from the group consisting of: a) a polypeptide comprising, consisting of and/or consisting essentially of an amino acid sequence as set forth in SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74,76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108,110,112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134,136, 138, : 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174,176,178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208,210,212,214, 216, 218, 220, 222, 224,226, 228, 230, 232, 234, 236, 238, 240, 242,244,246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, ; 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, SE 310,312,314,316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, b 344, 346, 348, 350, 352, 354, 356, 358, 360, 362 and/or 364 in any combination, including multiples of the same sequence, and/or encoded by a nucleic acid molecule described herein; b) a polypeptide comprising, consisting of and/or consisting 5 essentially of a fragment of an amino acid sequence as set forth in SEQ ID NO:2, 4, 6, 8,10, 12,14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, oo 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, BN 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170,172,174, 176, 178, 180, 182, 184, 186, 188,190, 192, 194, 196, 198, 200,
© WO 2005/084411 PCT/US2005/007594 202, 204, 206, 208, 210, 212,214,216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, .
SE 270,272,274,276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, lo 304, 306, 308, 310, 312, 314,316, 318, 320, 322, 324,326, 328, 330, 332, 334, 336, 338,340, 342, 344, 346,348, 350, 352, 354, 356, 358, 360, 362 and/or 364 in any * combination, including multiples of the same sequence, and/or encoded by a nucleic i acid molecule described herein; ¢) a polypeptide comprising, consisting of and/or “ _ consisting essentially of an amino acid sequence having at least 90% sequence : identity with an.amino acid sequence as set forth in SEQ ID NO:2, 4,6, 8, 10, 12, 14, 16, 18,20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108,110,112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174,176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 200, 208,210, 212, 214,216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242,244,246, 248, 250,252, 254, 256, 258, 260, 262, 264, 266, 268,270, 272, 274, 276,278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, - 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362 and/or 364 in any combination, - 20 including multiples of the same sequence, and/or encoded by a nucleic acid molecule described herein; d) a polypeptide encoded by a nucleotide sequence having at least 90% sequence identity to a nucleotide sequence as set forth in SEQ ID NOS:1, 3, 5, 7, oo 9,11,13,15,17, 19,21, 23,25,27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57,59, 61, 63,65,67,69,71, 73,75, 77,79, 81, 83, 85, 87, 89, 91, 93, 95,97, 99, - 101,103, 105, 107,109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, : 135,137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175,177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217,219, 221, 223, 225, 227, 229, 231, 233, 235, | : 237,239, 241, 243, 245, 247, 249, 251, 253, 255, 257,259, 261, 263, 265, 267, 269, 271,273,275,277, 279, 281, 283, 285, 287, 289, 291, 293, 295, 297, 299, 301, 303, ] 305,307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361 and/or 363 in any ~ combination; and e) a polypeptide encoded by a nucleotide sequence as set forth in :
SEQID NOS:1,3,5,7,9, 11, 13, 15,17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, .
43,45,47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 7s, 77, 79, 81, 83, 85, 87, 89,91, 93,95,97,99, 101, 103, 105, 107,109, 111,113, 115, 117, 119, 121, 123, 125,127,129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, ) 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193,195,197, 199, 201,203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227,229, 231,233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 293, - 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, : 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361 » 10 and/or 363 in any combination.
Also provided is a polypeptide of this invention further comprising one or : more heterologous amino acid sequences, and antibodies that selectively bind to the polypeptides described herein. : - Additionally provided are methods for producing a polypeptide, said method comprising culturing the cell of this invention under conditions in which a nucleic acid encoding the polypeptide is expressed, said polypeptide being selected from the -group consisting of: a) a polypeptide comprising an amino acid sequence of SEQ ID
NO:2,4,6, 8,10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94,96,98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, ’ 130, 132,134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168,170,172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214,216, 218, 220, 222, 224, 226, 228, 230, ; 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, : 266, 268, 270,272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, } 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362 and/or 364 in any combination, including multiples of the same sequence, and/or encoded by a - nucleic acid molecule described herein; b) a polypeptide comprising a fragment of an : ) 30 amino acid sequence as set forth in SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, : oo 24,26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70,72,74, 76,78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112,114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, ) 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168,170,172, 174, 176, 178, . :
180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214,216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272,274,276, 278, 280, . - 282,284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308,310, 312, 314, a 316,318, 320,322, 324,326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, ) 350, 352, 354, 356, 358, 360, 362 and/or 364 in any combination, including multiples : of the same sequence, and/or encoded by a nucleic acid molecule described herein; ¢) | . a polypeptide comprising an amino acid sequence having at least 90% sequence : identity with an amino acid sequence as set forth in SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18,20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70,72, 74,76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212,214,216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242,244, 246, 248, 250, 252, 254, 256, 258,260, 262, 264, 266, 268, 270, 272, 274, 276,278, 280, 282, 284, 286, 288,290,292, 204, 296, 298, 300, 302, 304, 306, 308, | - 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344,346, 348, 350, 352, 354, 356, 358, 360, 362 and/or 364 in any combination, including multiples of the same sequence, and/or encoded by a nucleic acid molecule ! described herein; d) a polypeptide encoded by a nucleotide sequence having at least 90% sequence identity to a nucleotide sequence as set forth in SEQ ID NOS:1, 3, 5, 7, 9,11, 13,15,17, 19, 21, 23, 25, 27,29, 31, 33, 35,37, 39,41,43,45,47,49,51,53, : 55,57, 59, 61, 63, 65, 67,69, 71,73,75, 77, 79, 81, 83, 85, 87, 89,91, 93, 95, 97, 99, : 101,103, 105,107,109, 111, 113,115,117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169,171, 173,175,177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 21 1,213, 215,217,219, 221, 223, 225, 227, 229, 231, 233, 235, . 237,239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, ) 30 271,273,275,277,279, 281, 283, 285, 287, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311,313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361 and/or 363 in any combination; and €) a polypeptide encoded by a nucleotide sequence as set forth in )
SEQIDNOS:1,3,5,7,9, 11, 13,15,17, 19, 21, 23, 25, 27,29, 31, 33, 35,37, 39, 41, 10 j -
43,45,47,49, 51, 53, 55,57, 59, 61, 63, 65, 67,69, 71,73, 75, 77,79, 81, 83, 85, 87, : 89,91, 93,95, 97,99, 101, 103, 105, 107, 109, 111, 113, 115,117,119, 121, 123, 125,127,129, 131,133, 135,137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, : - 159,161, 163, 165,167, 169, 171,173, 175, 177, 179, 181, 183; 185, 187, 189, 191, : 193,195,197, 199,201,203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, ) : 227,229,231, 233,235,237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, : 261,263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291,293, Cs 295,297,299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, _ 329,331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361 and/or 363 in any combination.
Also provided are methods for detecting the presence of a polypeptide in a : ‘sample, said method comprising contacting the sample with a compound that selectively binds to a polypeptide and determining whether the compound binds to the . polypeptide in the sample; wherein said polypeptide is selected from the group consisting of: a) a polypeptide encoded by a nucleotide sequence as set forth in SEQ . - IDNOS:1,3,5,7,9,11, 13, 15,17, 19, 21, 23, 25,27, 29, 31, 33, 35, 37, 39, 41, 43, a 45,47, 49, 51, 53, 55, 57,59, 61, 63, 65, 67,69, 71,73, 75, 77, 79, 81, 83, 85, 87, 89, 91,93,95,97,99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, : 127,129,131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, - 20 161,163, 165,167,169,171, 173,175,177, 179, 181, 183, 185, 187, 189, 191, 193,
Co 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231,233, 235,237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263,265, 267,269, 271,273, 275, 277, 279, 281, 283, 285, 287, 289,291, 293, 295, ‘ : 297,299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, ) 331,333, 335,337,339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361 and/or 363 in any combination; b) a polypeptide comprising a fragment of an amino acid sequence encoded by a nucleic acid sequence as set forth in SEQ ID NOS:1, 3, 5, 7, 9, 11,13, 15,17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45,47, 49, 51, 53, 55, 3 57,59, 61,63, 65,67,69,71, 73,75,77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, - 30 101,103, 105,107,109, 111, 113,115,117, 119, 121, 123, 125, 127,129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, ) 169, 171,173, 175,177,179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, ~ 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229,231, 233, 235, 237,239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269,
271,273, 275,277, 279, 281, 283, 285, 287, 289, 291, 293, 295, 297, 299, 301, 303, oo 305,307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361 and/or 363 in any - | combination; c) a polypeptide encoded by a nucleotide sequence having at least 90% sequence identity to a nucleotide sequence as set forth in SEQ ID NOS:1,3,5,7,9, ) 11,13,15,17, 19,21, 23, 25, 27,29, 31, 33, 35, 37,39, 41, 43, 45,47, 49, 51, 53, 55, 57,59, 61, 63,65,67,69,71,73,75,77,79, 81, 83, 85, 87, 89, 91,93, 95, 97, 99, . 101,103, 105,107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, oo 135, 137, 139, 141, 143. 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169,171,173,175,177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207,209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237,239, 241,243,245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271,273,275,277,279, 281, 283, 285, 287, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317,319, 321, 323, 325, 327, 329, 331, 333, 335,337, 339,341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361 and/or 363 in any combination; d) a polypeptide comprising an amino acid sequence having at least | : oo 90% sequence identity to an amino acid sequence as set forth in SEQ ID NO:2, 4, 6, 8,10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, oo 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88,90, 92, 94, 96, 98, 100,102,104, 106,108,110, 112,114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168,170, 172,174,176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, : 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270,272,274, 276,278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312,314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362 and/or 364 in any combination; and €) a polypeptide comprising an amino acid sequence as set forth in 3
SEQ ID NO:2,4, 6,8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, ” 30 44,46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118,120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220,222, 224, 226, :
228,230, 232,234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, oo 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, - 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362 and/or 364 in any combination. ’ - Additionally provided are methods for detecting the presence of a polypeptide in a sample, said method comprising contacting the sample with a compound that + selectively binds to a polypeptide and determining whether the compound binds to the
EE polypeptide in the sample of the invention, wherein the compound that binds to the polypeptide is an antibody. Also provided is a kit comprising a compound for use in the methods of the invention and instructions for use. | :
The present invention also provides methods for detecting the presence of a nucleic acid molecule and/or fragments thereof of this invention in a sample, comprising: a) contacting the sample with a nucleic acid probe or primer that selectively hybridizes to the nucleic acid molecule and/or fragment thereof; and b) _ determining whether the nucleic acid probe or primer hybridizes. to a nucleic acid molecule in the sample, thereby detecting the presence of a nucleic acid molecule and/or fragment thereof of this invention in the sample. Also provided are methods for detecting the presence of a nucleic acid molecule and/or fragment of the invention in a sample wherein the sample comprises mRNA molecules and is contacted with a oo 7 nucleic acid probe. Additionally provided herein is a Kit comprising a compound that : selectively hybridizes to a nucleic acid of the invention, and instructions for use.
Additionally provided are methods for 1) modifying the ability of an organism . to transport a carbohydrate into or out of a cell; 2) modifying the ability of an B organism to accumulate a carbohydrate; 3) modifying the ability of an organism to utilize a carbohydrate as an energy source; 4) modifying the ability of an organism to produce a modified carbohydrate; 5) modifying the flavor of a food product fermented by a microorganism; 6) modifying the texture of a food product fermented by a - microorganism; 7) modifying the ability of an organism to survive food processing - 30 and storage conditions; 8) modifying the ability of a microorganism to survive in a gastro-intestinal (GI) tract; 9) modifying the ability of an organism to transport a drug ) into or out of a cell; and 10) modifying the ability of an organism to produce a ~ carbohydrate, comprising introducing into said organism and/or microorganism a : vector comprising at least one nucleotide sequence of this invention and/or at least one nucleotide sequence selected from the group consisting of: a) a nucleotide sequence as set forth in SEQ ID NOS:1, 3,5, 7,9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29,31, 33, 35,37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67,69, 71, 73, :
SEE 75,717, 79, 81,83, 85, 87, 89,91, 93, 95,97, 99, 101, 103, 105, 107,109, 111, 113, . 115,117,119, 121, 123,7125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, - 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, oo 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, . 217,219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, LL 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 2717, 279, 281, 283, : 285,287, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, Co 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361 and/or 363 in any combination; b) a nucleotide sequence comprising a fragment of a nucleotide sequence as set forth in SEQ ID NOS:1, 3,5, 7, : 9,11,13,15,17,19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41,43, 45,47, 49, 51, 53, 55,57,59, 61, 63, 65, 67,69,71,73,75,77,79, 81, 83, 85, 87, 89,91, 93,95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135,137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169,171, 173, 175,177,179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229,231,233, 235, 237,239,241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271,273,275,277, 279, 281, 283, 285, 287, 289, 291, 293, 295, 297, 299, 301, 303, 305,307,309,311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361 and/or 363 in any ” combination, wherein said fragment encodes a polypeptide that retains activity; c) a nucleotide sequence that is at least 90% identical to the sequence as set forth in SEQ : ID NOS:1,3,5,7,9, 11, 13,15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45,47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71,73, 75, 77, 79, 81, 83, 85, 87, 89, : 91, 93, 95,97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, : 127,129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, . 30 161,163, 165,167,169, 171, 173,175,177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, : 229,231,233, 235,237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, ~ 263, 265, 267, 269, 271, 273, 275,277, 279, 281, 283, 285, 287, 289, 291, 293, 295, oo - 297,299, 301, 303, 305, 307, 309,311, 313, 315, 317, 319, 321, 323, 325, 327, 329,
’ © WO 2005/084411 PCT/US2005/007594 : 331,333,335, 337,339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361 and/or 363 in any combination, wherein said nucleotide sequence encodes a polypeptide that retains activity; and d) a nucleotide sequence encoding a polypeptide comprising an . amino acid sequence having at least 90% sequence identity to-an amino-acid sequence C0 as set forth in SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34,
To 36, 38,40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68,70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110,112,114, 116, 118, . : 120,122,124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154,156, 158, 160, 162, 164, 166, 168, 170,172, 174,176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222,224,226, 228,230, 232, 234, 236, 238, 240, 242,244,246, 248, 250, 252, 254, oC 256,258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, : 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, : 324,326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358,360, 362 and/or 364 in any combination, wherein said polypeptide retains activity; and e) a nucleotide sequence encoding a polypeptide comprising an amino - acid sequence as set forth in SEQ ID-NO:2, 4,6,8,10,12, 14,16, 18, 20, 22, 24, 26, 128,30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74,76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114,116,118, 120,122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, ' 148, 150, 152, 154,156, 158, 160, 162, 164, 166, 168,170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, -216,218,220,222,224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252,254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, : 284,286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362 and/or 364 in any combination.
Further provided herein is 1) a Lactobacillus acidophilus bacterial strain with : a modified ability to transport a carbohydrate into or out of a cell as compared to a - 30 wild-type Lactobacillus acidophilus; 2) a Lactobacillus acidophilus bacterial strain with a modified ability to accumulate a carbohydrate, as compared to a wild-type ) Lactobacillus acidophilus; 3) a Lactobacillus acidophilus bacterial strain with a modified ability to utilize a carbohydrate as an energy source, as compared to a wild- - ) type Lactobacillus acidophilus, 4) a Lactobacillus acidophilus bacterial strain that -
provides a food product with a modified flavor as a result of fermentation, as compared to a wild-type Lactobacillus acidophilus; 5) a Lactobacillus acidophilus bacterial strain that provides a food product with a modified texture as a result of . fermentation, as compared to a wild-type Lactobacillus acidophilus; 6) a
Lactobacillus acidophilus bacterial strain with a modified ability 0 produce a ’ carbohydrate, as compared to a wild-type Lactobacillus acidophilus; 7) a
Lactobacillus acidophilus bacterial strain with a modified ability. to survive food - processing and storage conditions, as compared to a wild-type Lactobacillus ’ : acidophilus; and 8) a Lactobacillus acidophilus bacterial strain with a modified ability to survive in a GI tract, as compared to a wild-type Lactobacillus acidophilus, : wherein said modified ability, flavor and/or texture is due to expression of at least one carbohydrate utilization-related polypeptide as set forth in SEQ IDNO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34; 36, 38, 40,42, 44, 46, 48, 50, 52, 54, 56, : 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96,98, 100, 102, 104, 106, 108, 110, 112,114,116, 118, 120, 122, 124, 126, 128, 130, 132, 134, os 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, -170, 172, 174, 176,178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212,214,216, 218, 220, 222, 224, 226, 228, 230, 232,234, 236, 238,240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, - 272,274,276,278, 280, 282,284, 286, 288, 290, 292, 294, 296, 298; 300, 302, 304, 306, 308, 310, 312,314,316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336,338, - ] 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362 and/or 364 in any : combination. :
Additionally provided is a Lactobacillus acidophilus bacterial strain with a : modified ability to survive contact with an antimicrobial polypeptide or toxin, as compared to a wild-type Lactobacillus acidophilus, wherein said modified ability is due to expression of at least one multidrug transport polypeptide as set forth in even
SEQ ID NOs: 78-88, 92-94, 124-126, 132, 282-288 ,308 and/or 312-322. | ;
Also provided is a plant, a plant cell and/or a seed of a plant, having stably : ” 30 incorporated into its genome a DNA construct comprising at least one nucleotide sequence of this invention and/or at least one nucleotide sequence of this invention, . ] selected from the group consisting of: a) a nucleotide sequence as set forth in any of : SEQ ID NOs:1-363, singly and/or in any combination, or a complement thereof; b) a ) nucleotide sequence having at least 90% sequence identity to a nucleotide sequence as :
set forth in any of SEQ ID NOs:1-363, singly and/or in any combination, ora complement thereof; c) a nucleotide sequence comprising a fragment of a nucleotide sequence as set forth in any of SEQ ID NOs:1-363, singly and/or in any combination, - or a complement thereof; d) a nucleotide sequence that encodes a polypeptide ’ comprising an amino acid sequence as set forth in any of SEQ ID NOs:2-364; ¢) a nucleotide sequence that encodes a polypeptide comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence as set forth in any of "
SEQ ID NOs:2-364 and f) a nucleotide sequence that hybridizes under stringent conditions to any of a)—e). | : co 0 . i oo oo :
Figure 1. Genetic loci of interest. The layouts of the loci discussed in the text . : are shown: man, glucose-mannose locus; fru, fructose locus; suc, sucrose locus; fos,
FOS locus; raff, raffinose locus; Lac, lactose-galactose loci; tre, trehalose locus; CCR, : carbon catabolite loci. : :
Figure 2. Carbohydrate utilization in Lactobacillus acidophilus. This diagram LL shows carbohydrate transporters and hydrolases as predicted by transcriptional profiles. Protein names and EC numbers are specified for each element. PTS transporters are shown in black. GPH transporters are shown in light gray. ABC , 20 transporters are shown in dark gray. oo
The present invention relates to carbohydrate utilization-related and multidrug : transport molecules from Lactobacillus acidophilus. Nucleotide and amino acid sequences of the carbohydrate utilization-related and multidrug transport molecules : are provided. The sequences are useful for modifying microorganisms, cells and plants for enhanced properties. ]
As used herein, “a,” “an” and “the” can be plural or singular as used i ] throughout the specification and claims. For example “a” cell can mean a single cell or a multiplicity of cells. - Also as used herein, “and/or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of - : combinations when interpreted in the alternative ("or").
By “carbohydrate utilization-related” molecules or genes is meant novel sequences from Lactobacillus acidophilus that encode proteins involved in the oo utilization of carbohydrate molecules, including, but not limited to, the synthesis, . transport, or degradation of carbohydrates. By “multidrug transporter” molecules is ’ meant those that are involved in the transport of antimicrobial polypeptides such as - bacteriocins, or other drugs or toxins. See Table 1 for specific carbohydrate utilization-related and multidrug transporter molecules of the present invention. The . + full-length gene sequences are referred to as “carbohydrate utilization-related sequences” or “multidrug transporter sequences,” showing that they have similarity to carbohydrate utilization-related genes or multidrug transporter genes, respectively.
The invention further provides fragments and variants of these carbohydrate utilization related sequences or multidrug transporter sequences, which can also be . used to practice methods of the present invention. oo : :
By “carbohydrate” is meant an organic compound containing carbon, : hydrogen, and oxygen, usually in the ratio 1:2:1. Carbohydrates include, but are not . : limited to, sugars, starches, celluloses, and gums. As used herein, the terms “gene” : and “recombinant gene” refer to nucleic acids comprising an open reading frame, particularly those encoding a carbohydrate utilization-related protein or a multidrug transporter protein. Isolated nucleic acids of the present invention comprise nucleic acid sequences encoding carbohydrate utilization-related proteins or multidrug transporter proteins, nucleic acid sequences encoding the amino acid sequences set forth in even numbered SEQ ID NOS:2-364, the nucleic acid sequences set forth in odd numbered SEQ ID NOS:1-363, and variants and fragments thereof. The present invention also encompasses antisense nucleic acids, as described below. :
In addition, isolated polypeptides and proteins having carbohydrate utilization- : related activity or multidrug transporter activity, and variants and fragments thereof, : are encompassed, as well as methods for producing those polypeptides. For purposes of the present invention, the terms “protein” and “polypeptide” are used | : interchangeably. The polypeptides of the present invention have carbohydrate : 30 utilization-related protein activity or multidrug transporter activity. Carbohydrate utilization-related protein activity or multidrug transporter activity refers to a ) biological or functional activity as determined in vivo or in vitro according to standard assay techniques. These activities include, but are not limited to, the ability to synthesize a carbohydrate, the ability to transport a carbohydrate into or out of a cell, ‘
the ability to degrade a carbohydrate, the ability to regulate the concentration of a carbohydrate in a cell, the ability to bind a carbohydrate, and the ability to transport a : drug or toxin into or out of a cell. - The structures of the various types of bacterial transporters are well known in oo So the art. The ATP-binding cassette (ABC) superfamily (PFAM Accession No.
PF00005) of transporters consists of proteins with four core domains (Higgins et al. (1986) Nature 323:448-450; Hyde ef al. (1990) Nature 346:362-365; Higgins (2001) .
Res. Microbiol. 152:205-210). Typically there are two transmembrane domains ) (PFAM Accession No. PF00664) with six membrane-spanning alpha helices per domain, and two ATP-binding domains that contain the core amino acids by which the transporters are defined (Higgins (2001) supra.), as well as the other conserved oo motifs including the Walker A and Walker B motifs (Walker et al. (1982) EMBO J. 1:945-951; Prosite Ref. No. PDOC00185). -
ABC transporter proteins of the present invention include those in SEQ ID NOS:40, 42, 44, 48, 52, 54, 56, 58, 62, 64, 66, 68, 70, 72,74, 110,112, 114, 116, 122, 124, 126, 128, 130, 132, 134, 136, 144, 146, 148, 152, 154, 160, 236, 262,274,278, 280, 294, 296, 298, 300, 302, 306, 338, 340, and 360. SEQ ID NOS:126 and 144 are members of the ABC transporter transmembrane region family (PFAM Accession No.
PF00664).
The TOBE domain (Transport-associated OB) (PFAM Accession No.
AE PF03459) always occurs as a dimer as the C-terminal strand of each domain is supplied by the partner (Koonin ef al. (2000) Adv. Protein Chem. 54:245-75). It is probably involved in the recognition of small ligands such as molybdenum and : sulfate. It is found in ABC transporters immediately after the ATPase domain. TOBE - domain proteins of the present invention include those in SEQ ID NO:110. : :
The secondary transport system proteins include the galactoside-pentose- hexuronide group of translocators (Poolman et al. (1996) Mol. Microbiol. 19:911- : 922). These proteins generally consist of a hydrophobic domain comprising twelve vu membrane spanning domains and a carboxyterminal enzyme IIA domain (Poolman ef - 30 al. (1989) J. Bacteriol. 171:244-253).
The phosphotransferase system (PTS) catalyzes the phosphorylation of sugar substrates during their translocation across the cell membrane. The mechanism involves the transfer of a phosphoryl! group from phosphoenolpyruvate (PEP) via enzyme 1 (EI) (Prosite Ref. No. PDOC00527) to enzyme II (El) of the PTS system 3 oo WO 2005/084411 : : PCT/US2005/007594 (Prosite Ref. Nos. PDOCO00528; PDOC00795), which in turn transfers it to a phosphocarrier protein (HPr) (Prosite Ref. No. PDOC00318) (PFAM Accession No.
PF00381). The HPr protein contains two conserved phosphorylation sites, a histidine residue at the amino-terminal side that is phosphorylated by Enzyme I, and a serine k } 5 residue at the carboxy-terminal side of the protein that may be phosphorylated by an oo
ATP-dependent protein kinase (de Vos (1996) Antonie van Leeuwenhoek 70:223~ 242). SEQ ID NO:178 is a member of the PTS HPr component phosphorylation site -- .. family (PFAM Accession No. PF00381).
The sugar-specific permease (enzyme II) of the PTS system consists of at least three structurally distinct domains (IIA, IIB, and IIC) which can either be fused together in a single polypeptide chain or exist as two or three interactive chains. The
IIA domain carries the first permease-specific phosphorylation site, a histidine that is ob phosphorylated by phospho-HPr. The second domain (IIB) is phosphorylated by phospho-IIA on a cysteinyl or histidyl residue, depending on the permease. Finally, the phosphoryl group is transferred from the IIB domain to the sugar substrate in a oo | process catalyzed by the I1C domain; this . is coupled to the transmembrane : : transport of the sugar. Phosphoenolpyruvate-dependent sugar phosphotransferase system, EIIA 1 family (PFAM Accession No. PF00358) proteins of the present "invention include those in SEQ ID NOS:6, 12, 14, 34, 102, 104, 174, 176, 268, and : 20 290. Phosphoenolpyruvate-dependent sugar phosphotransferase system, EIIA 2 (PFAM Accession No. PF00359) proteins of the present invention include that in
SEQ ID NO:36. SEQ ID NO:36 is also a member of the PTS system, Fructose specific IIB subunit (PFAM Accession No. PF02379). Phosphotransferase system,
EIIB family (PFAM Accession No. PF00367) proteins of the present invention : 25 include those in SEQ ID NOS:12, 14, 32, 34, 102, 104, 268, and 290.
Phosphotransferase system, EIIC family (PFAM Accession No. PF02378) proteins of the present invention include those in SEQ ID NOS:12, 14, 16, 28, 30, 32, 34, 36, 102, 104, 174, 268, 270, 272, and 290.
The lactose/cellobiose-specific family is one of four structurally and 30 functionally distinct groups. The IIA PTS system enzymes (PFAM Accession No. ] ~ PF02255) normally function as a homotrimer, stabilized by a centrally located metal . ion. PTS system, Lactose/Cellobiose specific IIA subunit family proteins of the present invention include that in SEQ ID NO:4. The Lactose/Cellobiose specific IIB : subunit family (PFAM Accession No. PF£2302) are cytoplasmic enzymes. The fold of IIB cellobiose shows similar structure to mammalian tyrosine phosphatases. PTS system, Lactose/Cellobiose specific IIB subunit proteins of the present invention include that in SEQ ID NO:170.
The mannose family is unique in several respects among PTS permease families. It is the only PTS family in which members possess ald protein; itisthe only PTS family in which the IIB constituent is phosphorylated on a histidyl rather than a cysteyl residue; and, its permease members exhibit broad specificity for a range . of sugars, rather than being specific for just one or a few sugars. SEQ ID NOS:20 and oo i
EE 264 are members of the PTS system fructose IIA component (PFAM Accession No. PF03610) family. SEQ ID NO:168 is a member of the PTS system Ce mannose/fructose/sorbose family IID component (PFAM Accession No. PF03613).
SEQ ID NO:264 is a member of the PTS system sorbose subfamily IIB component (PFAM Accession No. PF03830). SEQ ID NO:166 is a member of the PTS system sorbose-specific lic component family (PFAM Accession No. PF 03609). ” : 15 A number of enzymes that catalyze the transfer of a phosphoryl group from oo ~~ phosphoenolpyruvate (PEP) via a phospho-histidine intermediate have been shown to be structurally related (Reizer ef al. (1993) Protein Sci. 2:506-21). All these enzymes share the same catalytic mechanism: they bind PEP and transfer the phosphoryl group : from itto a histidine residue. The sequence around that residue is highly conserved.
The PEP-utilizing enzyme, TIM barrel domain (PFAM Accession No. PF02896) is } often found associated with the pyruvate phosphate dikinase, PEP/pyruvate-binding domain (INTERPRO:IPR002192) at its N-terminus and the PEP-utilizing enzyme mobile domain (PFAM Accession No. PF00391). The PEP-utilizing enzyme, mobile 3 domain is a “swiveling” 3/B/a domain that is thought to be mobile in all proteins known to contain it (Cosenza et al. (2002) J. Mol. Biol. 318:1417-32). It is often found associated with the pyruvate phosphate dikinase, PEP/pyruvate-binding domain (INTERPRO:IPR002192) at its N-terminus. PEP-utilizing enzyme, TIM barrel domain proteins of the present invention include that in SEQ ID NO:180. PEP- : utilizing enzyme mobile domain proteins of the present invention include those in
SEQID NOS:180 and 258. PEP-utilizing enzyme, N-terminal family (PFAM ] Accession No. PF05524) proteins of the present invention include that in SEQ ID
NO:180. : ~
Members of the major facilitator super family (MFS) of multidrug transporters have either 12 or 14 transmembrane segments. Members of the small multidrug oo resistance family (SMR) of multidrug transporters are thought to form a tightly ) packed four-helix antiparallel bundle. They confer resistance to a wide range of toxic compounds by removing them from the cells. Members of the resistance nodulation- cell division family (RND) contain a single N-terminal transmembrane segment and a ] large C-terminal periplasmic domain (Putman ef al. (2000) Microbiol. Mol. Biol. .
Reviews 64:672-693). Conserved motifs within each of these types of multidrug transporters and also throughout the multidrug transporters of the MFS, SMR, and
RND families, as well as specific proteins from various bacteria (with Accession
Nos.) have been described (Putman er al. (2000) supra). Multidrug transporter proteins of the present invention include those in SEQ ID NOS:78, 80, 82, 84, 86, 88, 92.94, 282, 284, 286, 288, and 322. : : - The Sugar (and other) transporter family (PFAM Accession No. PFO0083)isa member of the Major Facilitator Superfamily clan. The MFS transporters are single-
So polypeptide secondary carriers capable only of transporting small solutes in response : to chemiosmotic ion gradients. All currently recognized MFS permeases retain the : two six-transmembrane segment (TMS) units within a single polypeptide chain, although in 3 of the 17 MFS families, an additional two TMSs are found (Paulson ef al (1996) Microbiol. Rev. 60:575-608). Moreover, the well-conserved MFS specific ’ * motif between TMS2 and TMS3 and the related but less well conserved motif between TMS8 and TMS9 (Henderson and Maiden (1990) Philos. Trans. R. Soc.
Lond. B. Biol. Sci. 326:391-410) prove to be a characteristic of virtually all of the more than 300 MFS proteins identified. Sugar (and other) transporter proteins of the : 25 present invention include those in SEQ ID NOS:80 and 282.
Bacterial binding protein-dependent transport systems are multicomponent systems typically composed of a periplasmic substrate-binding protein, one or two . reciprocally homologous integral inner-membrane proteins (PFAM Accession No. i
PF00528) and one or two peripheral membrane ATP-binding proteins that couple ) 30 energy to the active transport system. The integral inner-membrane proteins ] translocate the substrate across the membrane. It has been shown that most of these i proteins contain a conserved region located about 80 to 100 residues from their C- terminal extremity (Dassa and Hofnung (1985) EMBO J. 4:2287-93; Saurin et al. oo Co (1994) Mol. Microbiol. 12:993-1004). This region seems to be located in a :
cytoplasmic loop between two transmembrane domains (Pearce ef al. (1992) Mol.
Microbiol. 6:47-57). These proteins can be classified into seven families which have been respectively termed: araH, cysTW, fecCD, hisMQ, livHM, malFG and oppBC. ’ Binding-protein-dependent transport system inner membrane component proteins-of © : . the present invention include those in SEQ ID NOS:42, 44, 62, 64,112, 114, and 294.
The Branched-chain amino acid transport system/permease component family (PFAM ) Accession No. PF02653) is a large family mainly comprising high-affinity branched- y chain amino acid transporter proteins. Also found with in this family are proteins from the galactose transport system permease and a ribose transport system. Branched- . 10 chain amino acid transport system/permease component proteins of the present ol invention include those in SEQ ID NOS:54, 72, and 74. :
SEQ ID NO:184 is a member of the HPr Serine kinase N terminus family (PFAM Accession No. PF02603), as well as a member of the HPr Serine kinase C terminus family (PFAM Accession No. PF07475). The N terminus family represents : 15 the N-terminal region of Hpr Serine/threonine kinase PtsK. The C terminus family represents the C terminal kinase domain of Hpr Serine/threonine kinase PtsK. This - kinase is the sensor in a multicomponent phosphorelay system in control of carbon catabolic repression in bacteria (Marquez ef al. (2002) Proc. Natl. Acad. Sci. U.S.A. 99:3458-63). This kinase is unusual in that it recognizes the tertiary structure of its target and is a member of a novel family unrelated to any previously described protein ’ + phosphorylating enzymes. X-ray analysis of the full-length crystalline enzyme from ~~
Staphylococcus xylosus at a resolution of 1.95 A shows the enzyme to consist of two : clearly separated domains that are assembled in a hexameric structure resembling a : three-bladed propeller. The blades are formed by two N-terminal domains each, and ; the compact central hub assembles the C-terminal kinase domains (Reizer er al. (1998) Mol. Microbiol. 27:1157-69). :
The Periplasmic binding proteins and sugar binding domain of the Lacl family (PFAM Accession No. PF00532) includes the periplasmic binding proteins, and the .
Lacl family transcriptional regulators. The periplasmic binding proteins are the primary receptors for chemotaxis and transport of many sugar based solutes. The Lacl 3 family of proteins consists of transcriptional regulators related to the lac repressor. In this case, generally the sugar binding domain binds a sugar that changes the DNA i. binding activity of the repressor domain (lac). Periplasmic binding proteins and
: sugar binding domain of the Lac] family proteins of the present invention include those in SEQ ID NOS:38 and 98. a | Bacterial high affinity transport systems are involved in active transport of ) solutes across the cytoplasmic membrane. The protein components of these traffic -5 systems include one or two transmembrane protein components, one or two membrane-associated ATP-binding proteins and a high affinity periplasmic solute- EE binding protein (PFAM Accession No. PF01547). In Gram-positive bacteria, which . oo are surrounded by a single membrane and therefore have.no periplasmic region, the : equivalent proteins are bound to the membrane via an N-terminal lipid anchor. These homologue proteins do not play an integral role in the transport process per se, but probably serve as receptors to trigger or initiate translocation of the solute through the : membrane by binding to external sites of the integral membrane proteins of the efflux system. In addition at least some solute-binding proteins function in the initiation of : : sensory transduction pathways. Bacterial extracellular solute-binding proteins of the present invention include those in SEQ ID NOS:40, 66, 116, 262,274, and 296.
Ce _ The sugar transport protein family (PFAM Accession No. PF06800) is a family of bacterial sugar transporters approximately 300 residues long. Members include glucose uptake proteins (Fiegler ef al. (1999) J. Bacteriol. 181:4929-36), ribose transport proteins, and several putative and hypothetical membrane proteins probably involved in sugar transport across bacterial membranes. Sugar transport ’ proteins of the present invention include that in SEQ ID NO:234.
MIP (Major Intrinsic Protein) family proteins (PF AM Accession No.
PF00230) exhibit essentially two distinct types of channel properties: (1) specific : water transport by the aquaporins, and (2) small neutral solutes transport, such as : glycerol by the glycerol facilitators (Froger er al. (1998) Protein Sci. 7:1458-68). :
MIP family proteins are thought to contain 6 TM domains. Sequence analysis suggests that the proteins may have arisen through tandem, intragenic duplication from an ancestral protein that contained 3 TM domains (Wistow ef al. (1991) Trends :
Biochem. Sci. 16:170-1). a. 30 General transport proteins of the present invention include those in SEQ ID ] NOS:76, 90, 96, and 194.
The nucleic acid and protein compositions encompassed by the present invention are isolated or substantially purified. By “isolated” or “substantially : purified” is meant that the nucleic acid or protein molecules, or biologically active fragments or variants thereof, are substantially or essentially free from components normally found in association with the nucleic acid or protein in its natural state. Such components include other cellular material, culture medium from recombinant -- production, and/or various chemicals used in chemically synthesizing the proteins or nucleic acids. Preferably, an “isolated” nucleic acid of the present invention is free of . - nucleic acid sequences that flank the nucleic acid of interest in the genomic DNA of the organism from which the nucleic acid was obtained (such as coding sequences v present at the 5' or 3' ends). However, the molecule may include some additional bases or moieties that do not deleteriously affect the basic characteristics of the : 10 composition. For example, in various embodiments, the isolated nucleic acid contains : less than 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleic acid sequence normally associated with the genomic DNA in the cells from which it was obtained.
Similarly, a substantially purified protein has less than about 30%, 20%, 10%, 5%, or 1% (by dry weight) of contaminating protein, or non-carbohydrate utilization-related ~ 15 protein. When the protein is recombinantly produced, preferably culture medium
Co represents less than 30%, 20%, 10%, or 5% of the volume of the protein preparation, : and when the protein is produced chemically, preferably the preparations have less than about 30%, 20%, 10%, or 5% (by dry weight) of chemical precursors, or non- : carbohydrate utilization-related chemicals.
The compositions and methods of the present invention can be used to ’ modulate the function of the carbohydrate utilization-related or multidrug transporter . molecules of Lactobacillus acidophilus. By “modulate,” “alter,” or “modify” is meant the up- or downregulation of a target biological activity. Proteins of the invention are ) useful in modifying the biological activities of lactic acid bacteria, and also in modifying the nutritional or health-promoting characteristics of foods fermented by } such bacteria. Nucleotide molecules of the invention are useful in modulating carbohydrate utilization-related or multidrug transporter protein expression by lactic acid bacteria. Up- or downregulation of expression from a nucleic acid of the present = invention is encompassed. Upregulation may be accomplished, for example, by . 30 providing multiple gene copies, modulating expression by modifying regulatory elements, promoting transcriptional or translational mechanisms, or other means.
Downregulation may be accomplished, for example, by using known antisense and : gene silencing techniques. : a
© WO 2005/084411 | PCT/US2005/007594
By “lactic acid bacteria” is meant bacteria from a genus selected from the : following: Aerococcus, Carnobacterium, Enterococcus, Lactococcus, Lactobacillus,
Leuconostoc, Oenococcus, Pediococcus, Streptococcus, Melissococcus, Alloiococcus, - Dolosigranulum, Lactosphaera, Tetragenococcus, Vagococcus, and Weissella (Holzapfel er al. (2001) Am. J. Clin. Nutr. 73:3655-373S; Bergey's Manual of : i Systematic Bacteriology, Vol. 2 (Williams and Wilkins, Baltimore; (1986)) pp. 1075— 1079). _ }
The polypeptides of the present invention or microbes expressing them are useful as nutritional additives or supplements, and as additives in dairy and : fermentation processing. The nucleic acid sequences, encoded polypeptides, and microorganisms expressing them are useful in the manufacture of milk-derived products, such as cheeses, yogurt, fermented milk products, sour milks, and buttermilk. Microorganisms that express polypeptides of the invention may be : probiotic organisms. By “probiotic” is meant a live microorganism that survives passage through the gastrointestinal tract and has a beneficial effect on the subject. By - “subject” is meant an organism that comes into contact with a microorganism : expressing a protein of the present invention. Subject may refer to humans and other animals. : :
In addition to the carbohydrate utilization-related and multidrug transporter nucleotide sequences and fragments and variants thereof as disclosed herein, the ' nucleic acids of the current invention also encompass homologous nucleic acid sequences identified and isolated from other organisms or cells by hybridization with entire or partial sequences obtained from the carbohydrate utilization-related and multidrug transporter nucleotide sequences or variants and fragments thereof as disclosed herein.
Fragments and Variants
The invention provides isolated nucleic acids comprising nucleotide sequences ; encoding carbohydrate utilization-related and multidrug transporter proteins, as well as the carbohydrate utilization-related and multidrug transporter proteins encoded - thereby. By “carbohydrate utilization-related protein” is meant a protein having an : amino acid sequence as set forth in even numbered SEQ ID NOS:2-364. Fragments and variants of these nucleotide sequences and encoded proteins are also provided. By Co
“fragment” of a nucleotide sequence or protein is meant a portion of the nucleotide or amino acid sequence.
Fragments of the nucleic acids disclosed herein can be used as hybridization . - probes to identify carbohydrate utilization-related-encoding nucleic acids or multidrug transporter-encoding nucleic acids, or can be used as primers in amplification ] ) protocols [e.g., polymerase chain reaction (PCR)] or mutation of carbohydrate utilization-related or multidrug transporter nucleic acids. Fragments of nucleic acids . of this invention can also be bound to a physical substrate to comprise what may be } considered a macro- or microarray (see, for example, U.S. Patent No. 5,837,832; U.S. Co ~ 10 Patent No. 5,861,242; WO 89/10977, WO 89/11548; WO 93/17126; U.S. Patent No. 6,309,823). Such arrays or “chips” of nucleic acids may be used to study gene E expression or to identify nucleic acids with sufficient identity to the target sequences. ) The present invention further provides a nucleic acid array or chip, i.e., a multitude of nucleic acids (e.2z., DNA) as molecular probes precisely organized or arrayed on a solid support, which allow for the sequencing of genes, the study of } mutations contained therein and/or the analysis of the expression of genes, as such : arrays and chips are currently of interest given their very small size and their high capacity in terms of number of analyses.
The function of these nucleic acid arrays/chips is based on molecular probes, mainly oligonucieotides, which are attached to a carrier having a size of generally a ’ few square centimeters or more, as desired. For an analysis, the carrier, such as in a
DNA array/chip, is coated with DNA probes (e.g., oligonucleotides) that are arranged at a predetermined location or position on the carrier. A sample containing a target nucleic acid and/or fragments thereof to be analyzed, for example DNA or RNA or - cDNA, that has been labeled beforehand, is contacted with the DNA array/chip 2 leading to the formation, through hybridization, of a duplex. After a washing step, analysis of the surface of the chip allows any hybridizations to be located by means of the signals emitted by the labeled target. A hybridization fingerprint results, which, by St computer processing, allows retrieval of information such as the expression of genes, a . 30 the presence of specific fragments in the sample, the determination of sequences and/or the identification of mutations. ] In one embodiment of this invention, hybridization between target nucleic acids and nucleic acids of the invention, used in the form of probes and deposited or .
synthesized in situ on a DNA chip/array, can be determined by means of fluorescence, radioactivity, electronic detection or the like, as are well known in the art. : In another embodiment, the nucleotide sequences of the invention can be used - | in the form of a DNA array/chip to carry out analyses of the expression of 3
Lactobacillus acidophilus genes. This analysis is based on DNA array/chips on ) which probes, chosen for their specificity to characterize a given gene or nucleotide © sequence, are present. The target sequences to be analyzed are labeled before being . : hybridized onto the chip. After washing, the labeled complexes are detected and N ]
So quantified, with the hybridizations being carried out at least in duplicate. Comparative analyses of the signal intensities obtained with respect to the same probe for different samples and/or for different probes with the same sample, allows, for example, for differential transcription of RNA derived from the sample.
In yet another embodiment, arrays/chips containing nucleotide sequences of the invention can comprise nucleotide sequences specific for other microorganisms, which allows for serial testing and rapid identification of the presence of a ‘microorganism in a sample. : : .
In a further embodiment, the principle of the DNA array/chip can also be used to produce protein arrays/chips on which the support has been coated with a polypeptide and/or an antibody of this invention, or arrays thereof, in place of the nucleic acid. These protein arrays/chips make it possible, for example, to analyze the. ’ biomolecular interactions induced by the affinity capture of targets onto a support coated, e.g., with proteins, by surface plasma resonance (SPR). The polypeptides or antibodies of this invention, capable of specifically binding antibodies or polypeptides ; derived from the sample to be analyzed, can be used in protein arrays/chips for the ~ detection and/or identification of proteins and/or peptides in a sample. :
Thus, the present invention provides a microarray or microchip comprising various nucleic acids of this invention in any combination, including repeats, as well as a microarray comprising various polypeptides of this invention in any combination, 2 including repeats. Also provided is a microarray comprising antibodies that ’ 30 specifically react with various polypeptides of this invention, in any combination, including repeats. .
By “nucleic acid” 1s meant DNA molecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g., mRNA) and analogs of the DNA or RNA generated using ) nucleotide analogs. The nucleic acid can be single-stranded or double-stranded, but is :
typically double-stranded DNA. A fragment of a nucleic acid encoding a carbohydrate utilization-related protein or a multidrug transporter protein may encode a protein ~ fragment that is biologically active, or it may be used as a hybridization probe or PCR s : primer as described.herein. A biologically active fragment of a polypeptide disclosed : . 5 herein can be prepared by isolating a portion of one of the nucleotide sequences of the ) invention, expressing the encoded portion of the protein (e.g., by recombinant expression in vitro), and assessing the activity of the encoded portion of the protein. . : Fragments of nucleic acids encoding carbohydrate utilization-related or multidrug a transporter proteins comprise at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, ] 10 100, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1600, 1700, - 1800, 1900, 2000, 2200, or 2500 contiguous nucleotides, including any number : between 5 and 2500 not specifically recited herein, or up to the total number of nucleotides present in a full-length carbohydrate utilization-related or multidrug ~ transporter nucleotide sequence as disclosed herein (for example, 432 for SEQ ID NO:1, 369 for SEQ ID NO:3, etc). :
Fragments of amino acid sequences include polypeptide fragments suitable for use as immunogens to raise anti-carbohydrate utilization-related or anti-multidrug transporter antibodies. Fragments include peptides comprising amino acid sequences sufficiently identical to or derived from the amino acid sequence of a carbohydrate ' utilization-related or multidrug transporter protein, or partial-length protein, of the invention and exhibiting at least one activity of a carbohydrate utilization-related or EE. multidrug transporter protein, but which include fewer amino acids than the full- E length proteins disclosed herein. Typically, biologically active portions comprise a B domain or motif with at least one activity of the carbohydrate utilization-related or ; multidrug transporter protein. A biologically active portion of a carbohydrate utilization-related or multidrug transporter protein can be a polypeptide that is, for example, 10, 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650 - contiguous amino acids in length, or any number between 10 and 650 not specifically ) 30 recited herein, up to the total number of amino acids present in a full-length protein of the current invention (for example, 144 for SEQ ID NO:2, 123 for SEQ ID NO:4, etc.). Such biologically active portions can be prepared by recombinant techniques : and evaluated for one or more of the functional activities of a native carbohydrate . -utilization-related or multidrug transporter protein. As used here, a fragment :
comprises at least 5 contiguous amino acids of any of even numbered SEQ ID ‘
NOS:2-364. The invention encompasses other fragments, however, such as any fragment in the protein greater than 6, 7, 8, or 9 amino acids. - Variants of the nucleotide and amino acid sequences are encompassed in the N present invention. By “variant” is meant a sufficiently identical sequence.
Accordingly, the invention encompasses isolated nucleic acids that are sufficiently } identical to the nucleotide sequences encoding carbohydrate utilization-related v proteins and multidrug transporter proteins in even numbered SEQ ID NOS:2-364, or nucleic acids that hybridize to a nucleic acid of odd numbered SEQ ID NOS:1-363, 3 or a complement thereof, under stringent conditions. Variants also include polypeptides encoded by the variant nucleotide sequences of the present Imvention. In addition, polypeptides of the current invention have an amino acid sequence that is sufficiently identical to an amino acid sequence set forth in even numbered SEQ ID + 110S:2-364. By “sufficiently identical” is meant that a first amino acid or nucleotide sequence contains a sufficient or minimal number of equivalent or identical amino ww. -... acid residues as compared to a second amino acid or nucleotide sequence, thus providing a common structural domain and/or indicating a common functional activity. Conservative variants include those sequences that differ due to the degeneracy of the genetic code. | In general, amino acid or nucleotide sequences that have at least about 45%, ’ 55%, or 65% identity, preferably at least about 70% or 75% identity, more preferably at least about 80%, 85% or 90%, most preferably at least about 91%, 92%, 93%, 94%, 95%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of the amino acid : sequences of even numbered SEQ ID NOS:2-364 or any of the nucleotide sequences oo of odd numbered SEQ ID NOS: 1-363, respectively, are defined herein as sufficiently identical. Variant proteins encompassed by the present invention are biologically active, that is they retain the desired biological activity of the native protein, that is, carbohydrate utilization-related activity or multidrug transporter activity as described : herein. A biologically active variant of a protein of the invention may differ from that - 30 protein by as few as 1-15 amino acid residues, as few as 1-10, such as 6-10, as few as 5, as few as 4, 3, 2, or even 1 amino acid residue. | . ) Naturally occurring variants may exist within a population (e.g., the
Lactobacillus acidophilus population). Such variants can be identified by using well- . known molecular biology techniques, such as the polymerase chain reaction (PCR), :
and hybridization as described below. Synthetically derived nucleotide sequences, for example, sequences generated by site-directed mutagenesis or PCR-mediated - mutagenesis, that still encode a carbohydrate utilization-related protein or multidrug : - | transporter protein, are also included as variants. One or more nucleotide or amino : 5 acid substitutions, additions, or deletions can be introduced into a nucleotide or amino
TL "acid sequence disclosed herein, such that the substitutions, additions, or deletions are introduced into the encoded protein. The additions (insertions) or deletions . (truncations) may be made at the N-terminal or C-terminal end of the native protein, or at one or more sites in the native protein. Similarly, a substitution of one or more
Co ~ 10 nucleotides or amino acids may be made at one or more sites in the native protein. =
For example, conservative amino acid substitutions may be made at one or : more predicted, preferably nonessential amino acid residues. A “nonessential” amino . acid residue is a residue that can be altered from the wild-type sequence of a protein without altering the biological activi, whereas an “essential” amino acid is required for biological activity. A “conservative amino acid substitution” is one in which the “amino acid residue is replaced with an amino acid residue with a similar side chain. :
Families of amino acid residues having similar side chains are known in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g. glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar . ’ side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, : histidine). Such substitutions would not be made for conserved amino acid residues, . or for amino acid residues residing within a conserved motif, where such residues are : essential for protein activity.
Alternatively, mutations can be made randomly along all or part of the length
Co of the carbohydrate utilization-related or multidrug transporter coding sequence, such i as by saturation mutagenesis. The mutants can be expressed recombinantly, and - 30 screened for those that retain biological activity by assaying for carbohydrate utilization-related or multidrug transporter activity using standard assay techniques. ) Methods for mutagenesis and nucleotide sequence alterations are known in the art. ~
See, for example, Kunkel (1985) Proc. Natl. Acad. Sci. USA 82:488-492; Kunkel et 3 al. (1987) Methods in Enzymol. Molecular Biology (MacMillan Publishing Company, :
New York) and the references sited therein. Obviously the mutations made in the a | DNA encoding the variant must not disrupt the reading frame and preferably will not create complementary regions that could produce secondary mRNA structure. See, EP - Patent Application Publication No. 75,444. Guidance as to appropriate amino acid :
C5 “stibstitutions that do not effect biological activity of the protein of interest may be ) found in the mode! of Dayhoff er al. (1978) Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found., Washington, D.C.), herein incorporated by reference. i
The deletions, insertions, and substitutions of the protein sequences ) encompassed herein are not expected to produce radical changes in the characteristics of the protein. However, when it is difficult to predict the exact effect of the . substitution, deletion, or insertion in advance of doing so, one skilled in the art will appreciate that the effect will be evaluated by routine screening assays. That is, the activity can be evaluated by comparing the activity of the modified sequence with the a “ activity of the original sequence. See the “Methods of Use” section below for examples of assays that may be used to measure carbohydrate utilization-related : : activity or multidrug transporter activity. -
Vanant nucleotide and amino acid sequences of the present invention also encompass sequences derived from mutagenic and recombinogenic procedures such as DNA shuffling. With such a procedure, one or more different carbohydrate } 20 utilization-related or multidrug transporter protein coding regions can be used to ’ create a new carbohydrate utilization-related protein or a new multidrug transporter protein possessing the desired properties. In this manner, libraries of recombinant polynucleotides are generated from a population of related sequence polynucleotides : comprising sequence regions that have substantial sequence identity and can be . homologously recombined in vitro or in vivo. For example, using this approach, : sequence motifs encoding a domain of interest may be shuffled between the : i carbohydrate utilization-related or multidrug transporter gene of the invention and other known carbohydrate utilization-related or multidrug transporter genes to obtain ) . a new gene coding for a protein with an improved property of interest, such as an - 30 increased Ky, in the case of an enzyme. Strategies for such DNA shuffling are known in the art. See, for example, Stemmer (1994) Proc. Natl. Acad. Sci. USA 91:10747- . 10751; Stemmer (1994) Nature 370:389-391; Crameri et al. (1997) Nature Biotech. 15:436—438; Moore et al. (1997) J. Mol. Biol. 272:336-347; Zhang et al. (1997) Proc. :
Natl. Acad. Sci. USA 94:4504-4509; Crameri et al. (1998) Nature 391:288-291; and : "U.S. Patent Nos. 5,605,793 and 5,837,458. : Variants of the carbohydrate utilization-related and multidrug transporter - proteins can function as either agonists (mimetics) or as antagonists. An agonist of the i protein can retain substantially the same, or a subset, of the biological activities of the . oC : naturally occurring form of the protein. An antagonist of the protein can inhibit one or more of the activities of the naturally occurring form of the protein by, for example, . : competitively binding to a downstream or upstream member of a cellular signaling cascade that includes the carbohydrate utilization-related or multidrug transporter
EE 10 protein. | Co . : | : Variants of a carbohydrate utilization-related or multidrug transporter protein : that function as either agonists or antagonists can be identified by screening combinatorial libraries of mutants, e.g., truncation mutants, of a carbohydrate - utilization-related or multidrug transporter protein for agonist or antagonist activity.
Co 15 In one embodiment, a variegated library of carbohydrate utilization-related variants is oo generated by combinatorial mutagenesis at the nucleic acid level and is encoded by a variegated gene library. A variegated library of carbohydrate utilization-related or multidrug transporter variants can be produced by, for example, enzymatically : ligating a mixture of synthetic oligonucleotides into gene sequences such that a | degenerate set of potential carbohydrate utilization-related or multidrug transporter ' sequences is expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g., for phage display) containing the set of carbohydrate utilization- : related or multidrug transporter sequences therein. There are a variety of methods that can be used to produce libraries of potential carbohydrate utilization-related or ‘multidrug transporter variants from a degenerate oligonucleotide sequence. Chemical : synthesis of a degenerate gene sequence can be performed in an automatic DNA synthesizer, and the synthetic gene then ligated into an appropriate expression vector. ;
Use of a degenerate set.of genes allows for the provision, in one mixture, of all of the : : sequences encoding the desired set of potential carbohydrate utilization-related or - 30 multidrug transporter sequences. Methods for synthesizing degenerate oligonucleotides are known in the art (see, e.g., Narang (1983) Tetrahedron 39:3; ) Itakura ef al. (1984) Annu. Rev. Biochem. 53:323; Itakura et al. (1984) Science © 198:1056; Ike ef al. (1983) Nucleic Acids Res. 11:477). oo
In addition, libraries of fragments of a carbohydrate utilization-related or : multidrug transporter protein coding sequence can be used to generate a variegated : * population of carbohydrate utilization-related or multidrug transporter fragments for . oo screening and subsequent selection of variants of a carbohydrate utilization-related or : _ 5 multidrug transporter protein. In one embodiment, a library of coding sequence - fragments can be generated by treating a double-stranded PCR fragment of a carbohydrate utilization-related or multidrug transporter coding sequence with a } nuclease under conditions wherein nicking occurs only about once per molecule, denaturing the double-stranded DNA, renaturing the DNA to form double-stranded
DNA which can include sense/antisense pairs from different nicked products, removing single-stranded portions from reformed duplexes by treatment with St nuclease, and ligating the resulting fragment library into an expression vector. By this method, one can derive an expression library that encodes N-terminal and internal fragments of various sizes of the carbohydrate utilization-related or multidrug transporter protein.
Several techniques are known in the art for screening gene products of : combinatorial libraries made by point mutations or truncation and for screening cDNA libraries for gene products having a selected property. Such techniques are adaptable for rapid screening of the gene libraries generated by the combinatorial mutagenesis of carbohydrate utilization-related or multidrug transporter proteins. The to most widely used techniques, which are amenable to high through-put analysis, for screening large gene libraries typically include cloning the gene library into replicable expression vectors, transforming appropriate cells with the resulting library of vectors, g and expressing the combinatorial genes under conditions in which detection of a desired activity facilitates isolation of the vector encoding the gene whose product was detected. Recursive ensemble mutagenesis (REM), a technique that enhances the frequency of functional mutants in the libraries, can be used in combination with the screening assays to identify carbohydrate utilization-related or multidrug transporter . variants (Arkin and Yourvan (1992) Proc. Natl. Acad. Sci. USA 89:7811-7815; - 30 Delgrave et al. (1993) Protein Engineering 6(3):327-331).
: Sequence Identity
The carbohydrate utilization-related and multidrug transporter sequences are members of families of molecules with conserved functional features. By “family” is meant two or more proteins or nucleic acids having sufficient nucleotide or amino ] 5 acid sequence identity. A family that contains deeply divergent groups may be : divided into subfamilies. A clan is a group of families that are thought to have common ancestry. Members of a clan often have a similar tertiary structure. By ; “sequence identity” is meant the nucleotide or amino acid residues that are the same when aligning two sequences for maximum correspondence over at least one : “10 specified comparison window. By “comparison window” is meant a contiguous segment of the two nucleotide or amino acid sequences for optimal alignment, | ; wherein the second sequence may contain additions or deletions (i.e., gaps) as compared to the first sequence. Generally, for nucleic acid alignments, the comparison window is at least 20 conti guous nucleotides in length, and optionally can be 30, 40, 50, 100, or longer. For amino acid sequence alignments, the comparison window is at least 6 contiguous amino acids in length, and optionally can be 10, 15, _ 20, 30, or longer. Those of skill in the art understand that to avoid a high similarity due to inclusion of gaps, a gap penalty is typically introduced and is subtracted from the number of matches. , 20 Family members may be from the same or different species, and can include homologues as well as distinct proteins. Often, members of a family display common functional characteristics. Homologues can be isolated based on their identity to the -
Lactobacillus acidophilus carbohydrate utilization-related or multidrug transporter : nucleic acid sequences disclosed herein using the cDNA, or a portion thereof, as a hybridization probe according to standard hybridization techniques under stringent hybridization conditions as disclosed below.
To determine the percent identity of two amino acid or nucleotide sequences, an alignment is performed. Percent identity of the two sequences is a function of the ; number of identical residues shared by the two sequences in the comparison window (ie. percent identity = number of identical residues/total number of residues x 100). } In one embodiment, the sequences are the same length. Methods similar to those mentioned below can be used to determine the percent identity between two | --
oo WO 2005/084411 PCT/US2005/007594 : sequences. The methods can be used with or without allowing gaps. Alignment may also be performed manually by inspection.
When amino acid sequences differ in conservative substitutions, the percent - identity may be adjusted upward to correct for the conservative nature of the substitution. Means for making this adjustment are known in the art. Typically the ) conservative substitution is scored as a partial, rather than a full mismatch, thereby } increasing the percentage sequence identity. .
Mathematical algorithms can be used to determine the percent identity of two oo sequences. Non-limiting examples of mathematical algorithms are the algorithm of : } 10 Karlin and Altschul a 990) Proc. Natl. Acad. Sci. USA 87:2264, modified as in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877; the algorithm of B
Myers and Miller (1988) CABIOS 4:11-17; the local alignment algorithm of Smith ef al. (1981) Adv. Appl. Math. 2:482; the global alignment algorithm of Needleman and
Wunsch (1970) J. Mol. Biol. 48:443-453; and the search-for-local-alignment method of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. USA 85:2444-2448. - Various computer implementations based on these mathematical algorithms have been designed to enable the determination of sequence identity. The BLAST programs of Altschul et al. (1990) J. Mol. Biol. 215:403 are based on the algorithm of
Karlin and Altschul (1990) supra. Searches to obtain nucleotide sequences that are homologous to nucleotide sequences of the present invention can be performed with ’ the BLASTN program, score = 100, wordlength = 12. To obtain amino acid sequences homologous to sequences encoding a protein or polypeptide of the current invention, the BLASTX program may be used, score = 50, wordlength = 3. Gapped alignments : may be obtained by using Gapped BLAST (in BLAST 2.0) as described in Altschul ef al. (1997) Nucleic Acids Res.-25:3389. To detect distant relationships between ’ : molecules, PSI-BLAST can be used. See, Altschul ef al. (1997) supra. For all of the
BLAST programs, the default parameters of the respective programs can be used.
Alignment may also be performed manually by inspection. :
Another program that can be used to determine percent sequence identity is - 30 the ALIGN program (version 2.0), which uses the mathematical algorithm of Myers and Miller (1988) supra. A PAM120 weight residue table, a gap length penalty of 12, - ! and a gap penalty of 4 can be used with this program when comparing amino acid sequences. :
In addition to the ALIGN and BLAST programs, the BESTFIT, GAP, FASTA and TFASTA programs are part of the GCG Wisconsin Genetics Software Package,
Version 10 (available from Accelrys Inc., 9685 Scranton Rd., San Diego, California, - USA), and can be used for performing sequence alignments. The preferred program is i
GAP version 10, which used the algorithm of Needleman and Wunsch (1 970) supra. -
Unless otherwise stated the sequence identity values provided herein refer to those : values obtained by using GAP Version 10 with the following parameters: % identity oo @ . and % similarity for a nucleotide sequence using GAP Weight of 50 and Length
So © Weight of 3, and the nwsgapdna.cmp scoring matrix; % identity and % similarity for an amino acid sequence using GAP Weight of 8 and Length Weight of 2, and the
BLOSUMBG62 scoring matrix; or any equivalent program thereof. By “equivalent program” is meant any sequence comparison program that, for any two sequences in « question, generates an alignment having identical nucleotide or amino acid residue : matches and an identical percent sequence identity when compared to the ) corresponding alignment generated by GAP Version 10. ~~ Alignment of a sequence in a database to a queried sequence produced by
BLASTN, FASTA, BLASTP or like algorithm is commonly described as a “hit.” Hits to one or more database sequences by a queried sequence produced by BLASTN,
FASTA, BLASTP or a similar algorithm, align and identify similar portions of a sequence. A hit to a database sequence generally represents an overlap over a fraction of the sequence length of the queried sequence, i.e., a portion or fragment of the : queried sequence. However, the overlap can represent the entire length of the queried sequence. The hits in an alignment to a queried sequence produced by BLASTN, .
FASTA, or BLASTP algorithms to sequences in a database are commonly arranged in i order of the degree of similarity and the length of sequence overlap. B
Polynucleotide and polypeptide hits aligned by BLASTN, FASTA, or
BLASTP algorithms to a queried sequence produce “Expect” values. The Expect value (E value) indicates the number of hits one can “expect” to see over a certain ot number of contiguous sequences at random when searching a database of a certain ) 30 size. The Expect value is used as a significance threshold for determining whether the hit to a database, such as the GenBank or the EMBL database, indicates actual similarity. For example, an E value of 0.1 assigned to a polynucleotide hit is oo interpreted as meaning that in a database of the size of the GenBank database, one might expect to see 0.1 matches over the aligned portion of the sequence with a Co similar score randomly. By this criterion, the aligned and matched portions of the polynucleotide sequences then have a probability of 90% of being the same. For sequences having an E value of 0.01 or less over aligned and matched portions, the 5 i probability of finding a match randomly in the GenBank database is 1% or less, using : the BLASTN or FASTA-algorithm. Co Co
According to an embodiment of this invention, “variant” polynucleotides and ] polypeptides of this invention, comprise sequences producing an E value of about > 0.01 or less when compared to the polynucleotide or polypeptide sequences of the present invention. That is, a variant polynucleotide or polypeptide is any sequence that has at least a 99% probability of being the same as the polynucleotide or polypeptide of the present invention, measured as having an E value of 0.01 or less using the BLASTN, FASTA, or BLASTP algorithms set at parameters described : herein. In other embodiments, a variant polynucleotide is a sequence having the same
Co number of, or fewer nucleic acids than a polynucleotide of the present invention that has at least a 99% probability of being the same as the polynucleotide of the present : invention, measured as having an E value of 0.01 or less using the BLASTN or
FASTA algorithms set at parameters described herein. Similarly, a variant polypeptide is a sequence having the same number of, or fewer amino acids than a polypeptide of the present invention that has at least a 99% probability of being the same as a polypeptide of the present invention, measured as having an E value of 0.01 or less using the BLASTP algorithm set at the parameters described herein.
As noted above, the percentage identity is determined by aligning sequences : using one of the BLASTN, FASTA, or BLASTP algorithms, set at the running - parameters described herein, and identifying the number of identical nucleic acids or ~ 25 amino acids over the aligned portions; dividing the number of identical nucleic acids ) or amino acids by the total number of nucleic acids or amino acids of the polynucleotide or polypeptide sequence of the present invention; and then multiplying ~ by 100 to determine the percent identity. For example, a polynucleotide of the present : invention having 220 nucleic acids has a hit to a polynucleotide sequence in the ) 30 GenBank database having 520 nucleic acids over a stretch of 23 nucleotides in the alignment produced by the BLASTN algorithm using the parameters described herein. i
The 23 nucleotide hit includes 21 identical nucleotides, one gap and one different ~ nucleotide. The percent identity of the polynucleotide of the present invention to the : hit in the GenBank library 1s thus 21/220 times 100, or 9.5%. The polynucleotide sequence in the GenBank database is thus not a variant of a polynucleotide of the present invention. : ) . Identification and Isolation of Homologous Sequences .
Carbohydrate utilization-related nucleotide sequences identified based on their . sequence identity to the carbohydrate utilization-related or multidrug transporter nucleotide sequences set forth herein or to fragments and variants thereof are : encompassed by the present invention. Methods such as PCR or hybridization canbe used to identify sequences from a cDNA or genomic library, for example that are ; substantially identical to a sequence of the invention. See, for example, Sambrook et al. (1989) Molecular Cloning: Laboratory Manual (2d ed., Cold Spring Harbor oo Laboratory Press, Plainview, New York) and Innis, ef al. (1990) PCR Protocols: A
Guide to Methods and Applications (Academic Press, New York). Methods for ) construction of such cDNA and genomic libraries are generally known in the art and arealso disclosed in the above reference. - In hybridization techniques, the hybridization probes may be genomic DNA - . fragments, cDNA fragments, RNA fragments, or other oligonucleotides, and may consist of all or part of a known nucleotide sequence disclosed herein. In addition, they may be labeled with a detectable group such as **P, or any other detectable marker, such as other radioisotopes, a fluorescent compound, an enzyme, or an enzyme co-factor. Probes for hybridization may be made by labeling synthetic oligonucleotides based on the known carbohydrate utilization-related or multidrug transporter nucleotide sequences disclosed herein. Degenerate primers designed on the basis of conserved nucleotides or amino acid residues in a known carbohydrate utilization-related or multidrug transporter nucleotide sequence or encoded amino R acid sequence can additionally be used. The hybridization probe typically comprises a region of nucleotide sequence that hybridizes under stringent conditions to at least about 10, preferably about 20, more preferably about 50, 75, 100, 125, 150, 175, 200, A 250, 300, 350, or 400 consecutive nucleotides of a nucleotide sequence of the
Co 30 invention or a fragment or variant thereof. To achieve specific hybridization under a } variety of conditions, such probes include sequences that are unique among carbohydrate utilization-related or multidrug transporter protein sequences. .
Preparation of probes for hybridization is generally known in the art and is disclosed -
in Sambrook ef al. (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold
Spring Harbor Laboratory Press, Plainview, New York), herein incorporated by reference.
In one embodiment, the entire nucleotide sequence encoding a carbohydrate ) utilization-related or multidrug transporter protein is used as a probe to identify novel carbohydrate utilization-related or multidrug transporter sequences and messenger
RNAs. In another embodiment, the probe is a fragment of a nucleotide sequence .
Lo disclosed herein. In some embodiments, the nucleotide sequence that hybridizes under stringent conditions to the probe can be at least about 300, 325, 350, 375, 400, 425, 450, 500, 550, 600, 650, 700, 800, 900, 1000, 1200, 1400, 1600, 1800, or 2000 nucleotides in length.
Substantially identical sequences will hybridize to each other-under stringent conditions. By “stringent conditions” is meant conditions under which a probe will hybridize to its target sequence to a detectably greater degiee than to other sequences (e.g. at least 2-fold over background). Generally, stringent conditions encompass . those conditions for hybridization and washing under which nucleotides having at - least about 60%, 65%, 70%, preferably 75% sequence identity typically remain hybridized to each other. Stringent conditions are known in the art and can be found in Current Protocols in Molecular Biology (John Wiley & Sons, New York (1989)), 6.3.1-6.3.6. Hybridization typically occurs for less than about 24 hours, usually about ’ 4 to about 12 hours.
Stringent conditions are sequence dependent and will differ in different circumstances. Full-length or partial nucleic acid sequences may be used to obtain : homologues and orthologs encompassed by the present invention. By “orthologs” is meant genes derived from a common ancestral gene and which are found in different species as a result of speciation. Genes found in different species are considered orthologs when their nucleotide sequences and/or their encoded protein sequences share substantial identity as defined elsewhere herein. Functions of orthologs are often ; highly conserved among species. ’ 30 When using probes, stringent conditions will be those in which the salt concentration is less than about 1.5 M Na ion, typically about 0.01 to 1.0 M Na ion : concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about ~ 30°C for short probes (e.g., 10 to 50 nucleotides) and at least about 60°C for long : probes (e.g., greater than 50 nucleotides).
The post-hybridization washes are instrumental in controlling specificity. The two critical factors are ionic strength and temperature of the final wash solution. For the detection of sequences that hybridize to a full-length or approximately full-length ] - target sequence, the temperature under stringent conditions is selected to be about 5°C - ) : ~ 5 lower than the thermal melting point (Tr) for the specific sequence at a defined ionic oo strength and pH. However, stringent conditions would encompass temperatures in the oo range of 1°C to 20°C lower than the Tr, depending on the desired degree of 5 stringency as otherwise qualified herein. For DNA-DNA hybrids, the Tp, can be determined using the equation of Meinkoth and Wahl (1984) Anal Biochem. 138:267-284: Ty, = 81.5°C + 16.6 (logM) + 0.41 (%GC) — 0.61 (% form) — 500/L; where M is the molarity of monovalent cations, %GC is the percentage of guanosine . and cytosine nucleotides in the DNA, % form is the percentage of formamide in the hybridization solution, and L is the length of the hybrid in base pairs. The Ty, is the temperature (under defined ionic strength and pH) at which 50% of a complementary target sequence hybridizes to a perfectly matched probe.
The ability to detect sequences with varying degrees of homology can be obtained by varying the stringency of the hybridization and/or washing conditions. To target sequences that are 100% identical (homologous probing), stringency conditions must be obtained that do not allow mismatching. By allowing mismatching of nucleotide residues to occur, sequences with a lower degree of similarity can be or detected (heterologous probing). For every 1% of mismatching, the Tp, is reduced about 1°C; therefore, hybridization and/or wash conditions can be manipulated to allow hybridization of sequences of a target percentage identity. For example, if ri sequences with >90% sequence identity are preferred, the Ty, can be decreased by EA 3 10°C. Two nucleotide sequences could be substantially identical, but fail to hybridize : to each other under stringent conditions, if the polypeptides they encode are substantially identical. This situation could arise, for example, if the maximum codon degeneracy of the genetic code is used to create a copy of a nucleic acid. :
Exemplary low stringency conditions include hybridization with a buffer i 30 solution of 30-35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulfate) at 37°C, and a wash in 1X to 2X SSC (20X SSC = 3.0 M NaCl/0.3 M trisodium citrate) at 50 to 55°C. Exemplary moderate stringency conditions include hybridization in 40 ~ : to 45% formamide, 1.0 M NaCl, 1% SDS at 37°C, and a wash in 0.5X to 1X SSC at : 55 to 60°C. Exemplary high stringency conditions include hybridization in 50% :
formamide, 1 M NaCl, 1% SDS at 37°C, and a wash in 0.1X SSC at 60 to 65°C.
Optionally, wash buffers may comprise about 0.1% to about 1% SDS. Duration of hybridization is generally less than about 24 hours, usually about 4 to about 12 hours. - An extensive guide to the hybridization of nucleic acids is found in Tijssen (1993) | Laboratory Techniques in Biochemistry and Molecular Biology — Hybridization with Co ) Nucleic Acid Probes, Part I, Chapter 2 (Elsevier, New York); and Ausubel ef al., eds. (1995) Current Protocols in Molecular Biology, Chapter 2 (Greene Publishing and . ~ Wiley-Interscience, New York). See Sambrook et al. (1989) Molecular Cloning: A
Laboratory Manual (2d ed.; Cold Spring Harbor Laboratory Press, Plainview, New
York).
In a PCR approach, oligonucleotide primers can be designed for use in PCR. . reactions to amplify corresponding DNA sequences from cDNA or genomic DNA extracted from any organism of interest. PCR primers are preferably at least about 10 nucleotides in length, and miost preferably at least about 20 nucleotides in length.
Methods for designing PCR primers and PCR cloning ae generally known in the art } and are disclosed in Sambrook et al. (1989) Molecular Cloning: A Laboratory :
Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, New York). See also Innis ef al., eds. (1990) PCR Protocols: A Guide to Methods and Applications (Academic Press, New York); Innis and Gelfand, eds. (1995) PCR Strategies (Academic Press, New York); and Innis and Gelfand, eds. (1999) PCR Methods ot "Manual (Academic Press, New York). Known methods of PCR include, but are not limited to, methods using paired primers, nested primers, single specific primers, degenerate primers, gene-specific primers, vector-specific primers, partially- : mismatched primers, and the like. oo -
Assays
Diagnostic assays to detect expression of the disclosed polypeptides and/or nucleic acids as well as their disclosed activity in a sample are disclosed. An exemplary method for detecting the presence or absence of a disclosed nucleic acid or protein comprising the disclosed polypeptide in a sample involves obtaining a sample ; from a food/dairy/feed product, starter culture (mother, seed, bulk/set, concentrated, dried, lyophilized, frozen), cultured food/dairy/feed product, dietary supplement, - bioprocessing fermentate, or a subject that has ingested a probiotic material, and -
contacting the sample with a compound or an agent capable of detecting the disclosed polypeptides or nucleic acids (e.g., an mRNA or genomic DNA comprising the disclosed nucleic acid or fragment thereof) such that the presence of the disclosed = sequence is detected in the sample. Results obtained with a sample from the food, : ) supplement, culture, product, or subject may be compared to results obtained with a sample from a control culture, product, or subject. :
One agent for detecting the mRN A or genomic DNA comprising a disclosed . oo nucleotide sequence is a labeled nucieic acid probe capable of hybridizing to the disclosed nucleotide sequence of the mRNA or genomic DNA. The nucleic acid probe : 10 can be, for example, a disclosed nucleic acid, such as a nucleic acid of odd numbered
SEQ ID NOS:1-363, or a portion thereof, such as a nucleic acid of at least 15, 30, 50, 100, 250, or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions to the mRNA or genomic DNA comprising the disclosed nucleic ~ acid sequence. Other suitable probes for use in the diagnostic assays of the invention are described herein. . One agent for detecting a protein comprising a disclosed polypeptide sequence : is an antibody capable of binding to the disclosed polypeptide, preferably an antibody with a detectable label. Antibodies can be polyclonal, or more preferably, monoclonal. An intact antibody, or a fragment thereof (e.g., Fab or F(ab'),) can be used. The term “labeled,” with regard to the probe or antibody, is meant to encompass direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled. Examples of : indirect labeling include detection of a primary antibody using a fluorescently labeled - secondary antibody and end-labeling of a DNA probe with biotin such that it can be detected with fluorescently labeled streptavidin.
The term “sample” is meant to include tissues, cells, and biological fluids oo present in or isolated from a subject, as well as cells from starter cultures or food : products carrying such cultures, or derived from the use of such cultures. That is, the ) 30 detection method of the invention can be used to detect mRNA, protein, or genomic
DNA comprising a disclosed sequence in a sample both in vitro and in vivo. In vitro techniques for detection of mRNA comprising a disclosed sequence include Northern ~ hybridizations and in situ hybridizations. In vitro techniques for detection of a protein . comprising a disclosed polypeptide include enzyme linked immunosorbent assays ®
© WO 2005/084411 PCTIUS2005/007594 (ELISAs), Western blots, immunoprecipitations, and immunofluorescence. In vitro techniques for detection of genomic DNA comprising the disclosed nucleotide sequences include Southern hybridizations. Furthermore, in vivo techniques for i detection of a protein comprising a disclosed polypeptide include introducing into a subject a labeled antibody against the disclosed polypeptide. For example, the oo antibody can be labeled with a radioactive marker whose presence and location in a . subject can be detected by standard imaging techniques. : [n one embodiment, the sample contains protein molecules from a test subject that has consumed a probiotic material. Alternatively, the sample can contain mRNA or genomic DNA from a starter culture.
The invention also encompasses kits for detecting the presence of disclosed : nucleic acids or proteins comprising disclosed polypeptides in a sample. Such kits can be used to determine if a microbe expressing a specific polypeptide of the invention is d present in a food product or starter culture, or in a subject that has consumed a : probiotic material. For example, the kit can comprise a labeled compound or agent capable of detecting a disclosed polypeptide or mRNA in a sample and means for determining the amount of a the disclosed polypeptide in the sample (e.g., an antibody that recognizes the disclosed polypeptide or an oligonucleotide probe that binds to
DNA encoding a disclosed polypeptide, e.g., even numbered SEQ ID NOS:2-364).
Kits can also include instructions detailing the use of such compounds.
For antibody-based kits, the kit can comprise, for example: (1) a first antibody (e.g., attached to a solid support) that binds to a disclosed polypeptide; and, optionally, (2) a second, different antibody that binds to the disclosed polypeptide or ¥ the first antibody and 1s conjugated to a detectable agent. For oligonucleotide-based kits, the kit can comprise, for example: (1) an oligonucleotide, e.g., a detectably ’ labeled oligonucleotide, that hybridizes to a disclosed nucleic acid sequence or (2) a pair of primers useful for amplifying a disclosed nucleic acid.
The kit can also comprise, e.g., a buffering agent, a preservative, or a protein : stabilizing agent. The kit can also comprise components necessary for detecting the
To 30 detectable agent (e.g., an enzyme or a substrate). The kit can also contain a control sample or a series of control samples that can be assayed and compared to the test . sample contained. Each component of the kit is usually enclosed within an individual ~ container, and all of the various containers are within a single package along with : instructions for use. }
In one embodiment, the kit comprises multiple probes in an array format, such as those described, for example, in U.S. Patent Nos. 5,412,087 and 5,545,531, and
International Publication No. WO 95/00530, herein incorporated by reference. Probes oo ] for use in the array may be synthesized either directly onto the surface of the array, as ’ disclosed in International Publication No. WO 95/00530, or prior to immobilization - onto the array surface (Gait, ed. (1984) Oligonucleotide Synthesis a Practical
Approach IRL Press, Oxford, England). The probes may be immobilized onto the . surface using techniques well known to one of skill in the art, such as those described : in U.S. Patent No. 5.41 2,087. Probes may be a nucleic acid or peptide sequence, preferably purified, or an antibody.
The arrays may be used to screen organisms, samples, or products for differences in their genomic, cDNA, polypeptide, or antibody content, including the : presence or absence of specific sequences or proteins, as well as the concentration of i those materials. Binding to a capture probe is detected, for example, by signal generated from a label attached to the nucleic acid comprising the disclosed nucleic : acid sequence, a polypeptide comprising the disclosed amino acid sequence, or an antibody. The method can include contacting the molecule comprising the disclosed nucleic acid, polypeptide, or antibody with a first array having a plurality of capture probes and a second array having a different plurality of capture probes. The results of each hybridization can be compared to analyze differences in expression between a to first and second sample. The first plurality of capture probes can be from acontrol sample, e.g., a wild type lactic acid bacteria, or control subject, e.g., a food, dietary : supplement, starter culture sample, or a biological fluid. The second plurality of E capture probes can be from an experimental sample, e.g., 2 mutant type lactic acid } bacteria, or subject that has consumed a probiotic material, e.g., a starter culture : sample or a biological fluid.
These assays may be especially useful in microbial selection and quality control procedures where the detection of unwanted materials is essential. The . detection of particular nucleotide sequences or polypeptides may also be useful in : } 30 determining the genetic composition of food, fermentation products, or industrial microbes, or microbes present in the digestive system of animals or humans that have - consumed probiotics.
Antisense Nucleotide Sequences
The present invention also encompasses antisense nucleic acids, i.e., ; : molecules that are complementary to a sense nucleic acid encoding a protein, e.g., : - complementary to the coding strand of a double-stranded cDNA molecule, or . 5S complementary to an mRNA sequence. ‘Accordingly, an antisense nucleic acid can ) hydrogen bond to a sense nucleic acid. The antisense nucleic acid can be : complementary to an entire carbohydrate utilization-related or multidrug transporter oT coding strand, or to only a portion thereof, e.g., all or part of the protein coding region (or open reading frame). An antisense nucleic acid can be antisense to a noncoding region of the coding strand of a nucleotide sequence encoding a carbohydrate | : utilization-related or multidrug transporter protein. The noncoding regions arc the 5' . and 3’ sequences that flank the coding region and are not translated into amino acids.
Antisense nucleotide sequences are useful in disrupting the expression of the target gene. Antisense constructions having 70%, preferably 80%, more preferably 85%, a 15 90% or 95% sequence identity to the corresponding sequence may be used.
Given the coding-strand sequence encoding a carbohydrate utilization-related or multidrug transporter protein disclosed herein (e.g., even numbered SEQ ID
NOS:2-364), antisense nucleic acids of the invention can be designed according to the rules of Watson and Crick base pairing. The antisense nucleic acid can be complementary to the entire coding region of carbohydrate utilization-related or multidrug transporter mRNA, but more preferably is an oligonucleotide that is . antisense to only a portion of the coding or noncoding region of carbohydrate utilization-related or multidrug transporter mRNA. For example, the antisense : oligonucleotide can be complementary to the region surrounding the translation start - site of carbohydrate utilization-related or multidrug transporter mRNA. An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50
Co nucleotides in length, or it can be 100, 200 nucleotides, or greater in length. An : antisense nucleic acid of the invention can be constructed using chemical synthesis and enzymatic ligation procedures known in the art. : : ’ 30 For example, an antisense nucleic acid (e.g., an antisense oligonucleotide) can be chemically synthesized using naturally occurring nucleotides or variously modified : nucleotides designed to increase the biological stability of the molecules or to increase - the physical stability of the duplex formed between the antisense and sense nucleic :
acids, including, but not limited to, for example e.g., phosphorothioate derivatives and acridine substituted nucleotides. Alternatively, the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been ” subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic h acid will be of an antisense orientation to a target nucleic acid of interest). - An antisense nucleic acid of the invention can be an a-anomeric nucleic acid. - : An a-anomeric nucleic acid forms specific double-stranded hybrids with : complementary RNA in which, contrary to the usual -units, the strands run parallel to each other (Gaultier et al. (1987) Nucleic Acids Res. 15:6625-6641). The antisense nucleic acid can also comprise a 2'-0-methylribonucleotide (Inoue er al. (1987)
Nucleic Acids Res. 15:6131-6148) or a chimeric RNA-DNA analogue (Inoue ef al. fe (1987) FEBS Lett. 215:327-330).
The invention also encompasses ribozymes, which are catalytic RNA oo _ molecules with ribonuclease activity that are capable of cleaving a single-stranded
I5 nucleic acid, such as an mRNA, to which they have a complementary region.
Ribozymes (e.g., hammerhead ribozymes (described in Haselhoff and Gerlach (1988)
Nature 334:585-591)) can be used to catalytically cleave carbohydrate utilization- related mRNA transcripts to thereby inhibit translation of carbohydrate utilization- related or multidrug transporter mRNA. A ribozyme having specificity for a carbohydrate utilization-related-encoding or multidrug transporter-encoding nucleic acid can be designed based upon the nucleotide sequence of a carbohydrate utilization-related or multidrug transporter cDNA disclosed herein (e.g., odd numbered SEQ ID NOS:1-363). See, e.g., Cech er al., U.S. Patent No. 4,987,071; and }
Cechetal, U.S. Patent No. 5,116,742. Alternatively, carbohydrate utilization-related or multidrug transporter mRNA can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules. See, e.g., Bartel and
Szostak (1993) Science 261:1411-1418.
The invention also encompasses nucleic acids that form triple helical : structures. For example, carbohydrate utilization-related or multidrug transporter gene : 30 expression can be inhibited by targeting nucleotide sequences complementary to the regulatory region of the carbohydrate utilization-related or multidrug transporter ) protein (e.g., the carbohydrate utilization-related or multidrug transporter promoter - and/or enhancers) to form triple helical structures that prevent transcription of the :
carbohydrate utilization-related or multidrug transporter gene in target cells. See generally, Helene (1991) Anticancer Drug Des. 6(6):569; Helene (1992) Ann. N. Y. ~
Acad. Sci. 660:27; and Maher (1992) Bioassays 14(12):807. - In some embodiments, the nucleic acids of the invention can be modified at 5S the base moiety, sugar moiety, or phosphate backbone to improve, e.g., the stability, a. hybridization, or solubility of the molecule. For example, the deoxyribose phosphate ] backbone of the nucleic acids can be modified to generate peptide nucleic acids (see \
Hyrup et al. (1996) Bioorganic & Medicinal Chemistry 4:5). As used herein, the terms “peptide nucleic acids” or “PNAs” refer to nucleic acid mimics, e.g., DNA © 10 mimics, in which the deoxyribose phosphate backbone is replaced by a pseudopeptide : - backbone and only the four natural nucleobases are retained. The neutral backbone of
PNA has been shown to allow for specific hybridization to DNA and RNA under ) conditions of low ionic strength. The synthesis of PNA oligomers can be performed using standard solid-phase peptide synthesis protocols as described, for example, in Hyrup et al. (1996) supra; Perry-O'Keefe et al. (1996) Proc. Natl. Acad. Sci. USA 93:14670. -
PNAS can be used as antisense or antigene agents for sequence-specific modulation of gene expression by, e.g., inducing transcription or translation arrest or inhibiting replication. PNAs of the invention can also be used, e.g, in the analysis of single base pair mutations in a gene by, e.g., PNA-directed PCR clamping; as . artificial restriction enzymes when used in combination with other enzymes, e.g., Si nucleases (Hyrup (1996) supra); or as probes or primers for DNA sequence and hybridization (Hyrup (1996) supra; Perry-O'Keefe et al. (1996) supra).
In another embodiment, PNAs of an carbohydrate utilization-related or multidrug transporter molecule can be modified, e.g., to enhance their stability, specificity, or cellular uptake, by attaching lipophilic or other helper groups to PNA, by the formation of PNA-DNA chimeras, or by the use of liposomes or other techniques of drug delivery known in the art. The synthesis of PNA-DNA chimeras : can be performed as described in Hyrup (1996) supra; Finn et al. (1996) Nucleic ” : 30 Acids Res. 24(17):3357-63; Mag et al. (1989) Nucleic Acids Res. 17:5973; and
Peterson et al. (1975) Bioorganic Med. Chem. Lett. 5:1119. :
Fusion Proteins oo
The invention also includes carbohydrate utilization-related or multidrug : transporter chimeric or fusion proteins. A carbohydrate utilization-related or . multidrug transporter “chimeric protein” or “fusion protein” comprises a carbohydrate JP ] 5 utilization-related or multidrug transporter polypeptide operably linked to a non- carbohydrate utilization-related or non-multidrug transporter polypeptide, respectively. A “carbohydrate utilization-related polypeptide” or a “multidrug f transporter polypeptide” refers to a polypeptide having an amino acid sequence corresponding to a carbohydrate utilization-related protein or a multidrug transporter protein, respectively; whereas a “non-carbohydrate utilization-related polypeptide” or - a “non-multidrug transporter polypeptide” refers to a polypeptide having an amino 2 acid sequence corresponding to a protein that is not substantially identical to the . carbohydrate utilization-related protein or multidrug transporter protein, respectively, ) and which is derived from the same or a different organism. Within a carbohydrate utilization-related or multidrug transporter fusion protein, the carbohydrate : utilization-related or multidrug transporter polypeptide can correspond to all ora ~~ : portion of a carbohydrate utilization-related or multidrug transporter protein, preferably including at least one biologically active portion of a carbohydrate utilization-related or multidrug transporter protein. Within the fusion protein, the term i 20 “operably linked” is meant to indicate that the carbohydrate utilization-related or multidrug transporter polypeptide and the non-carbohydrate utilization-related or multidrug transporter polypeptide are fused in-frame to each other. The non- carbohydrate utilization-related or multidrug transporter polypeptide can be fused to | : the N-terminus or C-terminus of the carbohydrate utilization-related or multidrug : transporter polypeptide. i
Expression of the linked coding sequences results in two linked heterologous _ amino acid sequences that form the fusion protein. The carrier sequence (the non- carbohydrate utilization-related or non-multidrug transporter polypeptide) can encode % a carrier polypeptide that potentiates or increases expression of the fusion protein in the bacterial host. The portion of the fusion protein encoded by the carrier sequence, ] L.e., the carrier polypeptide, may be a protein fragment, an entire functional moiety, or an entire protein sequence. The carrier region or polypeptide may additionally be -- : designed to be used in purifying the fusion protein, either with antibodies or with ;
affinity purification specific for that carrier polypeptide. Likewise, physical properties of the carrier polypeptide can be exploited to allow selective purification of the fusion protein. - Particular carrier polypeptides of interest include superoxide dismutase : (SOD), maltose-binding protein (MBP), glutathione-S-transferase (GST), an N- ) ~ terminal histidine (His) tag, and the like. This list is not meant to be limiting, as any carrier polypeptide that potentiates expression of the carbohydrate utilization-related . protein or multidrug resistance protein as a fusion protein can be used in the methods of the invention. =~
Co 10 In one embodiment, the fusion protein 1s a GST-carbohydrate utilization- ~ related fusion protein in which the carbohydrate utilization-related sequences are fused to. the C-terminus of the GST sequences. In another embodiment, the fusion protein is a carbohydrate utilization-related-immunoglobulin fusion protein in which : all or part of a carbohydrate utilization-related protein is fused to sequences derived from a member of the immunoglobulin protein family. In other embodiments, the fusion protein comprises a multidrug transporter protein of the present invention. The : oo carbohydrate utilization-related- or multidrug transporter-immunoglobulin fusion proteins of the invention can be used as immunogens to produce anti-carbohydrate utilization-related or anti-multidrug transporter-related antibodies in a subject, to purify carbohydrate utilization-related or multidrug transporter-related ligands, and in screening assays to identify molecules that inhibit the interaction of a carbohydrate utilization-related or multidrug transporter protein with a carbohydrate utilization- related or multidrug transporter ligand. :
One of skill in the art will secogian that the particular carrier polypeptide is chosen with the purification scheme in mind. For example, His tags, GST, and maltose-binding protein represent carrier polypeptides that have readily available : affinity columns to which they can be bound and eluted. Thus, where the carrier polypeptide is an N-terminal His tag such as hexahistidine (Hiss tag), the carbohydrate . utilization-related or multidrug transporter fusion protein can be purified using a : 30 matrix comprising a metal-chelating resin, for example, nickel nitrilotriacetic acid (Ni-NTA), nickel iminodiacetic acid (Ni-IDA), and cobalt-containing resin (Co- resin). See, for example, Steinert er al. (1997) QIAGEN News 4:11-15, herein incorporated by reference in its entirety. Where the carrier polypeptide is GST, the - carbohydrate utilization-related or multidrug transporter fusion protein can be purified using a matrix comprising glutathione-agarose beads (Sigma or Pharmacia Biotech); where the carrier polypeptide is a maltose-binding protein (MBP), the carbohydrate utilization-related or multidrug transporter fusion protein can be purified using a : matrix comprising an agarose resin derivatized with amylose. :
Preferably, a chimeric or fusion protein of the invention is produced by ) standard recombinant DNA techniques. For example, DNA fragments coding for the . different polypeptide sequences may be ligated together in-frame, or the fusion gene - can be synthesized, such as with automated DNA synthesizers. Alternatively, PCR amplification of gene fragments can be carried out using anchor primers that give rise ~*~ = 10 to complementary overhangs between two consecutive gene fragments, which can subsequently be annealed and re-amplified to generate a chimeric gene sequence (see, : e.g., Ausubel er al, eds. (1995) Current Protocols in Molecular Biology (Greene )
Publishing and Wiley-Interscience, New York). Moreover, a carbohydrate utilization- : related or multidrug transporter-encoding nucleic acid can be cloned into a Co commercially available expression vector such that it is linked in-frame to an existing fusion moiety. oo "The fusion protein expression vector is typically designed for ease of - removing the carrier polypeptide to allow the carbohydrate utilization-related or } multidrug transporter protein to retain the native biological activity associated with it.
Methods for cleavage of fusion proteins are known in the art. See, for example, ’ Ausubel et al., eds. (1998) Current Protocols in Molecular Biology (John Wiley & :
Sons, Inc.). Chemical cleavage of the fusion protein can be accomplished with - reagents such as cyanogen bromide, 2-(2-nitrophenylsulphenyl)-3-methyl-3’- . ; - bromoindolenine, hydroxylamine, or low pH. Chemical cleavage is oftén : accomplished under denaturing conditions to cleave otherwise insoluble fusion :
I oo proteins. So oo . . oo | Co Where separation of the carbohydrate utilization-related or multidrug
SET ) Co transporter polypeptide from the carrier polypeptide is desired and a cleavage site at CL . Lo
Cs ] i : } - BE - : E : the junction between these fused polypeptides is not naturally occurring, the fusion oo " Cel R - 30 | Construct can be designed to contain a specific protease cleavage site to facilitate = Co : oe BS oo So efaymatic cleavage and removal of the carrier polypeptide. In this manner, a linker
Ce + sequence comprising a coding sequence for a peptide that has a cleavage site specific oo SE for an enzyme of interest can be fused in-frame between the coding sequence for the carrier polypeptide (for example, MBP, GST, SOD, or an N-terminal His tag) and the : .
coding sequence for the carbohydrate utilization-related or multidrug transporter polypeptide. Suitable enzymes having specificity for cleavage sites include, but are not limited to, factor Xa, thrombin, enterokinase, remin, collagenase, and tobacco etch . : virus (TEV) protease. Cleavage sites for these enzymes are well known in the art.
Thus, for example, where factor Xa is to be used to cleave the carrier polypeptide from the carbohydrate utilization-related or multidrug transporter polypeptide, the . fusion construct can be designed to comprise a linker sequence encoding a factor Xa- : sensitive cleavage site, for example, the sequence IEGR (see, for example, Nagai and
Thogersen (1984) Nafure 309:810-812, Nagai and Thegersen (1987) Meth. Enzymol. 153:461-481, and Pryor and Leiting (1997) Protein Expr. Purif. 10(3):309-319, herein incorporated by reference). Where thrombin is to be used to cleave the carrier - polypeptide from the carbohydrate utilization-related or multidrug transporter polypeptide, the fusion construct can be designed to comprise a linker sequence Co - encoding a thrombin-sensitive cleavage site, for example the sequence LVPRGS or
VIAGR (see, for example, Pryor and Leiting (1997) Protein Expr. Purif. 10(3):309- 319, and Hong. er al. (1997) Chin. Med. Sci. J. 12(3):143-147, respectively, herein incorporated by reference). Cleavage sites for TEV protease are known in the art. See, : for example, the cleavage sites described in U.S. Patent No. 5,532,142, herein incorporated by reference in its entirety. See also the discussion in Ausubel ef al., eds. (1998) Current Protocols in Molecular Biology (John Wiley & Sons, Inc.), Chapter 16. | oo
Antibodies : :
An isolated polypeptide of the present invention can be used as an immunogen to generate antibodies that specifically bind carbohydrate utilization-related or multidrug transporter proteins, or stimulate production of antibodies in vivo. The full- length carbohydrate utilization-related or multidrug transporter protein can be used as } E an immunogen or, alternatively, antigenic peptide fragments of carbohydrate oo oo vo ~~ utilization-related or multidrug transporter proteins as described herein can be used. . : oo - © 30 The antigenic peptide of an carbohydrate utilization-related or multidrug transporter :
N protein comprises at least 8, preferably 10, 15, 20, or 30 amino acid residues of the amino acid sequences shown in even numbered SEQ ID NOS:1-320 and = encompasses an epitope of a carbohydrate utilization-related or multidrug transporter :
protein such that an antibody raised against the peptide forms a specific immune complex with the carbohydrate utilization-related or multidrug transporter protein. ”
Preferred epitopes encompassed by the antigenic peptide are regions of a Co - carbohydrate utilization-related or multidrug transporter protein that are located on the surface of the protein, e.g., hydrophilic regions.
Recombinant Expression Vectors and Cells Ca
The nucleic acids of the present invention may be included in vectors, preferably expression vectors. “Vector” refers to a nucleic acid capable of - transporting another nucleic acid to which it has been linked. Expression vectors : include one or more regulatory sequences and direct the expression of genes to which . they are operably linked. By ‘“‘operably linked” is meant that the nucleotide sequence 3 of interest is linked to the regulatory sequence(s) such that expression of the nucleotide sequence is allowed (e.g., in an in vitro transcription/translation system or in a cell when the vector is introduced into the cell). The term “regulatory sequence” _ .is meant to include controllable transcriptional promoters, operators, enhancers, transcriptional terminators, and other expression control elements such as translational control sequences (e.g., Shine-Dalgarno consensus sequence, initiation and termination codons). These regulatory sequences will differ, for example, depending © 20 onthe cell being used. oo ~~ The vectors can be autonomously replicated in a cell (episomal vectors), or : may be integrated into the genome of a cell, and replicated along with the host : genome (non-episomal mammalian vectors). Integrating vectors typically contain at Cor least one sequence homologous to the bacterial chromosome that allows for - recombination to occur between homologous DNA in the vector and the bacterial chromosome. Integrating vectors may also comprise bacteriophage or transposon
Co Co oo ‘sequences. Episomal vectors, or plasmids are circular double-stranded DNA loops into which additional DNA segments can be ligated. Plasmids capable of stable : maintenance in a host are generally the preferred form of expression vectors when oo | 30 using recombinant DNA techniques. | | 0 oo | The expression constructs or vectors encompassed in the present invention comprise a nucleic acid construct of the invention in a form suitable for expression of - the nucleic acid in a cell. Expression in prokaryotic cells and plant cells is encompassed in the present invention. It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of ’ the cell to be transformed, the level of expression of protein desired, etc. The . expression vectors of the invention can be introduced into cells to thereby produce : | 5 proteins or peptides, including fusion proteins or peptides, encoded by nucleic acids So : as described herein (e.g., carbohydrate utilization-related or multidrug transporter . proteins, mutant forms of carbohydrate utilization-related or multidrug transporter 5 proteins, fusion proteins, etc.). )
Bacterial Expression Vectors
Regulatory sequences include those that direct constitutive expression of a : nucleotide sequence as well as those that direct inducible expression of the nucleotide sequence only under certain environmental conditions. A bacterial promoter is any
DNA sequence capable of binding bacterial RNA polymerase and initiating the downstream (3') transcription of a coding sequence (e.g., structural gene) into mRNA. : A promoter will have a transcription initiation region, which is usually placed oo : proximal to the 5' end of the coding sequence. This transcription initiation region typically includes an RNA polymerase binding site and a transcription initiation site.
A bactenal promoter may also have a second domain called an operator, which may overlap an adjacent RNA polymerase binding site at which RNA synthesis begins. oo } The operator permits négative regulated (inducible) transcription, as a gene repressor protein may bind the operator and thereby inhibit transcription of a specific gene.
Constitutive expression may occur in the absence of negative regulatory elements, such as the operator. In addition, positive regulation may be achieved by a gene activator protein binding sequence, which, if present is usually proximal (5) to the Co } .
RNA polymerase binding sequence. | a
An example of a gene activator protein is the catabolite activator protein (CAP), which helps initiate transcription of the lac operon in Escherichia coli ; - (Raibaud et al. (1984) Annu. Rev. Genet. 18:173). Regulated expression may therefore i 30 be either positive or negative, thereby either enhancing or reducing transcription. oo : Other examples of positive and negative regulatory elements are well known in the : art. Various promoters that can be included in the protein expression system include, - but are not limited to, a T7/LacO hybrid promoter, a trp promoter, a T7 promoter, a | 3 oo Cs lac promoter, and a bacteriophage lambda promoter. Any suitable promoter can be used to carry out the present invention, including the native promoter or a heterologous promoter. Heterologous promoters may be constitutively active or } inducible. A non-limiting example of a heterologous promoter is given in US Patent )
No. 6,242,194. oo - Sequences encoding metabolic pathway enzymes provide particularly useful ‘ promoter sequences. Examples include promoter sequences derived from sugar 5 metabolizing enzymes, such as galactose, lactose (lac) (Chang et al. (1987) Nature oo 198:1056), and maltose. Additional examples include promoter sequences derived from biosynthetic enzymes such as tryptophan (trp) (Goeddel er al. (1980) Nucleic
Acids Res. 8:4057; Yelverton et al. (1981) Nucleic Acids Res. 9:731; U.S. Patent No. 4,738,921; EPO Publication Nos. 36,776 and 121,775). The beta-lactamase (bla) promoter system (Weissmann, (1981) “The Cloning of Interferon and Other :
Mistakes,” in Interferon 3 (ed. 1. Gresser); bacteriophage lambda PL (Shimatake ef al. (1981) Nature 292:128); the arabinose-inducible araB promoter (U.S. Patent No. 5,028,530); and T5 (U.S. Pat. No. 4,689,406) promoter systems also provide useful promoter sequences. See also Balbas (2001) Mol. Biotech. 19:251-267, where E. coli expression systems are discussed.
In addition, synthetic promoters that do not occur in nature also function as bacterial promoters. For example, wanscription activation sequences of one bacterial or bacteriophage promoter may be joined with the operon sequences of another bacterial or bacteriophage promoter, creating a synthetic hybrid promoter (U.S. Patent
No. 4,551,433). For example, the tac (Amann ef al. (1983) Gene 25:167; de Boer et | : al. (1983) Proc. Natl. Acad. Sci. 80:21) and trc (Brosius et al. (1985) J. Biol. Chem. : . 260:3539-3541) promoters are hybrid trp-lac promoters comprised of both trp - Co CE oo ~ promoter and lac — sequences that are regulated by the lac repressor. The tac promoter has the additional feature of being an inducible regulatory sequence. Thus, for example, expression of a coding sequence operably linked to the tac promoter can # be induced in a cell culture by adding isopropyl-1-thio-B-D-galactoside (IPTG). 0 - . ~ 30 Furthermore, a bacterial promoter can include naturally occurring promoters of non- bacterial origin that have the ability to bind bacterial RNA polymerase and initiate - transcription. A naturally occurring promoter of non-bacterial origin can also be ~ coupled with a compatible RNA polymerase to produce high levels of expression of = 55 oo some genes in prokaryotes. The bacteriophage T7 RNA polymerase/promoter system is an example of a coupled promoter system (Studier et al. (1986) J Mol. Biol. . 189:113; Tabor et al. (1985) Proc. Natl. Acad. Sci. 82:1074). In addition, a hybrid . promoter can also be comprised of a bacteriophage promoter and an E. coli operator k region (EPO Publication'No. 267,851).
So The vector may additionally contain a gene encoding the repressor (or . inducer) for that promoter. For example, an inducible vector of the present invention - may regulate transcription from the Lac operator (LacO) by expressing the gene encoding the Lacl repressor protein. Other examples include the use of the lexA gene to regulate expression of pRecA, and the use of trpO to regulate ptrp. Alleles of such genes that increase the extent of repression (e.g., laclq) or that modify the manner of induction (e.g., lambda CI857, rendering lambda pL thermo-inducible, or lambda CI+, rendering lambda pL. chemo-inducible) may be employed.
Co In addition to a functionirig promoter sequence, an efficient ribosome-binding site is also useful for the expression of the fusion construct. In prokaryotes, the ribosome binding site is called the Shine-Dalgarno (SD) sequence and includes an : initiation codon (ATG) and a sequence 3-9 nucleotides in length located 3-11 oo nucleotides upstream of the initiation codon (Shine ef al. (1975) Nature 254:34). The
SD sequence is thought to promote binding of mRNA to the ribosome by the pairing of bases between the SD sequence and the 3' end of bacterial 16S rRNA (Steitz et al. oF (1979) “Genetic Signals and Nucleotide Sequences in Messenger RNA,” in Biological
Regulation and Development: Gene Expression (ed. R. F. Goldberger, Plenum Press,
NY). : . Carbohydrate utilization-related proteins can also be secreted from the cell by So © 25 creating chimeric DNA molecules that encode a protein comprising a signal peptide oo iE - sequence fragment that provides for secretion of the carbohydrate utilization-related and multidrug transporter polypeptides in bacteria (U.S. Patent No. 4,336,336). The . signal sequence fragment typically encodes a si gnal peptide comprised of : hydrophobic amino acids that direct the secretion of the protein from the cell. The . 30 protein is either secreted into the growth media (Gram-positive bacteria) or into the periplasmic space, located between the inner and outer membrane of the cell (Gram- - negative bacteria). Preferably there are processing sites, which can be cleaved either ~
Co in vivo or in vitro, encoded between the signal peptide fragment and the carbohydrate . utilization-related or multidrug transporter protein.
DNA encoding suitable signal sequences can be derived from genes for secreted bacterial proteins, such as the £. coli outer membrane protein gene (ompA) (Masui et al. (1983) FEBS Lett. 151(1):159-164; Ghrayeb ef al. (1984) EMBO J. ] 3:2437-2442) and the E. coli alkaline phosphatase signal sequence (phoA) (Oka ef al. (1985) Proc. Natl. Acad- Sci. 82:7212). Other prokaryotic signals include, for . - example, the signal sequence from penicillinase, Ipp, or heat stable enterotoxin II leaders. "
Bacteria such as L. acidophilus generally utilize the start codon ATG, which } specifies the amino acid methionine (which is modified to-N-formylmethionine in prokaryotic organisms). Bacteria also recognize alternative start codons, such as the ) codons GTG and TTG, which code for valine and leucine, respectively. When they are used as the initiation codon, however, these codons direct the incorporation of methionine rather than of the amino acid they normally encode. Lactobacillus acidophilus NCFM recognizes these alternative start sites and incorporates methionine as the first amino acid.
Typically, transcription termination sequences recognized by bacteria are : regulatory regions located 3' to the translation stop codon and thus, together with the promoter, flank the coding sequence. These sequences direct the transcription of an mRNA that can be translated into the polypeptide encoded by the DNA. Transcription termination sequences frequently include DNA sequences (of about 50 nucleotides) that are capable of forming stem loop structures that aid in terminating transcription. .
Examples include transcription termination sequences derived from genes with strong promoters, such as the trp gene in £. coli as well as other biosynthetic genes. y
The expression vectors will have a plurality of restriction sites for insertion of B iE the carbohydrate utilization-related or multidrug wansporter sequence so that it is ; under transcriptional regulation of the regulatory regions. Selectable marker genes that ensure maintenance of the vector in the cell can also be included in the expression vector. Preferred selectable markers include those that confer resistance to drugs such i ) as ampicillin, chloramphenicol, erythromycin, kanamycin (neomycin), and . ) 30 tetracycline (Davies et al. (1978) Annu. Rev. Microbiol. 32:469). Selectable markers B may also allow a cell to grow on minimal medium, or in the presence of toxic - metabolite and may include biosynthetic genes, such as those in the histidine, ~ : tryptophan, and leucine biosynthetic pathways. .
The regulatory regions may be native (homologous), or may be foreign (heterologous) to the cell and/or the nucleotide sequence of the invention. The regulatory regions may also be natural or synthetic. Where the region is “foreign” or ] “heterologous” to the cell, it is meant that the region is not found in the native cell ] : into which the region is introduced. Where the region is “foreign” or “heterologous” - to the carbohydrate utilization-related or multidrug transporter nucleotide sequence of the invention, it 1s meant that the region is not the native or naturally occurring region . for the operably linked carbohydrate utilization-related or multidrug transporter nucleotide sequence of the invention. For example, the region may be derived from phage. While it may be preferable to express the sequences using heterologous : regulatory regions, native regions may be used. Such constructs would be expected in y some cases to alter expression levels of carbohydrate utilization-related or multidrug transporter proteins in the cell. Thus, the phenotype of the cell could be altered.
In preparing the expression cassette, the various DNA fragments may be manipulated, so as to provide for the DNA sequences in the proper orientation and, as appropriate, in the proper reading frame. Toward this end, adapters or linkers may be employed to join the DNA fragments or other manipulations may be involved to provide for convenient restriction sites, removal of superfluous DNA, removal of restriction sites, or the like. For this purpose, in vifro mutagenesis, primer repair, : 20 restriction, annealing, resubstitutions, e.g., transitions and transversions, may be ’ involved. | : ~The invention further provides a recombinant expression vector comprising a * DNA molecule of the invention cloned into the expression vector in an antisense SEE : - orientation. That is, the DNA molecule is operably linked to a regulatory sequence in | ) a manner that allows for expression (by transcription of the DNA molecule) of an
RNA molecule that is antisense to carbohydrate utilization-related or multidrug transporter mRNA. Regulatory sequences operably linked to a nucleic acid cloned in the antisense orientation can be chosen to direct the continuous or inducible :
NB expression of the antisense RNA molecule. The antisense expression vector can be in oo 30 the form of a recombinant plasmid or phagemid in which antisense nucleic acids are produced under the control of a high efficiency regulatory region, the activity of : - which can be determined by the cell type into which the vector is introduced. For a 3 discussion of the regulation of gene expression using antisense genes see Weintraub et al: (1986) Reviews - Trends in Genetics, Vol. 1(1).
: Alternatively, some of the above-described components can be put together in transformation vectors. Transformation vectors are typically comprised of a selectable market that is either maintained in a replicon or developed into an integrating vector, i as described above. - : - Plant Expression Vectors
For expression in plant cells, the expression cassettes will comprise a : transcriptional initiation region operably linked to a nucleotide sequence of the present invention. Various restriction sites may be included in these expression vectors to enable insertion of the nucleotide sequence under the transcriptional : oo regulation of the regulatory regions. Additionally, the expression cassette may contain selectable marker genes, including those genes that provide herbicide or antibiotic resistance, such as tetracycline resistance, hygromycin resistance, ampicillin resistance, or glyphosate resistance.
The expression cassette will include in the 5'-to-3' direction of transcription, a "transcriptional and translational initiation region, a nucleotide sequence of the : invention, and a transcriptional and translational termination region (i.e., termination region) functional in plants. The termination region may be native with the transcriptional initiation region comprising the promoter nucleotide sequence, may be
A 20 native with the nucleotide sequence of the invention, or may be derived from another : source. Convenient termination regions are known in the art and include, but are not limited to, a termination region from the Ti-plasmid of A. tumefaciens, such as the : octopine synthase and nopaline synthase termination regions. See also, Guerineau ef a al. (1991) Mol. Gen. Genet. 262:141-144; Proudfoot (1991) Cell 64:671-674; oo : Sanfacon ef al. (1991) Genes Dev. 5:141-149; Mogen et al. (1990) Plant Cell 2:1261-1272; Munroe ef al. (1990) Gene 91:151-158; Ballas er al. 1989) Nucleic
Acids Res. 17:7891-7903; and Joshi et al. (1987) Nucleic Acid Res. 15:9627-9639.
The expression cassette comprising a nucleotide sequence of the present invention may also contain at least one additional nucleotide sequence for a gene to be - 30 cotransformed into the organism. Alternatively, the additional sequence(s) may be provided on another expression cassette.
To The expression cassettes may additionally contain 5' non-translated leader -- sequences or 5' non-coding sequences. As used herein, “5' leader sequence,” :
“translation leader sequence,” or “5' non-coding sequence” refer to that DNA sequence portion of a gene between the promoter and coding sequence that is transcribed into RNA and is present in the fully processed mRNA upstream (5') of the : - translation start codon. A 5' non-translated leader sequence is usually characterized as 3 that portion of the mRNA molecule that most typically extends from the 5' CAP site oC to the AUG protein translation initiation codon. The translation leader sequence may ) affect processing of the primary transcript to mRNA, mRNA stability or translation . efficiency (Turner ef al. (1995) Molecular Biotechnology 3:225). Thus, translation ! © leader sequences play an important role in the regulation of gene expression. 1 0 Translation leaders are known in the art and include but are not limited to: picornavirus leaders, for example, EMCV leader (Encephalomyocarditis 5' noncoding region) (Elroy-Stein et al. (1989) Proc. Nat. Acad. Sci. USA 86:6126-6130): potyvirus leaders, for example, TEV leader (Tobacco Etch Virus) (Allison ef al. (1986) Virology 154:9-20); MDMV leader (Maize Dwarf Mosaic Virus); human immunoglobulin heavy-chain binding protein (BiP) (Macejak ef al. (1991) Nature 353:90-94); untranslated leader from the coat protein mRNA of alfalfa mosaic virus : (AMV RNA 4) (Jobling et al. (1987) Nature 325:622-625); tobacco mosaic virus leader (TMV) (Gallie er al. (1989) Molecular Biology of RNA, pages 237-256); and maize chlorotic mottle virus leader (MCMV) (Lommel ef al. (1991) Virology 81:382- 385). ’ * Other methods known to enhance translation and/or mRNA stability can also be utilized, for example, introns, such as the maize ubiquitin intron (Christensen and
Quail (1996) Transgenic Res. 5:213-218 and Christensen ef al. (1992) Plant . :
Molecular Biology 18:675-689) or the maize Adhl intron (Kyozuka ef al. (1991) Mol.
Gen. Genet. 228:40-48 and Kyozuka ef al. (1990) Maydica 35:353-357), and the like.
Various intron sequences have been shown to enhance expression, particularly in monocotyledonous cells. The introns of the maize Adhl gene have been found to ‘significantly enhance the expression of the wild-type gene under its cognate promoter : when introduced into maize cells. Intron 1 was found to be particularly effective and - 30 - enhanced expression in fusion constructs with the chloramphenicol acetyltransferase gene (Callis er al. (1987) Genes Develop. 1:1183-1200). In the same experimental ) system, the intron from the maize bronzel gene had a similar effect in enhancing expression. The Adhl intron has also been shown to enhance CAT expression 12-fold E (Mascarenhas et al. (1990) Plant Mol. Biol. 6:913-920). Intron sequences have §
© WO 2005/084411 | PCT/US2005/007594 routinely been incorporated into plant transformation vectors, typically within the : non-translated leader.
The expression cassette comprising a promoter sequence of the present } invention may additionally contain a 3' non-coding sequence. A “3' non-coding : 5 sequence” or “3' non-translated region” refers to a nucleotide sequence located 3' - (downstream) to a coding sequence and includes polyadenylation signal sequences and other sequences encoding regulatory signals capable of affecting the addition of - polyadenylic acid tracts to the 3’ end of the mRNA precursor. A 3' non-translated : region comprises a region of the mRNA generally beginning with the translation termination codon and extending at least beyond the polyadenylation site. Non- translated sequences located in the 3' end of a gene have been found to influence gene expression levels. Ingelbrecht er al. (see, Plant Cell, 1:671-680, 1989) evaluated the importance of these elements and found large differences in expression in stable plants depending on the source of the 3' non-trauslated region. Using 3' non-translated regions associated with octopine synthase, 2S seed protein from Arabidopsis, small subunit of rbcS from Arabidopsis, extension from carrot, and chalcone synthase from
Antirrhinium, a 60-fold difference was observed between the best-expressing construct (which contained the rbcS 3' non-translated region) and the lowest- expressing construct (which contained the chalcone synthase 3' region).
Transcription levels may also be increased by the utilization of enhancers in ’ combination with the promoter regions of the invention. Enhancers are nucleotide : sequences that act to increase the expression of a promoter region. Enhancers are known in the art and include the SV40 enhancer region, the 35S enhancer element, : : and the like. | : oo
In preparing the expression cassette, the various DNA fragments may be manipulated, so as to provide for the DNA sequences in the proper orientation and, as appropriate, in the proper reading frame. Adapters or linkers may be employed to join the DNA fragments or other manipulations may be involved to provide for convenient 3 restriction sites. Restriction sites may be added or removed, superfluous DNA may be CT . 30 removed, or other modifications may be made to the sequences of the invention. For this purpose, in vitro mutagenesis, primer repair, restriction, annealing, : : resubstitutions, for example, transitions and transversions, may be involved. : In addition to selectable markers that provide resistance to antibiotics or “herbicides, as described above, other genes that could serve utility in the recovery of transgenic events but might not be required in the final product would include, but are not limited to, GUS (b-glucoronidase; Jefferson (1987) Plant Mol. Biol. Rep. 5:387), So
GFP (green florescence protein; Chalfie er al. (1994) Science 263:802), luciferase . (Riggs et al. (1987) Nucleic Acids Res. 15(19):8115 and Luchrsen ef al. (1992) -
Methods Enzymol. 216:397-414), and the maize genes encoding for anthocyanin _— production (Ludwig ef al. (1990) Science 247:449).
The nucleic acids of the present invention are useful in methods directed to : expressing a nucleotide sequence in a plant. This may be accomplished by transforming a plant cell of interest with an expression cassette comprising a promoter operably linked to a nucleotide sequence identified herein, and regenerating a stably transformed plant from said plant cell. The expression cassette comprising the : promoter sequence operably linked to a nucleotide sequence of the present invention can be used to transform any plant. In this manner, genetically modified, i.e. transgenic or transformed, plants, plant cells, plant tissue, seed, root, and the like can be obtained. . Microbial or Bacterial Cells
The production of bacteria containing heterologous phage resistance genes, the preparation of starter cultures of such bacteria, and methods of fermenting substrates, , . 20 particularly food substrates such as milk, may be carried out in accordance with : © known techniques. :
By “introducing” as it pertains to nucleic acids is meant introduction into prokaryotic or eukaryotic cells via conventional transformation or transfection : techniques, or by phage-mediated infection. As used herein, the terms “transformation,” “transduction,” “conjugation,” and “protoplast fusion’ are meant to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a cell, including calcium phosphate or calcium chloride co- precipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation.
Suitable methods for transforming or transfecting cells can be found in Sambrook et ’ 30 al (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor
Laboratory Press, Plainview, New York) and other laboratory manuals. By “introducing” as it pertains to polypeptides or microorganisms of the invention, is -
meant introduction into a host by ingestion, topical application, nasal, suppository, urogenital, or oral application of the polypeptide or microorganism.
Bacterial cells used to produce the carbohydrate utilization-related or . multidrug transporter polypeptides of this invention are cultured in suitable media, as described generally in Sambrook et al. (1989) Molecular Cloning, A Laboratory - Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, New York.
Bacterial strains encompassed by the present invention include those that are . biologically pure cultures of a bacterium comprising at least one nucleotide or amino acid sequence of the present invention. These strains include: a Lactobacillus : : acidophilus bacterial strain with a modified ability to transport a carbohydrate into or out of a cell as compared to a wild-type Lactobacillus acidophilus, wherein the modified ability is due to expression of at least one carbohydrate utilization-related polypeptide of the present invention; a Lactobacillus acidophilus bacterial strain with a a modified ability to accumulate a carbohydrate, as compared to a wild-type
Lactobacillus acidophilus, wherein the modified ability is due to expression of at least one carbohydrate utilization-related polypeptide of the present invention; a
Lactobacillus acidophilus bacterial strain with a modified ability to utilizea carbohydrate as an energy source, as compared to a wild-type Lactobacillus acidophilus, wherein the modified ability is due to expression of at least one : carbohydrate utilization-related polypeptide of the present invention; a Lactobacillus : acidophilus bacterial strain that provides a food product with a modified flavor as a result of fermentation, as compared to a wild-type Lactobacillus acidophilus, wherein the modified flavor is due to expression of a carbohydrate utilization-related ; . polypeptide of the present invention; a Lactobacillus acidophilus bacterial strain that provides a food product with a modified texture as a result of fermentation, as : compared to a wild-type Lactobacillus acidophilus, wherein the modified texture is } due to expression of a carbohydrate utilization-related polypeptide of the present invention; a Lactobacillus acidophilus bacterial strain with a modified ability to : produce a carbohydrate, as compared to a wild-type Lactobacillus acidophilus, : . 30 wherein the ability is due to expression of at least one carbohydrate utilization-related . polypeptide of the present invention; a Lactobacillus acidophilus bacterial strain with : a modified ability to survive food processing and storage conditions, as compared to a wild-type Lactobacillus acidophilus, wherein the modified ability is due to expression ; -of at least one carbohydrate utilization-related polypeptide of the present invention; a -
Lactobacillus acidophilus bacterial strain with a modified ability to survive in a GI tract, as compared to a wild-type Lactobacillus acidophilus, wherein the modified ability is due to expression of at least one carbohydrate utilization-related polypeptide i of the present invention; a Lactobacillus acidophilus bacterial strain with a modified ability to survive contact with an antimicrobial polypeptide or toxin, as compared to a - wild-type Lactobacillus acidophilus, wherein the modified ability is due to expression . of at least one multidrug transport polypeptide of the present invention. . - Transgenic Plants and Plant Cells
As used herein, the terms “transformed plant” and “transgenic plant” refer to a A plant that comprises within its genome a heterologous polynucleotide. Generally, the heterologous polynucleotide is stably integrated within the genome of a transgenic or oo transformed plant such that the polynucleotide is passed on to successive generations.
The heterologous polynucleotide may be integrated into the genome alone or as part of arecombinant expression cassette. It is to be understood that as used herein the term “transgenic” includes any cell, cell line, callus, tissue, plant part, or plant the genotype of which has been altered by the presence of heterologous. nucleic acid including those transgenics initially so altered as well as those created by sexual ‘ crosses or asexual propagation from the initial transgenic. The term “transgenic” as k 20 used herein does not encompass the alteration of the genome (chromosomal or extra- oo : chromosomal) by conventional plant breeding methods or by naturally occurring E events such as random cross-fertilization, non-recombinant viral infection, non- recombinant bacterial transformation, non-recombinant transposition, or spontaneous ) mutation.
A transgenic “event” is produced by transformation of plant cells with a heterologous DNA construct, including a nucleic acid expression cassette that ’ comprises a transgene of interest, the regeneration of a population of plants resulting from the insertion of the transgene into the genome of the plant, and selection of a : particular plant characterized by insertion into a particular genome location. An event : ) 30 is characterized phenotypically by the expression of the transgene. At the genetic level, an event is part of the genetic makeup of a plant. The term “event” also refers to ' progeny produced by a sexual outcross between the transformant and another variety -. that includes the heterologous DNA.
As used herein, the term “plant” includes whole plants, plant organs (e.g., leaves, stems, roots, etc.), seeds, plant cells, and progeny of same. Parts of transgenic . plants within the scope of the invention are to be understood to comprise, for example, plant cells, protoplasts, tissues, callus, embryos as well as flowers, pollen, : anthers, stems, fruits, ovules, leaves, or roots originating in transgenic plants or their } progeny previously transformed with a DNA molecule of the invention, and therefore consisting at least in part of transgenic cells. | . : As used herein, the term “plant cell” includes, without limitation, seeds suspension cultures, embryos, meristematic regions, callus tissue, leaves, roots, ~ 10 shoots, gametophytes, sporophytes, pollen, and microspores. The class of plants that Co can be used in the methods of the invention is generally as broad as the class of higher oo plants amenable to transformation techniques, including both monocotyledonous and dicotyledonous plants. | ;
The present invention may be used for transformation of any plant species, = including, but not limited to, monocots and dicots. Examples of plants of interest include, but are not limited to, corn (Zea mays), Brassica sp. (e.g., B. napus, B. rapa, B.
Juncea), particularly those Brassica species useful as sources of seed oil, alfalfa (Medicago sativa), rice (Oryza sativa), rye (Secale cereale), sorghum (Sorghum bicolor,
Sorghum vulgare), millet (e.g., pearl millet (Pennisetum glaucum), proso millet 20° (Panicum miliaceum), foxtail millet (Setaria italica), finger millet (Eleusine coracana)), ' sunflower (Helianthus annuus), safflower (Carthamus tinctorius), wheat (Triticum . aestivum), soybean (Glycine max), tobacco (Nicotiana tabacum), potato (Solanum : tuberosum), peanuts (Arachis hypogaea), cotton (Gossypium barbadense, Gossypium - hirsutum), sweet potato (Jpomoea batatus), cassava (Manihot esculenta), coffee (Coffea BS spp.), coconut (Cocos nucifera), pineapple (4nanas comosus), citrus trees (Citrus spp.), a cocoa (Theobroma cacao), tea (Camellia sinensis), banana (Musa spp.), avocado (Persea americana), fig (Ficus casica), guava (Psidium guajava), mango (Mangifera indica), olive (Olea europaea), papaya (Carica papaya), cashew (Anacardium i : occidentale), macadamia (Macadamia integrifolia), almond (Prunus amygdalus), sugar ] 30 beets (Beta vulgaris), sugarcane (Saccharum spp.), oats, barley, vegetables, omamentals, and conifers. . Vegetables include tomatoes (Lycopersicon esculentum), lettuce (e.g., Lactuca : sativa), green beans (Phaseolus vulgaris), lima beans (Phaseolus limensis), peas (Lathyrus spp.), and members of the genus Cucumis such as cucumber (C. sativus), os cantaloupe (C. cantalupensis), and musk melon (C. melo). Omamentals include azalea : (Rhododendron spp.), hydrangea (Macrophylla hydrangea), hibiscus (Hibiscus rosasanensis), roses (Rosa spp.), tulips (Tulipa spp.), daffodils (Narcissus spp.), petunias ] (Petunia hybrida), camation (Dianthus caryophyllus), poinsettia (Euphorbia ) pulcherrima), and chrysanthemum. Conifers that may be employed in practicing the - present invention include, for example, pines such as loblolly pine (Pinus taeda), slash : pine (Pinus elliotii), ponderosa pine (Pinus ponderosa), lodgepole pine (Pinus contorta), and Monterey pine (Pinus radiata), Douglas-fir (Pseudotsuga menziesii); Westen . hemlock (Tsuga canadensis), Sitka spruce (Picea glauca; redwood (Sequoia oo sempervirens); true firs such as silver fir (Abies amabilis) and balsam fir (Abies balsamea); and cedars such as Western red cedar (Thuja plicata) and Alaska yellow- cedar (Chamaecyparis nootkatensis).
The methods of the invention do not depend on a particular method for : oo introducing a nucleotide construct to a plant, only that the nucleotide construct gains access to the interior of at least one cell of the plant. Methods for introducing nucleotide constructs into plants are known in the art including, but not limited to, ‘ stable transformation methods, transient transformation methods, and virus-mediated k methods.
By “transient transformation” it is meant that a nucleotide construct introduced : into a plant does not integrate into the genome of the plant. By “stable transformation” it is meant that the nucleotide construct introduced into a plant : : integrates into the genome of the plant and is capable of being inherited by progeny thereof. “Primary transformant” and “TO generation” transgenic plants are of the same genetic generation as the tissue that was initially transformed (i.e., not having gone through meiosis and fertilization since transformation). “Secondary transformants” and “Tl, T2, T3, and subsequent generations” refer to transgenic plants derived from primary transformants through one or more meiotic and fertilization cycles. They may be derived by self-fertilization of primary or secondary transformants or crosses of ; primary or secondary transformants with other transformed or untransformed plants. : ) 30 Transformation protocols as well as protocols for introducing nucleotide sequences into plants may vary depending on the type of plant or plant cell, i.e., : - monocot or dicot, targeted for transformation. The nucleotide constructs of the © invention may be introduced into plants by any method known in the art, including, but not limited to, contacting the plants with a virus or viral nucleic acids (see, for : :
example, U.S. Patent Nos. 5,889,191, 5,889,190, 5,866,785, 5,589,367, and 5,316,931; herein incorporated by reference), microinjection (Crossway er al. (1986)
Biotechniques 4:320-334), electroporation (Riggs ef al. (1986) Proc. Natl. Acad. Sci. ] USA 83:5602-5606, Agrobacterium-mediated transformation (U.S. Patent Nos. ’ 5S 5,981,840 and 5,563,055), direct gene transfer (Paszkowski ef al. (1984). EMBO J. ~*~ - 3:2717-2722), and ballistic particle acceleration (see, for example U.S. Patent Nos. : 4,945,050; 5,879,918; 5,886,244; and 5,932,782); all of which are herein incorporated 4 by reference. - Co )
The transformed cells may be grown into plants with methods known in the art. See, for example, McCormick ef al. (1986) Plant Cell Reports 5:831-84. These no plants may then be grown, and either pollinated with the same transformed strain or ) different strains, and the resulting hybrid having expression of the desired phenotypic characteristic identified. Two or more generations may be grown to ensure that - - * expression of the desired phenotypic characteristic is stably maintained and inherited Co and then seeds may be harvested to ensure expression of the desired phenotypic characteristic has been achieved. Thus as used herein, “transformed seeds” refers to seeds that contain the nucleotide construct stably integrated into the plant genome.
Methods of Use k 20 Methods are provided for modifying expression of carbohydrate utilization- related or multidrug transporter genes or proteins of an organism. In one embodiment, properties of microorganisms used in fermentation are modified to provide strains : able to utilize alternative carbohydrates for energy or carbon sources. These : oo modifications may result in a new ability to synthesize, transport, accumulate, or : degrade a carbohydrate. Alternatively, these modifications may result in the ability to E survive contact with antimicrobial polypeptides, including antibiotics and toxins.
These new abilities may also allow the microorganisms to better survive stressful
Lo . conditions, such as the digestive tract or those found during food processing and ES storage, which will increase the utility of these microorganisms in fermenting various . 30 foods, as well as allowing them to provide longer-lasting probiotic activity after ingestion. These new abilities may also allow the microorganisms to generate different flavors or textures in a product upon fermentation. In addition, the new - abilities may enable a bacterium to produce a modified carbohydrate, | :
exopolysaccharide, or cell surface polysaccharide. In another embodiment, the properties of plants are modified to provide similar abilities. These abilities are provided by the nucleotide and amino acid sequences disclosed in the present ] invention. i. 5 In general, the methods comprise introducing or overexpressing one or more oo
SI proteins involved in carbohydrate utilization or multidrug resistance. By “introducing” is meant that the protein of interest is expressed in a modified cell when i. it wag not expressed in an unmodified cell. By “overexpressing” is meant that the protein of interest is expressed in an increased amount in the modified organism compared to its production in the unmodified wild-type organism. Homofermentative : lactic acid bacteria, in particular, have a relatively simple metabolism, with almost no overlap between energy metabolism and biosynthesis metabolism, making them ideal targets for metabolic engineering (Hugenholz and Kleerebezem (1999) Current Opin. | -
Biotech. 10:492-497). The expression of bacterial genes in plants is well known in the art. See, for example, Shewmaker ef al. (1994) Plant Physiol. 104:1159-1166; Shen i. et al. (1997) Plant Physiol. 113:1177-1183; Blaszczyk et al. (1999) Plant J. 20:237— 243. | :
Expression of one or more carbohydrate utilization-related or multidrug : ‘transporter proteins may allow for an organism to have a modified ability to transport a carbohydrate or an antimicrobial polypeptide such as a bacteriocin into or out of a ) cell. Transport-related carbohydrate utilization proteins or multidrug transporter proteins comprise ABC transporter system components including substrate-binding proteins (for example HisJ and MalE), membrane-associated components such as permeases (for example LacF and LacG), and cytoplasmic proteins such as ATP- binding proteins (for example msmK). Transport-related carbohydrate utilization EB proteins or multidrug transporter proteins also comprise secondary transport system proteins such as those in the major facilitator superfamily (MFS) and the glycoside/pentoside/hexuronide family. Group translocation system proteins are also . included, including enzyme I, enzyme IL, and HPr proteins. ~ ) 30 Carbohydrate utilization-related proteins also include dehydrogenases.
Aldehyde dehydrogenases (EC:1.2.1.3 and EC:1.2.1.5) (PFAM Accession No. . - PF00171) are enzymes that oxidize a wide variety of aliphatic and aromatic aldehydes using NADP as a cofactor. In mammals at least four different forms of the enzyme are ) known (Hempel ef al. (1989) Biochemistry 28:1160-7): class-1 (or Ald C) a tetrameric )
cytosolic enzyme, class-2 (or Ald M) a tetrameric mitochondrial enzyme, class-3 (or
Ald D) a dimeric cytosolic enzyme, and class-4 a microsomal enzyme. Aldehyde ; dehydrogenases have also been sequenced from fungal and bacterial species. A ] number of enzymes are known to be evolutionary related to aldehyde dehydrogenases. }
A glutamic acid and a cysteine residue have been implicated in the catalytic activity - of mammalian aldehyde dehydrogenase. These residues are conserved in all the
SL enzymes of this family. Aldehyde dehydrogenase proteins of the present invention - include that in SEQ ID NO:228. D-isomer specific 2-hydroxyacid dehydrogenase, catalytic domain (PFAM Accession No. PF00389) proteins of the present invention include those in SEQ ID NO:242. D-isomer specific 2-hydroxyacid dehydrogenase,
NAD binding domain (PFAM Accession No. PF02826) proteins of the present invention include those in SEQ ID NO:242. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) plays an important role in glycolysis and gluconeogenesis : by reversibly catalyzing the oxidation and phosphorylation of D-glyceraldehyde-3- phosphate to 1,3-diphospho-glycerate (Huang et al- (1989) J. Mol. Biol. 206:411-24).
The enzyme exists as a tetramer of identical subunits, each containing 2 conserved functional domains: an-NAD-binding domain, and a highly conserved catalytic domain. SEQ ID NO:248 has a Glyceraldehyde 3-phosphate dehydrogenase, C- terminal domain (PFAM Accession No. PF02800), as well as a Glyceraldehyde 3- phosphate dehydrogenase, NAD binding domain (PFAM Accession No. PF00044). g . L-lactate dehydrogenases are metabolic enzymes that catalyze the conversion of L- lactate to pyruvate, the last step in anaerobic glycolysis. Malate dehydrogenases catalyze the interconversion of malate to oxaloacetate. The enzyme participates in the : citric acid cycle. Lactate/malate dehydrogenase, alpha/beta C-terminal domain - (PFAM Accession No. PF02866) and lactate/malate dehydrogenase, NAD binding domain (PFAM Accession No. PF00056) proteins of the present invention include those in SEQ ID NOS:222 and 246. Methods for measuring dehydrogenase activity oo are well known in the art (see, for example, Ercolani ef al. (1988) J. Biol. Chem. % 2631533541). i 30 Carbohydrate utilization-related proteins also include O-Glycosyl hydrolases.
O-Glycosyl hydrolases (EC 3.2.1.-) are a widespread group of enzymes that hydrolyze - - the glycosidic bond between two or more carbohydrates, or between a carbohydrate and a non-carbohydrate moiety. Assays to measure hydrolase activity are well known : ‘in the art (see, for example, Avigad and Bauer (1966) Methods Enzymol. 8:621-628,; :
Neumann and Lampen (1967) Biochemistry 6:468-475; Henry and Darbyshire (1980) : Phytochemistry 19:1017-1020).
Alpha amylase, catalytic domain proteins (PFAM Accession No. PF00128) . are classified as family 13 of the glycosyl hydrolases. Alpha amylase, catalytic } domain proteins of the present invention include those in SEQ ID NOS:108, 196, 240, : : 292 and 332. Co
The Beta-galactosidase family (PFAM Accession No. PF02449) belongs to the 4 glycosyl hydrolase 42 family. The enzyme catalyses the hydrolysis of terminal, non- } reducing terminal beta-D-galactosidase residues. Beta-galactosidase proteins of the present invention include that in SEQ ID NO:21 0. The Beta galactosidase small chain,
C terminal domain (PFAM Accession No. PF02930) is found in the carboxy-terminal : portion of the small chain of dimeric beta-galactosidases (EC:3.2.1.23). The Beta . galactosidase small chain, N terminal domain (PFAM Accession No. PF02929) is found in the amino-terminal portion of the small chain of dimeric beta-galactosidases (EC:3.2.1.23). These domains are also found in single chain beta-galactosidase. Beta galactosidase small chain, C terminal proteins of the present invention include that in
SEQ ID NO:214. Beta galactosidase small chain, N terminal domain proteins of the "present invention include that in SEQ ID NO:214. oo
Glycoside hydrolase family 1 (PFAM Accession No. PF00232) comprises . 20 enzymes with a number of known activities; B-glucosidase (EC:3.2.1.21); B- galactosidase (EC:3.2.1.23); 6-phospho-f-galactosidase (EC:3.2.1.85); 6-phospho-p- glucosidase (EC:3.2.1.86); lactase-phiorizin hydrolase (EC:3.2.1.6), (EC:3.2.1.108);
B-mannosidase (EC:3.2.1.25); myrosinase (EC:3.2.1.147). Glycoside hydrolase Le
Co family. proteins of the present invention include those in SEQ ID NOS:10 and 220.
Glycoside hydrolase family 2 comprises enzymes with several known activities; B-galactosidase (EC:3.2.1.23); B-mannosidase (EC:3.2.1.25); B- glucuronidase (EC:3.2.1.31). These enzymes contain a conserved glutamic acid : residue that has been shown, in Escherichia coli lacZ, to be the general acid/base - catalyst in the active site of the enzyme (Gebler er al. (1992) J. Biol. Chem. Co - 30 267:11126-30). The Glycosyl hydrolases family 2, immunoglobulin-like beta- sandwich domain (PFAM Accession No. PF00703) describes the immunoglobulin- ) -like B-sandwich domain. The sugar binding domain (PFAM Accession No. PF02837) - has a jelly-roll fold. Beta-galactosidase from E. coli has a TIM-barel-like core )
(PFAM Accession No. PF02836) surrounded by four other largely p domains. SEQ }
ID NO:212 has each of these domains. .
Glycoside hydrolase family 31 (PFAM Accession No. PF01055) comprises oo ". enzymes with several known activities; a-glucosidase (EC:3.2.1.20), o-galactosidase oo B 5 (EC:3.2.1.22), ghucoamylase (EC:3.2.1.3), sucrase-isomaltase (EC:3.2.1 48) oo oo (BC:3.2.110); a-xylosidase (EC:3.2.1); and a-glucan lyase (EC:4.2.2.13). Glycoside hydrolase family 31 groups a number of glycosyl hydrolases on the basis of sequence | : similarities (Henrissat (1991) Biochem. J. 280:309-16; Naim ef al. (1991) FEBS Lett. 294:109-12). An aspartic acid has been implicated in the catalytic activity of sucrase, isomaltase, and lysosomal a-glucosidase (Hermans ef al. (1991) J. Biol. Chem. 266:13507-12). Glycoside hydrolase family 31 proteins of the present invention include that in SEQ ID NO:226. | :
Glycoside hydrolase family 32 (PFAM Accession No. PF00251) comprises : : enzymes with several known activities; invertase (EC:3.2.1.26); inulinase (EC:3.2.1.7); levanase (EC:3.2.1.65); exo-inulinase (EC:3.2.1.80); sucrose:sucrose 1- fructosyltransferase (EC:2.4.1.99); and fructan:fructan 1-fructosyltransferase (EC2.4. 1.100). Glycoside hydrolase family 32 proteins of the present invention include those in SEQ ID NOS:46 and 100.
Enzymes containing the Isoamylase N-terminal domain (PFAM Accession
No. PF02922) belong to family 13 of the glycosyl hydrolases. This domain is found in a range of enzymes that act on branched substrates i.e. isoamylase, pullulanase and branching enzyme. Isoamylase hydrolyses 1,6-a-D-glucosidic branch linkages in glycogen, amylopectin and dextrin; 1,4-a-glucan branching enzyme functions in the K formation of 1,6-glucosidic linkages of glycogen; and pullulanase is a starch- ; debranching enzyme: Isoamylase N-terminal domain proteins of the present invention include that in SEQ ID NO:240. . RR . SL EA : | a i Alpha-galactosidase (EC:3.2. 1.22) (melibiase) (PFAM Accession No. IEEE oo = - -PF02065) catalyzes the hydrolysis of melibiose into galactose and glucose (Dey and A
Pridham (1972) Adv. Enzymol. Relat. Areas Mol. Biol 36:91-130). Alpha- - 30 galactosidase is present in a variety of organisms. There is'a considerable degree of similarity in the sequence of a-galactosidase from various eukaryotic species.
Escherichia coli o-galactosidase (gene melA), which requires NAD and magnesium a. as cofactors, is not structurally related to the eukaryotic enzymes; by contrast, an :
Escherichia coli plasmid encoded a-galactosidase (gene rafA) contains a region of about 50 amino-acids which is similar to a domain of the eukaryotic a-galactosidases (Aslanidis ef al. (1989) J. Bacteriol. 171:6753-63). Melibiase proteins of the present . invention include that in SEQ ID NO:198. }
Carbohydrate utilization-related proteins also include, but are not limited to,
RR the following types of enzymes: | Co oo oo Aldose 1-epimerase (EC:5.1.3.3) (mutarotase) (PFAM Accession No. -
PF01263) is the enzyme responsible for the anomeric interconversion of D-glucose and other aldoses between their a- and 3-forms. Methods to measure aldose 1- : epimerase activity are well known in the art (see, for example, Majumdar ef al. (2004)
Eur. J. Biochem. 271:753-9). Aldose 1-epimerase proteins of the present invention include that in SEQ ID NO:200.
Enolase (2-phospho-D-glycerate hydrolase) is an essential glycolytic enzyme that catalyses the interconversion of 2-phosphoglycerate and phosphoenolpyruvate. :
Methods to measure phosphopyruvate hydratase activity are well known in the art ) (see, for example, Fox et al: (1995) Plant Physiol. 109:433-43). SEQ ID NO:254 has an Enolase, C-terminal TIM barrel domain (PFAM Accession No. PF00113) and an + v= = . -.. Enolase, N-terminal domain (PFAM Accession No. PF(03952).
Fructose-bisphosphate aldolase (EC:4.1.2.13) is a glycolytic enzyme that catalyzes the reversible aldol cleavage or condensation of fructose-1,6-bisphosphate : into dihydroxyacetone-phosphate and glyceraldehyde 3-phosphate. There are two oo classes of fructose-bisphosphate aldolases with different catalytic mechanisms. Class- - . II aldolases (PFAM Accession No. PF01116) (Marsh and Lebherz (1992) Trends :
Biochem. Sci. 17:110-3), mainly found in prokaryotes and fungi, are homodimeric
N 25 enzymes, which require a divalent metal ion, generally zinc, for their activity. This coo 0 family also includes the Escherichia coli galactitol operon protein, gatY, which oe IR on catalyzes the transformation of tagatose 1,6-bisphosphate intd glycerone phosphate oo a oo and D-glyceraldehyde 3-phosphate; and Escherichia coli N-acetyl galactosamine , operon protein, agaY, which catalyzes the same reaction. There are two histidine Co - 30 residues in the first half of the sequence of these enzymes that have been shown to be involved in binding a zinc ion. Methods for measuring fructose-bisphosphate aldolase : ) activity are well known in the art (see, for example, Alefounder ef al. (1989) Biochem. ~
J. 257:529-534). Fructose-bisphosphate aldolase class Il proteins of the present invention include that in SEQ ID NO:260.
Galactose-1-phosphate uridyl transferase catalyzes the conversion of UTP and - a-D-galactosel-phosphate to UDP-glucose and pyrophosphate during galactose 5 . metabolism. The C-terminal domain (PFAM Accession No. PF02744) describes the
C terminal of Galactose-1-phosphate uridyl transferase. The N-terminal domain ~~ - (PFAM Accession No. PF01 087) describes the N terminal of Galactose-1-phosphate xv uridy! transferase. SEQ ID NO:202 has both of these domains. Methods for : measuring UTP-hexose-1-phosphate uridylyliransferase activity are well known in the art (see, for example, Lobelle-Rich and Reeves (1983) Mol. Biochem. Parasitol. 7:173-182). | :
The galacto- (EC:2.7.1.6), homoserine (EC:2.7.1.39), mevalonate (EC:2.7.1.36) and phosphomevalonate (EC:2.7.4.2) kinases contain, in their N- terminal section, a conserved Gly/Ser-rich region which is probably involved in the binding of ATP. SEQ ID NO:204 is a member of the GHMP Kinases putative ATP- binding protein family (PFAM Accession No. PF00288). Methods for measuring : kinase activity are well known in the art (see, for example, Tsay and Robinson (1991)
Mol. Cell Biol 11:620-31).
NAD dependent epimerase/dehydratase family (PFAM Accession No. PF01370) proteins utilize NAD as a cofactor. The proteins in this family use : nucleotide-sugar substrates for a variety of chemical reactions (Thoden ef al. (1997)
Biochemistry 36:6294-304). NAD dependent epimerase/dehydratase proteins of the present invention include that in SEQ ID NO:216. :
Lantibiotic and non-lantibiotic bacteriocins are synthesized as precursor : peptides containing N-terminal extensions (leader peptides), which are cleaved off E during maturation. Most non-lantibiotics and also some lantibiotics have leader _ - peptides of the so-called double-glycine type. These leader peptides share consensus sequences and also a common processing site with two conserved glycine residues-in - 3 positions -1 and -2. The double- glycine-type leader peptides are unrelated to the N- terminal signal sequences that direct proteins across the cytoplasmic membrane via the sec pathway. Their processing sites are also different from typical signal peptidase cleavage sites, suggesting that a different processing enzyme is involved. Peptide ~ bacteriocins are exported across the cytoplasmic membrane by a dedicated ATP- :
binding cassette (ABC) transporter. The ABC transporter is the maturation protease and its proteolytic domain resides in the N-terminal part of the protein (Havarstein ef al. (1995) Mol. Microbiol. 16:229-40). The peptidase C39 family (PFAM Accession . No. PF03412) domain is found in a wide range of ABC transporters. However, the h : 5 presumed catalytic cysteine and histidine are not conserved in all members of this : ) . family. Peptidase C39 family proteins of the present invention include that in SEQ ID .
NO:144. The activity of peptidases can be evaluated by measuring hydrolyzing i activity (see, for example, Sasaki ef al. (1995) J. Dairy Res. 62:601-610, and i
Machuga and Ives (1984) Biochim. Biophys. Acta 789:26-36).
The pfkB family carbohydrate kinase family (PFAM Accession No. PF00294) includes a variety of carbohydrate and pyrimidine kinases. The family includes . phosphomethylpyrimidine kinase (EC:2.7.4.7), fructokinase (EC:2.7.1.4), and : ribokinase (EC:2.7.1.15) (gene rbsK). This enzyme is part of the Thiamine : pyrophosphate (TPP) synthesis pathway, TPP is an essential cofactor for many enzymes. Methods for measuring kinase activity are well known in the art (see, for
B example, Sato ef al. (1993) J. Gen. Microbiol. 139:921-7). pfkB family carbohydrate : a kinase proteins of the present invention include those in SEQ ID NOS:60, 186, 224, and 238.
The enzyme-catalyzed transfer of a phosphoryl group from ATP 1s an important reaction in a wide variety of biological processes. One enzyme that utilizes ’ "this reaction is phosphofructokinase (PFK) (PFAM Accession No. PF00365), which catalyses the phosphorylation of fructose-6-phosphate to fructose-1,6- bisphosphate, a key regulatory step in the glycolytic pathway. PFK is about 300 amino acids in length, and structural studies of the bacterial enzyme have shown it comprises two | 3 similar (a/B) lobes: one involved in ATP binding and the other housing both the | : substrate-binding site and the allosteric site (a regulatory binding site distinct from the active site, but that affects enzyme activity). The identical tetramer subunits adopt two different conformations: in a ‘closed’ state, the bound magnesium ion bridges the phosphoryl groups of the enzyme products (ADP and fructose-1,6- bisphosphate); and ’ 30 in an ‘open’ state, the magnesium ion binds only the ADP, as the two products are now further apart. These conformations are thought to be successive stages of a reaction
Co pathway that requires subunit closure to bring the two molecules sufficiently close to ~ react (Shirakihara and Evans (1988) J. Mol. Biol. 204:973-94). Methods for measuring 6-phosphofructokinase activity are well known in the art (see, for example,
Wegener and Krause (2002) Biochem. Soc. Trans. 30:264-70). Phosphofructokinase proteins of the present invention include that in SEQ ID NO:256. . | Phosphoglucose isomerase (EC:5.3.1.9) (PGI) (PFAM Accession No.
PF 00342) 1s ‘a dimeric enzyme that catalyses the reversible isomerization of glucose- oo : 6-phosphate and fructose-6-phosphate. PGI 1s involved in different pathways: in most higher organisms it is involved in glycolysis; in mammals it is involved in - gluconeogenesis; in plants in carbohydrate biosynthesis; in some bacteria it provides a gateway for fructose into the Entner-Doudouroff pathway. The multifunctional protein, PGI, is also known as neuroleukin (a neurotrophic factor that mediates the differentiation of neurons), autocrine motility factor (a tumor-secreted cytokine that regulates cell motility), differentiation and maturation mediator and myofibril-bound serine proteinase inhibitor, and has different roles inside and outside the cell. In the cytoplasm, it catalyses the second step in glycolysis, while outside the cell it serves as a nerve growth factor and cytokine. Methods to measure glucose-6-phosphate
Co isomerase activity are well known in the art (see, for example, Nozue et al. (1996)
DNA Seq. 6:127:35). Phosphoglucose isomerase proteins of the present invention include that in SEQ ID NO:252.
Phosphoglycerate kinase (EC:2.7.2.3) (PGK) (PFAM Accession No.
PF00162) is an enzyme that catalyses the formation of ATP to ADP and vice versa. In . | “the second step of the second phase in glycolysis, 1 3-diphosphoglycerate is converted to3-phosphoglycerate, forming one molecule of ATP. If the reverse were to occur, one molecule of ADP would be formed. This reaction is essential in most cells for the : generation of ATP in aerobes, for fermentation in anaerobes and for carbon fixation in = plants. PGK is found in all living organisms and its sequence has been highly conserved throughout evolution. The enzyme exists as a monomer containing two oo nearly equal-sized domains that correspond to the N- and C-termini of the protein (the last 15 C-terminal residues loop back into the N-terminal domain). 3- N phosphoglycerate (3-PG) binds to the N-terminal, while the nucleotide substrates, ) ; 30 MgATP or MgADP, bind to the C-terminal domain of the enzyme. This extended two-domain structure is associated with large-scale ‘hinge-bending' conformational . changes, similar to those found in hexokinase (Kumar et al. (1999) Cell Biochem. ~
Biophys. 31:141-64). At the core of each domain is a 6-stranded parallel B-sheet surrounded by a-helices. Domain 1 has a parallel B-sheet of six strands with an order of 342156, while domain 2 has a parallel B-sheet of six strands with an order of 321456. Analysis of the reversible unfolding of yeast phosphoglycerate kinase leads : } to the conclusion that the two lobes are capable of folding independently, consistent } with the presence of intermediates on the folding pathway with a single domain oo - folded (Yon er al. (1990) Biochimie 72:417-29). Methods to measure : _ phosphoglycerate kinase activity are well known in the art (see, for example, Pal ef al. 3 (2004) Biochim. Biophys. Acta. 1699:277-80) Phosphoglyceate kinase proteins of the oo present invention include that in’ SEQ ID NO:250.
Phosphoglycerate mutase (EC:5.4.2.1) (PGAM) and bisphosphoglycerate mutase (EC:5.4.2.4) (BPGM) are structurally related enzymes that catalyze reactions ) involving the transfer of phospho groups between the three carbon atoms of phosphoglycerate. Both enzymes can catalyze three different reactions with different specificities, the isomerization of 2-phosphoglycerate (2-PGA) to 3-phosphoglycerate : 15 (3-PGA) with 2,3-diphosphoglycerate (2,3-DPG) as the primer of the reaction, the
Co synthesis of 2,3-DPG from 1,3-DPG with 3-PGA as a primer and the degradation of . - 2,3-DPG to 3-PGA (phosphatase EC:3.1.3.13 activity). A number of other proteins oo ~ including, the bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6- bisphosphatase that catalyses both the synthesis and the degradation of fructose-2,6- : | bisphosphate and bacterial a-ribazole-5"-phosphate phosphatase, which is involved in
Co cobalamin biosynthesis, belong to this family. Methods to measure the catalytic Co activity of these enzymes are well known in the art (see, for example, Rigden ef al. oo (2001) Protein Sci. 10:183 5-46). Phosphoglycerate mutase family (PFAM Accession : : a No. PF00300) proteins of the present invention include that in SEQ ID NO:244.
Pyruvate kinase (EC:2.7.1.40) (PK) catalyses the final step in glycolysis, the conversion of phosphoenolpyruvate to pyruvate with concomitant phosphorylation of
ADP to ATP. Most bacteria and lower eukaryotes have one form of this enzyme, except in certain bacteria, such as Escherichia coli, that have two isozymes. All isozymes appear to be tetramers of identical subunits of about 500 residues. PK helps . 30 control the rate of glycolysis, along with phosphofructokinase and hexokinase.
Methods to measure pyruvate kinase activity are well known in the art (see, for : example, Boles et al. (1997) J. Bacteriol. 179:2987-93). Pyruvate kinase, alpha/beta N : domain (PFAM Accession No. PF02887) proteins of the present invention include :
that in SEQ ID NO:258. Pyruvate kinase, barrel domain (PFAM Accession No.
PF00224) proteins of the present invention include that in SEQ ID NO:258. : A number of enzymes require thiamine pyrophosphate (TPP) (vitamin B1) as ] a cofactor. It has been shown that some of these enzymes are structurally related (Green (1989) FEBS Lett. 246:1-5). The thiamine pyrophosphate enzyme, central . domain (PFAM Accession No. PF00205) contains a 2-fold Rossman fold. SEQ ID
NO:232 has a thiamine pyrophosphate enzyme, central domain, as well as a thiamine oo - pyrophosphate enzyme, N-terminal TPP binding domain (PFAM Accession No.
PF02776). : So
The enzyme 3 beta-hydroxysteroid dehydrogenase/S-ene-4-ene isomerase (3 beta-HSD) catalyzes the oxidation and isomerization of 5-ene-3 beta- hydroxypregnene and 5-ene-hydroxyandrostene steroid precursors into the corresponding 4-ene-ketosteroids necessary for the formation of all classes of steroid hormones. 3-beta hydroxysteroid dehydrogenase/isomerase family (PFAM Accession
No. PF01073) proteins of the present invention include that in SEQ ID NO:216. :
Methods to measure 3-beta-hydroxy-delta5-steroid dehydrogenase activity are well : known in the art (see, for example, Moisan er al. (1999) J Clin Endocrinol Metab. 84:4410-25).
Dihydroxyacetone kinase (glycerone kinase) (EC:2.7.1.29) catalyses the phosphorylation of glycerone in the presence of ATP to glycerone phosphate in the ! glycerol utilization pathway. The Dak] domain (PFAM Accession No. PF02733) is the kinase domain of the dihydroxyacetone kinase family. Dak1 domain proteins of the present invention include that in SEQ ID NO:190. The DAK?2 domain (PFAM - Accession No. PF02734) is the predicted phosphatase domain of the dihydroxyacetone kinase family. Dak2 domain proteins of the present invention : include that in SEQ ID NO:192 Methods to measure glycerone Kinase activity are } well known in the art (see, for example, Sellinger and Miller (1957) Fed. Proc. 16:245-246). : : a
The biosynthesis of disaccharides, oligosaccharides and polysaccharides =~ = ) 30 involves the action of hundreds of different glycosyltransferases. These are enzymes that catalyze the transfer of sugar moieties from activated donor molecules to specific - acceptor molecules, forming glycosidic bonds. A classification of glycosyltransferases ~ using nucleotide diphospho-sugar, nucleotide monophospho-sugar and sugar ) : phosphates (EC:2.4.1) and related proteins into distinct sequence based families has 8 been described. The same three-dimensional fold is expected to occur within each of the families. Because 3-D structures are better conserved than sequences, several of oo the families defined on the basis of sequence similarities may have similar 3-D
SN structures and therefore form ‘clans’. Members of the Glycosyl transferases group 1 oo family (PFAM Accession No. PF00534) transfer UDP, ADP, GDP or CMP linked - sugars. The bacterial enzymes are involved in various biosynthetic processes that . include exopolysaccharide biosynthesis, lipopolysaccharide core biosynthesis and the - biosynthesis of the slime polysaccharide colanic acid. Glycosyl transferase group 1 proteins of the present invention include that in SEQ ID NO:328.
Kinase proteins of the present invention include those in SEQ ID NOS:224, 230, 190, 192, 186, and 238. Hydrolysis proteins of the present invention include SE those in SEQ ID NOS:10, 46, 50, 60, 100, 108, 196, 198, 200, 202, 204, 210, 212, 214,216, 218, 220, 222, 226, 276, 292, 342, 344, 346, 356, 358, 362, and 364.
Proteins of the present invention involved in metabolism include those in SEQ ID
NOS:60, 186, 200, 202, 216, 218, 228, 342, 344, 346, 356, and 358. Glycolysis proteins of the present invention include those in SEQ ID NOS:242, 244, 246, 248, 250, 252, 254, 256, 258, 260. Glycogen metabolism proteins of the present invention include those in SEQ ID NOS:240, 324, 326, 328, 330, and 332. Proteins of the present invention involved in EPS metabolism include those in SEQ ID NOS:348, 350,352, and 354.
Methods are known in the art for cloning and expressing carbohydrate oo utilization-related proteins in microorganisms and plants, and for assessing function : of those proteins (see, for example, de Vos (1996) Antonie van Leeuwenhoek 70:223— - 242; Yeo er al. (2000) Mol. Cells 10:263-268; Goddijn ef al. (1997) Plant Physiol. 113:181-190). Function for primary and secondary transport system-related proteins may be assessed, for example, by enzyme assays, fermentation assays, and transport . assays. Function for group translocation system-related proteins may be assessed, for oo example, by sugar phosphorylation assays. See, for example, Russell ef al. (Russell ef : al. (1 992) J. Biol. Chem. 267:4631—4637), where genes from a primary transport ; 30 system (msm) in Streptococcus mutans are identified and expressed in E. coli; Leong-
Morgenthaler ef al. (Leong-Morgenthaler ef al. (1991) J. Bacteriol. 173:1951-1957, : - where two genes from a secondary transport system (lactose) from Lactobacillus ~ bulgaricus were cloned and expressed in E. coli; Vaughan ef al. (Vaughan ef al. | : : : (1996) Appl. Env. Microbiol. 62:1574-1582), where a secondary transport system
(lacS) gene from Leuconostoc lactis was cloned and expressed in E. coli: de Vos et al. (de Vos et al. (1990) J. Biol. Chem. 265:22554-22560), where two PTS system genes
Jrom Lactococcus lactis were identified, cloned and expressed in E.coli and ) Lactobacillus lactis; Sato et al. (Sato et al. (1989) J. Bacteriol. 171 :263-271), where the scr4 gene from Streptococcus mutans was cloned into E. coli and found to exhibit - sucrose PTS activity; Alpert and Chassy (Alpert and Chassy (1990) J. Biol. Chem. 265:22561-22568), where the gene coding for the lactose-specific Enzyme II of :
Lactobacillus casei was cloned and expressed in E. coli; Boyd er al. (Boyd et al. (1994) Infect. Immun. 62:1156-1165), where the genes that encode HPr and Enzyme I of the PTS transport system of Streptococcus mutans were cloned and expressed in E. coli; Garg et al. (Garg et al. (2002) Proc. Natl. Acad. Sci. USA 99:15898-15903), where the overexpression of E. coli trehalose biosynthetic genes ofs4 and otsB led to - increased tolerance of the transgenic plants to abiotic stress, and enhanced productivity; and Grinius and Goldberg (Grinius and Goldberg (1994) J. Biol. Chem. 269:29998-30004), where a multidrug resistance protein was expressed and demonstrated to function as a drug pump. : :
Expression of one or more carbohydrate utilization-related proteins may allow for an organism to have a modified ability to accumulate a carbohydrate in the cytoplasm of a cell. For example, introducing or overexpressing an enzyme involved in sugar catabolism without expressing a relevant transport protein may lead to an ot accumulation of that carbohydrate in the cytoplasm. Alternatively, introduction or ’ So overexpression of a carbohydrate transport-related protein may lead to enhanced transport of the carbohydrate into the external environment. Methods are known in the ; art for introducing or expressing carbohydrate-related genes in organisms. . 025 Accumulation of a carbohydrate in a cell may be assessed, for example, by | oo " E 7 3 oo : CL chromatographic methods Or enzyme assays. See, for example, Chaillou er al. (1998) iJ Bacteriol. 180:4011-8014 and Goddijn et al. (1997) supra.
Expression of one or more carbohydrate utilization-related proteins may allow 3 for an organism to have a modified ability to utilize or produce a carbohydrate as an . 30 energy source. Methods are known in the art for cloning and expressing carbohydrate utilization-related proteins in organisms, and for assessing function of those proteins = (see, for example, de Vos (1996) Antonie van Leeuwenhoek 70:223-242; Hugenholz ~ : et al. (2002) Antonie van Leeuwenhoek 82:217-235). For example, the genes for : lactose metabolism may be introduced into a bacterium to improve the utilization of oO lactose, and to produce a product more acceptable to lactose-intolerant people (Hugenholz et al. (2002) supra). Further modifications may be made in these modified bacteria, such as blocking glucose metabolism so that glucose is not i _ -degraded, but is released from the cell into the medium, thereby providing natural | ) *
S sweetness. See, for example (Hugenholz ef al. (2002) supra). Altematively, the genes - for galactose metabolism -as well as the gene for a-phosphoglucomutase may be | IR introduced, to improve the galactose-fermenting capability of the microorganism, ~ thereby aiding in preventing the consumption of high levels of galactose, which could : cause health problems (Hugenholz et al. (2002) supra; Hirasuka and Li (1992) J. 10 Stud. Alcohol 62:397-402). One gene associated with galactose metabolism is o- galactosidase, the expression of which may be useful for removing raffinose-type sugars from fermented products, since monogastric animals cannot degrade them (Hugenholz et al (2002) supra). Expression of the bacterial gene for mannitol-1- phosphate dehydrogenase (mtID) in tobacco plants successfully resulted in the 15 synthesis and accumulation of mannitol (Tarczynski ef al. (1992) Proc. Natl Acad.
Sci. USA 89:2600-2604). -
Function of the various carbohydrate-related proteins may be assessed, for example, by microbial growth assays, transport assays, enzyme assays, or analysis by chromatography methods and NMR. See, for example, Djordjevic et al. (2001) J. 20 Bacteriol. 183:3224-3236; Chaillou er al. (1998) J. Bacteriol. 180:4011—4014; and : Tarczynski et al. (1992) supra. oo
Generally, permeases, membrane-associated enzymes, and regulators such as transcriptional repressors or antiterminators may need to be expressed in the cell for . optimal utilization of a carbohydrate. The function of transcriptional antiterminators ) ) 25 may be assayed by antitermination activity in a reporter system (see, for example, EERE
I oe | Alpert and Siebers (1997) J. Bacteriol. 179:1555-1562). The function of repressors } such as lacR may be assessed by enzyme activity or growth assays (see, for example, van Rooijen et al. (1993) Protein Eng. 6:201-206; van Rooijen and de Vos (1990) J. oY
Biol. Chem. 265:18499-18503). ; 30 | Bacterial regulatory proteins of the present invention include those in SEQ ID
NOS:8, 38, 98, 104, 118, 150, 178, 180, 182, 184, 188, 266, 290, 304, and 336. : ) Bacterial regulatory protein, lacl family (PFAM Accession No. PF00356) 3 proteins of the present invention include those in SEQ ID NOS:38, 98 and 182. :
Bacterial regulatory protein, gntR family (PFAM Accession No. PF00392) of the present invention include that in SEQ ID NO:106. Bacterial regulatory helix-turn- } helix proteins, AraC family (PFAM Accession No. PF001 65) proteins of the present So ) invention include that in SEQ ID NO:118. Bacterial regulatory proteins, deoR family : (PFAM Accession No. PF00455) proteins of the present invention include that in
RE SEQ ID NO:188 and 336. oo oo
The PRD domain (for PTS Regulation Domain) (PFAM Accession No. :
PF00874), is the phosphorylatable regulatory domain found in bacterial transcriptional antiterminator of the BglG family as well as in activators such as MtIR and LevR. The PRD domain is phosphorylated on a conserved histidine residue. PRD- containing proteins are involved in the regulation of catabolic operons in Gram+ and
Gram- bacteria and are often characterized by a short N-terminal effector domain that binds to either RNA (CAT-RBD for antiterminators (CAT_RBD)) or DNA (for : : activators), and a duplicated PRD module which is phosphorylated on conserved
I5 histidines by the sugar phosphotransferase system (PTS) in response to the availability of carbon source. The phosphorylations are thought to modify the stability of the dimeric proteins and thereby the RNA- or DNA-binding activity of the effector ~ domain. This is a family of bacterial proteins related to the Escherichia coli bglG protein. E. coli bglG protein mediates the positive regulation of the B-glucoside (bgl) operon by functioning as a transcriptional antiterminator (Houman ef al. (1990) Cell ’ 62:1 153-63). BglG is an RNA-binding protein that recognizes a specific sequence located just upstream of two termination sites within the operon. The activity of bglG
SE 1S suppressed by its phosphorylation by bglF (EIl-bgl), the permease from the B- oo | ~ glucoside PTS system (Amster-Choder and Wright (1990) Science 249:540-2). BglG : . 25 is highly similar to other proteins, which also probably act as transcriptional 0 antiterminators. PRD domain-containing proteins of the present invention include those in SEQ ID NOS:8 and 266.
The AraC-like ligand binding domain family (PFAM Accession No. PF02311) 3 represents the arabinose-binding and dimerization domain of the bacterial gene . 30 regulatory protein AraC. The domain is found in conjunction with the helix-turn-helix : (HTH) DNA-binding motif HTH AraC. This domain is distantly related to the Cupin - domain. AraC-like ligand binding domain family proteins of the present invention ~ include that in SEQ ID NO:118. ;
~ The CAT RNA binding domain (PFAM Accession No. PF03123) is found at : . the amino terminus of a family of transcriptional antiterminator proteins. This domain - has been called the CAT (Co-AntiTerminator) domain. This domain forms a dimer in - | the known structure. Transcriptional antiterminators of the BglG/SacY family are . 5 regulatory proteins that mediate the induction of sugar metabolizing operons in Gram- i positive and Gram-negative bacteria. Upon activation, these proteins bind to specific . : targets in nascent mRNAs, thereby preventing abortive dissociation of the RNA . polymerase from the DNA template (Declerck ef al. (1999) J. Mol. Biol. 294:389- 402). CAT RNA binding domain proteins of the present invention include those in
SEQ ID NOS:8 and 266. | oo oo
SEQ ID NO:184 is a member of the HPr Serine kinase N terminus family (PFAM Accession No. PF02603), as well as a member of the HPr Serine kinase C terminus family (PFAM Accession No. PF07475). The N terminus family represents tiie N-terminal region of Hpr Serine/threonine kinase PtsK. The C terminus family represents the C terminal kinase domain of Hpr Serine/threonine kinase PtsK. This oo kinase is the sensor in a multicomponent phosphorelay system in control of carbon catabolic repression in bacteria (Marquez ef al. (2002) Proc. Natl. Acad. Sci. U.S.A. 99:3458-63). This kinase is unusual in that it recognizes the tertiary structure of its target and is a member of a novel family unrelated to any previously described protein = phosphorylating enzymes. X-ray analysis of the full-length crystalline enzyme from
Staphylococcus xylosus at a resolution of 1.95 A shows the enzyme to consist of two clearly separated domains that are assembled in a hexameric structure resembling a ~ three-bladed propeller. The blades are formed by two N-terminal domains each, and | So ‘the compact central hub assembles the C-terminal kinase domains (Reizer etal. So a (1998) Mol. Microbiol. 27:1157-69). "
The sequences of the present invention may also modify the ability of an - organism to alter the flavor or texture of a food product. Modification of glucose metabolism to produce alternative sugars is one approach that may lead to altered : flavor or textural characteristics. Disruption of the lactate dehydrogenase gene with . 30 the concomitant expression of genes from the mannitol or sorbitol operons results in the production of mannitol and sorbitol (Hugenholz er al. (2002) supra). Diacetyl . ) production during fermentation results in a butter aroma, which can be enhanced by ~ either disruption of lactate dehydrogenase or overexpression of NADH oxidase in -.
combination with disruption of a-acetolactate decarboxylase (Hugenholz and : + Kleerebezem, (1999) supra; Hugenholtz er al. (2000) Appl. Environ. Microbiol. 66:4112-4114) Alternatively, overproduction of a-acetolactate synthase or : ; - acetohydroxy acid synthase with disruption of a-acetolactate decarboxylase has resulted in increased diacetyl production (Swindell et al. (1996) Appl. Environ.
Microbiol. 62:2641-2643; Platteeuw et al. (1995) Appl. Environ. Microbiol. 61:3967- 3971). Overexpression of alanine dehydrogenase results in the production of alanine : - instead of lactic acid, providing a taste-enhancer.and sweetener in fermented foods (Hols er al. (1999) Nat. Biotechnol. 17:588-592). :
Methods for modifying the ability of an organism to produce a modified carbohydrate are also encompassed, comprising introducing at least one nucleotide : sequence of the present invention into an organism. Methods for producing modified _ . carbohydrates are also encompassed, and comprise contacting a carbohydrate to be : modified with a polypeptide of the present invention. Methods are known in the art for producing modified carbohydrates. See, for example Kim er al (2001) Biotechnol.
Prog. 17:208-210. oe
The sequences of the current invention may also modify the ability of an : organism to survive in a food system or the gastrointestinal tract of a mammal, or modify an organism’s stability and survival during food processing and storage. For : example, increased production of trehalose may result in prolonged freshness and taste of a fermented product (see, for example, www.nutracells.com). Trehalose also - may aid in the prevention of diseases that result from protein aggregation or Co oo
Cee pathological conformations of proteins, such as Creutzfeld-Jacob disease. In plants, : Co
Co accumulation of trehalose leads to protection against environmental stresses such as Co drought, salt, and cold (see, for example, Jang et al. (2003) Plant Physiol. 131:516— 524; Penna (2003) Trends Plant Sci. 8:35 5-357; Garg er al. (2002) Proc. Natl. Acad.
Science 99:15898-15903; Yeo et al. (2000) supra). In addition, plants have been transformed with fructosyltransferase genes, which enabled the plant to accumulate ‘ fructans to a high level (van der Meer et al. (1994) Plant Cell 6:561-570). In addition - 30 to having a role as a carbohydrate reserve, fructans may also provide tolerance to dry : and cold conditions (Pontis and del Campillo (1985) “Fructans” in Biochemistry of ) Storage Carbohydrates in Green Plants, Day and Dixon, eds. (London: Academic .
Press), pp. 810-816; Pilon-Smits ef al. (1995) Plant Physiol. 107:125-130). The
: bacterial gene mannitol-1-phosphate dehydrogenase has also been expressed in plants, resulting in the production of mannitol, which is thought to confer beneficial traits including osmoregulation and neutralization of hydroxyl radicals (Tarczynski et al. ; (1992) supra). .
IB) The multidrug transporter sequences of the invention may allow an organism ) to survive contact with an antimicrobial polypeptide or other toxin. This may be due . to an increased ability to transport a drug or toxin out of the cell. :
Variants of these nucleotide sequences are also encompassed, such as those : ) that retain or modify the ability to transport a carbohydrate or toxin into or out of a cell, and those that retain or modify the ability to accumulate or utilize a carbohydrate.
Methods for making and testing variants of carbohydrate utilization-related or multidrug transporter proteins are well known in the art. See, for example, Poolman er .al. (Poolman et al. (1996) Mol. Microbiol. 19:911-911), where variants of secondary transport system proteins (mellibiose and lactose) with altered substrate specificities : 15 were isolated or constructed and tested. In these mutants, sugar transport is uncoupled from cation symport. See also, for example, Djorovevic ef al. (2001) supra, where © mutant HPr proteins were constructed with altered regulatory activity; and Adams ef al. (Adams et al. (1994) J. Biol. Chem. 269:5666—5672), where cold-sensitive variants of the B-galactosidase gene from Lactobacillus delbriieckii subsp. bulgaricus were generated and characterized. These mutated genes had a reduced Vmax at low temperatures and therefore may be useful in preventing the acidification of fermented oo EB } .~ .~ products during cold storage (Mainzer el al. (1990) “Pathway engineeringof ~~ - | Lactobacillus bulgaricus for improved yoghurt,” in Yoghurt: Nutritional and Health oo : : . Properties, Chandan, ed., (National Yoghurt Association, Virginia, US), pp. 41-55.
See, also, Bettenbrock er al. (Bettenbrock ef al. (1999) J. Bacteriol. 181:225-230), where mutants with modified galactose-specified PTS genes were isolated. See also, van Rooijen er al. (1990) supra, where variants of the lacR repressor were isolated that had no effect on activity. See also Kroetz ef al., where polymorphism of the Cl human MDR1 gene was analyzed (Kroetz et al. (2003) Pharmacogenetics 13:481— . - 30 94), and Mitomo et al., where variants of the ABC transporter ABCG2 were analyzed (Mitomo et al. (2003) Biochem. J. 373:767-74). ) Any of the above modifications may be combined with other metabolic BN alterations that have been engineered or suggested in lactic acid bacteria. These :
include, B-vitamin production, such as folate (B11), riboflavin (B2), or cobalamin (B12), the production of polyols, or low-calorie sugars, that could replace sucrose, lactose, glucose, or fructose as sweeteners, the production of tagatose, another sucrose - replacement, the production of various exopolysaccharides, blocking glucose } metabolism to provide a natural sweetening effect, reduced production of galactose, } production of foods with lower levels of a-galactosides such as stachyose and raffinose, and increased production of trehalose, which has preserving properties for - foodstuffs and is potentially involved in disease prevention (Hugenholz et al. (2002) supra; van Roojen et al. (1991) J. Biol. Chem. 266:7176-7178).
SE 10 Methods are also provided for eliminating or modifying undesirable carbohydrates from a food or chemical product. The methods comprise contacting the product with a purified polypeptide of the present invention. Methods to assay for the : elimination or modification of carbohydrates are well known in the art. :
Table 1. Carbohydrate Utilization Proteins of the Present Invention :
ORF | SEQID
IDENTITY/FUNCTION COG # NO: : PTS system mannose-specific factor 452 1,2 3444 [IAB \ ~ Phosphotransferase system (PTS) : | 877 | 3,4 lichenan-specific enzyme IIA 1447 So component : . Beta-glucoside specific transport : 5,6 1 £ P P oo 2190 - protein 1574 EE Phospho-beta-glucosidase 2723
Beta-glucoside permease IIABC i : 1707 11,12 1263 : component : - PTS system, beta-glucosides-specific 725 13, 14 1263 :
IIABC component i Phosphotransferase system (PTS) 491] 15, 16 oo } 1455 - E protein, lichenan-specific enzyme 11C N
SEQ ID
IDENTITY/FUNCTION : COG
NO: :
EN I
1369 | 17,18 ~ Phosphotransferase system enzyme II 1455 : - BE Phosphotransferase system IIA 1684 19,20 2893 ‘ component }
PTS system enzyme [IBC component ) 146 21,22 } 1762 : (galactitol/fructose-specific) 23,24 PTS cellobiose-specific component IC | 1455 25,26 PTS cellobiose-specific enzyme IIC 1455 | }
Cellobiose-specific PTS system IC 884 | 27,28 1455 component
PTS system, cellobiose-specific 618 29,30 enzyme [IC
Phosphotransferase system (PTS) 31,32 1263 arbutin-like enzyme [IBC component
Sucrose-specific PTS system IBC : 1705 33,34 1263 component . | 1777 | 35,36. |. PTSsystemprotein oo : ABC transporter substrate-binding : 502 39, 40 1653 protein
ABC transporter membrane-spanning 503 41,42 “N75 permease - sugar transporter
ABC transporter membrane-spanning 504 43, 44 395 ] permease - sugar transport protein = 45, 46 Sucrose-6-phosphate hydrolase
Multiple sugar-binding transport ATP- 506 47,48 ] P & 8 P 3839 . . binding protein -
. WO. 2005/084411 PCT/US2005/007594
SEQ ID 1
IDENTITY/FUNCTION COG
NO: aE
Ribose ABC transporter(ribose-binding 1481 51,52 ] 1879 : . protein) : : 1482 | 53,54 Ribose ABC transporter (permease)
Ribose ABC transporter ATP binding 1483 | 55,56 . 1129 5 oo protein - 1484 | 57.58 Ribose permease (RbsD) 1485 | 59,60 Ribokinase (RbsK)
Maltose ABC transporter permease - 1864 | 61,62 } 3833 protein : Maltose ABC transporter permease 1865 63, 64 } : 1175 . protein
Maltose ABC transporter substrate 1866 65, 66 2182 : binding protein
Multiple sugar-binding transport ATP- 1867 | 67,68 pe sue sense 3839 binding protein 1944 | 69,70 Sugar ABC transporter protein : : Sugar ABC transporter permease : CL 1945 | 71,72 Co oo 4603 protein
Sugar ABC transporter permease 1946 73,74 Ca 1079 iN protein no : 81, 82 Drug-efflux transporter ’ 1446 85, 86 Drug-export protein 1471 | 87,88 Efflux protein | )
SEQ ID
IDENTITY/FUNCTION COG
NO: [ee 1621 91,92 Efflux transporter protein ’ ) : 1853 93,94 Drug-efflux transporter protein } 1917 95, 96 Polysaccharide transporter r 97,98 Sucrose operon regulatory protein 99, 100 Sucrose-6-phosphate hydrolase 101, 102 Phosphotransferase system enzyme II : Beta-glucoside-specific PTS system -1012 | 103, 104 1263 : [IABC component . oo -Trehalose operon transcriptional : 1013 105, 106 2188 repressor 1014 107, 108 Dextran glucosidase
ABC transporter ATP-binding protein - : 1439 109, 110 3839 multiple sugar Transport . Multiple sugar-binding transport CL 1440 111,112 P ¥ & P E 395. } oo
Set system permease protein - ABC transporter membrane-spanning oo 1441 113,114 1175 permease - Multiple sugars
Multiple sugar-binding protein So : 1442 115,116 P 8 Ep 1653 precursor : p
Raffinose operon transcriptional 1443 117, 118 2207
Cb regulatory protein Cele 119, 120 Carbohydrate-utilization-related 121, 122 ABC transporter bacteriocin : 123, 124 ABC transporter 131 | 125,126 ABC transporter | i 32 | 127,128 ABC transporter
ORF | SEQID
IDENTITY/FUNCTION COG : # NO: 1357 129, 130 ABC transporter ) 1358 131,132 ABC transporter 1681 137, 138 Carbohydrate-utilization-related 1793 | 139, 140 Carbohydrate-utilization-related hE 1794 | 141, 142 Carbohydrate-utilization-related 11913 | 151,152 ABC transporter | : 153,154 | ABC transporter , Los | ss, 156 Carbohydrate-utilization-related ] 157, 158 Carbohydrate-utilization-related oo 1939 159, 160 ABC transporter
Mannose-specific phosphotransferase 453 161, 162 ~ system component . PTS system mannose-specific factor 454 163, 164 [IAB oo
PTS system mannose-specific, factor 8 455 165, 166 3715 [IC - PTS system mannose-specific factor 456 167, 168 3716
IID
169, 170 PTS system enzyme II protein [| - -
SEQ ID
IDENTITY/FUNCTION COG
NO: : :
Phosphotransferase system sugar- 879 171,172 1455 . specific EII component Cw y } ) PTS system, beta-glucoside-specific . 1575 173,174 : 1263 : : enzyme II, ABC component : 1433 | 189, 190 dihydroxyacetone kinase 1434 | 191, 192 dihydroxyacetone kinase : 1436 193, 194 glycerol uptake facilitator oo
CL 1437 | 195,19 gtfAIl | oo 1461 | 207, 208 transcriptional regulator a 1467 211,212 beta-galactosidase 1468 | 213,214 beta-galactosidase -
ORF | SEQID
E me] 0 A [
CRE wn BE
EE En
ERE EE
SEQID
IDENTITY/FUNCTION COG
NO: 1641 | 261,262 glycerol 3-phosphate ABC transporter | : ‘Mannose; PTS system mannose- : 452 263,264 | 3444 i specific factor [IAB beta-glucoside; transcription oo 1479 265, 266 } 371 . antiterminator : beta-glucoside; PTS system, beta- 725 267,268 } 1263 oo glucosides-specific IABC component
Cellobiose; phosphotransferase system : 1369 269,270 1455 enzyme lI
Cellobiose; PTS cellobiose-specific - 227 271,272 1455 : : component 11 sugar transporter; ABC transporter 502 273,274 1653 substrate-binding protein rbsA; ribose ABC transporter ATP Co To : 1483 277,278 } Co 1129 binding protein oo 1484 | 279,280 ribose permease RbsD 281,282 multidrug transporter 283,284 multidrug transporter . 1471 285, 286 multidrug transporter , 1853 | 287,288 multidrug transporter treB; beta-glucoside; beta-glucoside- 1012 289, 290 i 1263 specific PTS system [IABC component Co
SEQ ID
IDENTITY/FUNCTION COG
NO: 1358 | 299,300 ABC transporter 1838 | 301,302 ABC transporter - transcriptional regulator (TetR/AcrR 1840 303,304 : 1309 family) 1913 | 305,306 ABC transporter 1938 307,308 Carbohydrate-utilization related protein : 309,310 multidrug transporter | ~ 311,312 multidrug transporter : 313,314 multidrug transporter 315,316 multidrug transporter 319,320 ABC multidrug transporter 1854 | 321,322 multidrug transporter lucose-1-phosphate 681 323,324 | & phosp : 448 © ' adenylyltransferase glgC :
Co lucose-1-phosphate
Co 682 325,326 8 PROSP 448 adenylyltransferase glgD ~ n 327,328 glycogen synthase 7 329,330 glycogen phosphorylase
E 331,332 amylopullulanase - | 1465 335,336 transcription repressor of galactosidase le ; Multiple sugar-binding ABC- : 1645 339, 340 3839 . transporter :
SEQID
IDENTITY/FUNCTION COG
NO: 341, 342 glycosyltransferase 1216 } 343, 344 galactosyl transferase ‘ : { 1733 345, 346 phospho-glucosyltransferase epsE 2148 ] 1738 355, 356 GTP-binding protein Hf1X 2262 : 1869 | 361,362 beta-phosphoglucomutase 1870 363, 364 maltose phosphorylase : 1554
The following examples are offered by way of illustration and not by way of bo limitation. B
Example 1. Gapped BlastP Results for amino acid sequences : :
A Gapped BlastP sequence alignment showed that SEQ [ID NO:2 (144 amino ; acids) has about 61% identity from amino acids 1-140 with a protein from Listeria innocua that is homologous to a PTS system mannose-specific factor IAB (Accession Nos. NP_469488.1; NC_003212), about 60% identity from amino acids 1- : 140 with a protein from Listeria monocytogenes that is homologous to a PTS system ; - mannose-specific factor [IAB (Accession Nos. NP_463629.1; NC_003210), about - 63% identity from amino acids 1-139 with a protein from Clostridium acetobutylicum that is a mannose-specific phosphotransferase system component [IAB (Accession .
Nos. NP_149230.1; NC_001988), about 62% identity from amino acids 1-139 with a : protein from Clostridium perfringens that is a PTS system protein (Accession Nos. ;
NP_561737.1; NC_003366), and about 50% identity from amino acids 2-141 with a ’
+ protein from Streptococcus pyogenes that is a mannose-specific phosphotransferase system component IIAB (Accession Nos. NP_269761.1; NC_002737).
A Gapped BlastP sequence alignment showed that SEQ ID NO:4 (123 amino ] acids) has about 60% identity from amino acids 20-109 with a protein from Listeria : innocua that is homologous to a phosphotransferase system (PTS) lichenan-specific - enzyme IIA component (Accession Nos. NP_471165.1; NC_003212), about 57% identity from amino acids 20-110 with a protein from Listeria innocua that is - homologous to a cellobiose phosphotransferase enzyme IIA component (Accession
Nos. NP_472161.1; NC_003212), about 46% identity from amino acids 1-112 with a protein from Lactococcus lactis subsp. lactis that is a cellobiose-specific PTS system [IA component (EC 2.7.1.69) (Accession Nos. NP 266570. 1; NC 002662), about i 44% identity from amino acids 9-112 with a protein from Bacillus halodurans that is . a PTS system, cellobiose-specific enzyme IIA component (Accession Nos. :
NP_241776.1; NC_002570), and about 51% identity from amino acids 16-112 with a protein from Streptococcus pyogenes that is homologous to a PTS enzyme 111 (Accession Nos. NP_607437.1; NC_003485). :
A Gapped BlastP sequence alignment showed that SEQ ID NO:6 (161 amino acids) has about 53% identity from amino acids 6-143 with a protein from
Enterococcus faecium that is a beta-glucoside specific transport protein (BglS) (Accession Nos. gblAAD28228.1; AF121254), about 48% identity from amino acids ) 13-159 with a protein from Streptococcus pneumoniae that is a PTS system, IIABC : component (Accession Nos. NP_345256.1; NC_003028), about 48% identity from amino acids 13-159 with a protein from Streptococcus pneumoniae that is a PTS - glucose-specific enzyme IIABC component (Accession Nos. NP_358262.1; : NC_003098), about 46% identity from amino acids 13-159 with a protein from )
Streptococcus pyogenes that is homologous to a PTS system, enzyme IIA component (Accession Nos. NP_608025.1; NC_003485), and about 46% identity from amino - acids 13-159 with a protein from Streptococcus pyogenes that is homologous to a PTS 2 system, enzyme IIA component (Accession Nos. NP_269950.1; NC_002737).
A Gapped BlastP sequence alignment showed that SEQ ID NO:8 (291 amino acids) has about 36% identity from amino acids 11-282 with a protein from Bacillus : . subtilis that is a transcription antiterminator (licT) (Accession No. : sp|P39805[LICT_BACSU), about 36% identity from amino acids 11-282 with a iy protein from Bacillus subtilis that is a transcriptional antiterminator (BglG family) -
(Accession Nos. NP_391787.1; NC_000964), about 37% identity from amino acids 11-282 with a protein from Escherichia coli that is involved in positive regulation of the bgl operon (Accession Nos. NP_418179.1; NC_000913), about 33% identity from . amino acids 11-282 with a protein from Erwinia chrysanthemi that is a beta-glucoside ] operon antiterminator (Accession No. sp|P26211|]ARBG_ERWCH), and about 34% ” identity from amino acids 9-288 with a protein from Clostridium acetobutylicum that is a transcriptional antiterminator (licT) (Accession Nos. NP_347062.1; NC_003030). :
A Gapped BlastP sequence alignment showed that SEQ ID NO:10 (480 amino oo acids) has about 59% identity from amino acids 8-473 with a protein from Listeria monocytogenes that is homologous to a phospho-beta-glucosidase (Accession Nos.
NP_463849.1; NC_003210), about 589% identity from amino acids 8-473 with a : protein from Listeria innocua that is homologous to a phospho-beta-glucosidase (Accession Nos. NP_469689.1; NC_003212), about 57% identity from amino acids 7- an 473 with a protein from Clostridium acetobutylicum that is a 6-phospho-beta-- glucosidase (NP _347379.1; NC_003030), about 57% identity from amino acids 8-473 with a protein from Clostridium longisporum that is a 6-phospho-beta-glucosidase (Accession No. sp|Q46130]JABGA_CLOLO), and about 55% identity from amino acids 1-473 with a protein from Bacillus subtilis that is a beta-glucosidase (Accession
Nos. NP_391805.1; NC_000964).
A Gapped BlastP sequence alignment showed that SEQ ID NO:12 (625 amino
Co acids) has about 38% identity from amino acids 1-624 with a protein from
Streptococcus pyogenes that 1s a beta-glucoside permease IIABC component (Accession : ~ Nos. NP_268836.1; NC_002737), about 38% identity from amino acids 1-624 with a gE protein from Streptococcus pyogenes that is a beta-glucoside permease [IABC component : (Accession Nos. NP_606826.1; NC_003485), about 38% identity from amino acids 1- 605 with a protein from Streptococcus pneumoniae that is a phosphotransferase system sugar-specific EII component (Accession Nos. NP_358099.1; NC_003098), about 38% . . identity from amino acids 1-605 with a protein from Streptococcus pheumoniae that is } _ a PTS system, beta-glucosides-specific IIABC component (Accession Nos. NP_345091.1;
NC_003028), and about 38% identity from amino acids 1-622 with a protein from
Bacillus halodurans that is a PTS system, beta-glucoside-specific enzyme IIABC : - component (Accession Nos. NP_241162.1; NC_002570). ~
A Gapped BlastP sequence alignment showed that SEQ ID NO:14 (675 amino acids) has about 50% identity from amino acids 17-648 with a protein from
Clostridium acetobutylicum that is a PTS system, beta-glucosides-specific [IABC ~ component (Accession Nos. NP_348035. 1; NC_003030), about 50% identity from ; amino acids 17-656 with a protein from Bacillus halodurans that is a PTS system, \ beta-glucoside-specific enzyme [IABC (Accession Nos. NP_241461.1; NC_002570), about 50% identity from amino acids 17-656 with a protein from Listeria - monocytogenes that is homologous to a PTS system, beta-glucosides specific enzyme
IIABC (Accession Nos. NP_463560.1; NC_003210), about 48% identity from amino E acids 17-654 with a protein from Clostridium longisporum that is a PTS-dependent enzyme II (Accession Nos. gb) AAC05713.1; L49336), and 48% identity from amino 10 . acids 13-654 with a protein from Streptococcus mutans that is a beta-glucoside- specific Ell permease (Accession Nos. gb|AAF89975.1; AF206272).
A Gapped BlastP sequence alignment showed that SEQ ID NO:16 (445 amino acids) has about 41% identity from amino acids 10-443 with a protein from Bacillus ’ _ subtilis that is a phosphotransferase system (PTS) protein, lichenan-specific enzyme
IC component (Accession Nos. NP_391737.1; NC_000964), about 42% identity from amino acids 14-442 with a protein from Bacillus subtilis that is homologous to a
PTS system IIBC component (ywbA) (Accession No. sp|P39584]YWBA BACSU), about 41% identity from amino acids 14-441 with a protein from Bacillus stearothermophilus that is a cellobiose phosphotransferase enzyme ile component + 20 (Accession No. sp|Q45400]PTCC_BACST), about 41% identity from amino acids 12- : ro 441 with a protein from Streptococcus pneumoniae that is a phosphotransferase : system sugar-specific EIl component (Accession Nos. NP_358015.1; NC 003098), and 40% identity from amino acids 12-441 with a protein from Streptococcus ~ pneumoniae that is a PTS system, cellobiose-specific IIC component (Accession Nos. NP_344993.1; NC _003028). : A Gapped BlastP sequence alignment showed that SEQ ID NO:18 (422 amino acids) has about 34% identity from amino acids 9-417 with a protein from Bacillus subtilis that is homologous to a phosphotransferase system enzyme IT (Accession Nos. 1. . NP_391718.1; NC_000964), about 33% identity from amino acids 17-414 with a ) 30 protein from Bacillus subtilis that is a phosphotransferase system (PTS) lichenan- : specific enzyme IIC component (Accession Nos. NP_391737.1; NC_000964), about - 34% identity from amino acids 10-417 with a protein from Bacillus 3 : stearothermophilus that is a cellobiose phosphotransferase enzyme IIC component (Accession No. sp|Q45400]PTCC_BACST), about 33% identity from amino acids 9- or
414 with a protein from Listeria innocua that is homologous to a PTS system, B cellobiose-specific [IC component (Accession Nos. NP_470241.1;NC_003212), and : 31% identity from amino acids 11-415 with a protein from Borrelia burgdorferi that . I isa PTS system, cellobiose-specific IIC component (celB) (Accession Nos. oT
NP_046990.1; NC_001903). - A Gapped BlastP sequence alignment showed that SEQ ID NO:20 (130 amino acids) has about 33% identity from amino acids 3-124 with a protein from Brucella . melitensis that is a phosphotransferase system ITA component (Accession Nos.
NP_540949.1; NC_003317), about 32% identity from amino acids 2-102 with a protein from Lactobacillus curvatus that is an EIIA-mannose protein (Accession Nos. gblAAB04153.1; U28163), about 32% identity from amino acids 3-96 with a protein : from Clostridium perfringens that is homologous to a PTS system protein (Accession
Nos. NP _563545.1; NC_003366), about 25% identity from amino acids 3-123 with a protein from Clostridium perfringens that is homologous to a PTS system protein (Accession Nos. NP_561737.1; NC_003366), and 25% identity from amino acids 3- 123 with a protein from Clostridium acetobutylicum that is a mannose-specific : phosphotransferase system component IIAB (Accession Nos. NP_149230.1;
NC_001988).
A Gapped BlastP sequence ahgnment showed that SEQ ID NO:22 (162 amino acids) has about 38% identity from amino acids 8-159 with a protein from : : ' - Clostridium acetobutylicum that is a PTS system enzyme [IBC component } | Co : (galactitol/fructose-specific) (Accession Nos. NP_349560.1; NC_003030), about 36% identity from amino acids 7-158 with a protein from Streptococcus pneumoniae that is a phosphotransferase system sugar-specific EIl component (Accession Nos.
NP_358156.1; NC_003098), about 36% identity from amino acids 7-158 with a protein from Streptococcus pneumoniae that is homologous to a PTS system IIA component (Accession Nos. NP_345152.1; NC_003028), about 38% identity from amino acids 20-134 with a protein from Streptococcus agalactiae that is a GatA ; protein (Accession Nos. gb|AAG09977.1; AF248038), and 33% identity from amino ) 30 acids 16-159 with a protein from Bacillus halodurans that is a PTS system, galactitol- specific enzyme IIA component (Accession Nos. NP_241058.1; NC_002570). : g A Gapped BlastP sequence alignment showed that SEQ ID NO:24 (466 amino acids) has about 47% identity from amino acids 30-461 with a protein from Co
Clostridium acetobutylicum that is a PTS cellobiose-specific component [IC B
(Accession NP_347026.1; NC_003030), about 45% identity from amino acids 26-465 : with a protein from Lactococcus lactis subsp. lactis that is a cellobiose-specific PTS : system ne component (EC 2.7.1.69) (Accession Nos. NP_266974.1; NC_002662), about 46% identity from amino acids 82-465 with a protein from: Lactococcus lactis subsp. lactis that is a cellobiose-specific PTS system IC component (EC 2.7.1.69) , (Accession Nos. NP_266572.1; NC 002662), about 41% identity from amino acids 34-466 with a protein from Streptococcus pyogenes that is homologous to a PTS . system, enzyme IIC component (Accession Nos. NP_269994.1; NC_002737), and a 40% identity from amino acids 34-466 with a protein from Streptococcus pyogenes that is homologous to a PTS system, enzyme 1IC component (Accession Nos.
NP_608069.1; NC_003485).
A Gapped BlastP sequence alignment showed that SEQ ID NO:26 (428 amino acids) has about 28% identity from amino acids 25-420 with a protein from Listeria : innocua that is homologous to a PTS cellobiose-specific enzyme IC (Accession
NP_472233.1; NC_003212), about 27% identity from amino acids 115-415 with a protein from Lactobacillus casei that is a LacE protein (Accession Nos. : emb|CAB02556.1: 280834), about 26% identity from amino acids 137-425 with a protein from Listeria innocua that is homologous to a PTS system, cellobiose-specific enzyme IC (Accession Nos. NP_472184.1; NC_003212), about 26% identity from amino acids 137-425 with a protein from Listeria monocytogenes that is homologous or h to a PTS system, cellobiose-specific enzyme IIC (Accession Nos. NP_466230.1;
NC_003210), and 26% identity from amino acids 115-415 with a protein from
Lactobacillus casei that is a phosphotransferase system enzyme II (EC 2.7.1.69) J (Accession No. pir||B23697). y :
A Gapped BlastP sequence alignment showed that SEQ ID NO:28 (475 amino : acids) has about 57% identity from amino acids 10-471 with a protein from
Lactococcus lactis subsp. lactis that is a cellobiose-specific PTS system IC component (EC 2.7.1.69) (Accession Nos. NP_266974.1; NC_002662), about 45% z identity from amino acids 71-475 with a protein from Lactococcus lactis subsp. lactis thats a cellobiose-specific PTS system IIC component (EC 2.7.1.69) (Accession oo Nos. NP_266572.1; NC_002662), about 42% identity from amino acids 13-470 with a . protein from Clostridium acetobutylicum that is a PTS cellobiose-specific component [IC (Accession Nos. NP_347026.1; NC_003030), about 41% identity from amino acids 17-468 with a protein from Streptococcus pyogenes that is homologous to a PTS :
system, enzyme [IC component (Accession Nos. NP_269994.1; NC_002737), and 41% identity from amino acids 17-468 with a protein from Streptococcus pyogenes that is homologous to a PTS system, enzyme IIC component (Accession Nos. on } | NP_608069.1] (NC_003485). aE
A Gapped BlastP sequence alignment showed that SEQ ID NO:30 (441 amino
EK acids) has about 46% identity from amino acids 1-428 with a protein from Listeria : innocua that is homologous to a PTS system, cellobiose-specific enzyme 1IC , (Accession Nos. NP_472184.1; NC_003212), about 46% identity from amino acids 1- 428 with a protein from Listeria monocytogenes that is homologous to a PTS system, celiobiose-specific enzyme IIC (Accession Nos. NP_466230.1; NC_003210), about : 39% identity from amino acids 10-427 with a protein from Streptococcus pyogenes that is homologous to a PTS system IIC component (Accession Nos. NP_607435.1;
NC_003485), about 36% identity from amino acids 1-428 with a protein from :
Lactococcus lactis subsp. lactis that is a cellobiose-specific PTS system [IC component (EC 2.7.1.69) (Accession Nos. NP_266330.1; NC_002662), and 31% identity from amino acids 1-421 with a protein from Listeria monocytogenes that is homologous to a cellobiose phosphotransferase enzyme [IC component (Accession
Nos. NP_466206.1; NC_003210).
A Gapped BlastP sequence alignment showed that SEQ ID NO:32 (626 amino
B 20 acids) has about 54% identity from amino acids 1-332 with a protein from Bacillus ~~ subtilis that is a phosphotransferase system (PTS) arbutin-like enzyme IIBC component (Accession Nos. NP_388701.1; NC_000964), about 51% identity from amino acids 2-530 with a protein from Clostridium perfringens that is a PTS arbutin- like enzyme [IBC component (Accession Nos. NP_561112.1; NC_003366), about 52% identity from amino acids 1-533 with a protein from Fusobacterium mortiferum that isa PTS protein EIl (Accession Nos. gb|AAB63014.2; U81185), about 51% : identity from amino acids 1-533 with a protein from Clostridium acetobutylicum that is a MalP protein (Accession Nos. gb|JAAK69555.1; AF290982), and 51% identity * from amino acids 1-533 with a protein from Clostridium acetobutylicum that is a PTS system, arbutin-like HBC component (Accession Nos. NP_347171.1; NC_003030).
A Gapped BlastP sequence alignment showed that SEQ ID NO:34 (663 amino : acids) has about 58% identity from amino acids 1-456 with a protein from ~
Lactococcus lactis subsp. lactis that is a sucrose-specific PTS system [IBC component (EC2.7.1.69) (Accession Nos. NP_267287.1; NC_002662), about 54% identity from
- amino acids 5-471 with a protein from Staphylococcus aureus subsp. aureus that is : homologous to a sucrose phosphotransferase enzyme II (Accession Nos.
NP _373429.1; NC_002745), about 46% identity from amino acids 5-472 with a ‘ protein from Bacillus halodurans that is a PTS system, sucrose phosphotransferase : enzyme [IBC componerit (Accession Nos. NP_244441.1; NC_002570), about 39% ) | identity from amino acids 4-468 with a protein from Salmonella enterica subsp. enterica serovar Typhi that 1s homologous to a PTS system 1IBC component 5 (Accession Nos. NP_457099.1; NC_003198), and 39% identity from amino acids 4- a. 468 with a protein from Salmonella typhimurium that 1s homologous to a phosphotransferase system IIB component (Accession Nos. NP_461505.1;
NC_003197). : oo
A Gapped BlastP sequence alignment showed that SEQ ID NO:36 (665 amino acids) has about 44% identity from amino acids 1-661 with a protein from
Clostridium perfringens that is a PTS system protein (Accession Nos. NP_561500.1; - .
NC_003366), about 46% identity from amino acids 1-657 with a protein from oo Streptococcus pyogenes that is homologous to a fructose-specific enzyme II, PTS system BC component (Accession Nos. NP_269062.1; NC_002737), about 46% _identity from amino acids 1-657 with a protein from Streptococcus pyogenes that is homologous to a fructose-specific enzyme II, PTS system BC component (Accession
Nos. NP_607065.1; NC_003485), about 45% identity from amino acids 1-657 with a protein from Lactococcus lactis subsp. lactis that is a fructose-specific PTS system enzyme IBC component (EC 2.7.1.69) (Accession Nos. NP_267115.1; NC_002662), and 43% identity from amino acids 1-660 with a protein from Bacillus halodurans oo ; that is a PTS system, fructose-specific enzyme [IBC component (Accession Nos.
NP_241694.1; NC_002570). 3
A Gapped BlastP sequence alignment showed that SEQ ID NO:38 (334 amino acids) has about 48% identity from amino acids 4-334 with a protein from
Streptococcus pneumoniae that is a sucrose operon repressor (Scr operon regulatory © protein) (Accession Nos. NP_359213.1; NC_003098), about 46% identity from amino acids 4-334 with a protein from Streptococcus pneumoniae that is a sugar-binding transcriptional regulator in the Lac] family (Accession Nos. NP_346232.1; ) ~ NC_003028), about 35% identity from amino acids 13-332 with a protein from ~
Pediococcus pentosaceus that is a sucrose operon repressor (Scr operon regulatory : protein) (Accession No. sp{P43472|SCRR_PEDPE), about 35% identity from amino
- acids 10-334 with a protein from Bacillus halodurans that is a transcriptional repressor of the ribose operon (Accession Nos. NP_244594.1; NC 002570), and 35% identity from amino acids 10-332 with a protein from Streptococcus pneumoniae that . is a sucrose operon repressor (Accession Nos. NP_346162.1; NC _003028).
A Gapped BlastP sequence alignment showed that SEQ 1D NO:40 (415 amino i acids) has about 50% identity from amino acids 3-415 with a protein from .
Streptococcus pneumoniae that is an ABC transporter substrate-binding protein . (Accession Nos. NP_359212.1; NC_003098), about 27% identity from amino acids 19-389 with a protein from Agrobacterium tumefaciens that is a sugar binding protein (Accession Nos. NP_535638.1; NC_003306), about 25% identity from amino acids : 11-396 with a protein from Nostoc sp. PCC 7120 that is an ABC transporter sugar : binding protein (Accession Nos. NP_488317.1; NC _003272), about 26% identity from amino acids 76-353 with a protein from Streptomyces coelicolor that is : : homologous to a sugar transport sugar binding protein (Accession Nos. : emb|CAB95275.1; AL359779), and 26% identity from amino acids 1-324 with a protein from Listeria innocua that is homologous to a sugar ABC transporter, periplasmic sugar-binding protein (Accession Nos. NP_470104.1; NC_003212).
A Gapped BlastP sequence alignment showed that SEQ ID NO:42 (294 amino acids) has about 56% identity from amino acids 10-285 with a protein from
Streptococcus pneumoniae that is an ABC transporter membrane-spanning permease - sugar transporter (Accession Nos. NP_359211.1; NC_003098), about 38% identity "from amino acids 7-285 with a protein from Listeria monocytogenes that is homologous to a sugar ABC transporter permease protein (Accession Nos. :
NP _464293.1; NC_003210), about 38% identity from amino acids 7-285 with a protein from Listeria innocua that is homologous to a sugar ABC transporter permease protein (Accession Nos. NP_470102.1; NC 003212), about 36% identity from amino acids 12-286 with a protein from Synechocystis sp. PCC 6803 that is a lactose transport system permease protein (LacF) (Accession Nos. NP 440703.1; ;
NC 000911), and 36% identity from amino acids 11-281 with a protein from Xylella . 30 fastidiosa that is a ABC transporter sugar permease (Accession Nos. NP_299726.1; 'NC_002488).
To A Gapped BlastP sequence alignment showed that SEQ ID NO:44 (285 amino ~ acids) has about 59% identity from amino acids 12-285 with a protein from ) :
Streptococcus pneumoniae that is an ABC transporter membrane-spanning permease -
sugar transport protein (Accession Nos. NP_359210.1; NC_003098), about 32% identity from amino acids 30-281 with a protein from Agrobacterium tumefaciens (Accession Nos. NP_356672.1; NC_003063), about 32% identity from amino acids . 30-281 with a protein from Agrobacterium tumefaciens that is an ABC transporter, membrane spanning protein [sugar] (Accession Nos. NP_534455.1; NC_003305), i about 33% identity from amino acids 10-281 with a protein from Listeria monocytogenes that is homologous to a sugar ABC transporter, permease protein - (Accession Nos. NP_463711.1; NC _003210), and 34% identity from amino acids 13- 281 with a protein from Listeria innocua that is homologous to a sugar ABC : 10 transporter, permease protein (Accession Nos. NP_469564.1; NC 003212).
A Gapped BlastP sequence alignment showed that SEQ ID NO:46 (430 amino acids) has about 36% identity from amino acids 2-429 with a protein from
Streptococcus pneumoniae that is a sucrose-6-phosphate hydrolase (Accession Nos. :
NP_359209.1; NC_003098), about 36% identity from amino acids 2-429 with a protein from Streptococcus pneumoniae that is homologous to a sucrose-6-phosphate hydrolase (Accession Nos. NP_346228.1; NC_003028), about 36% identity from amino acids 18-373 with a protein from Thermotoga maritima that is a beta- : fructosidase (Accession Nos. NP_229215.1; NC_000853), about 31% identity from amino acids 21-405 with a protein from Zymomonas mobilis that is a beta- - fructofuranosidase (EC 3.2.1.26) (Accession No. pir|[JU0460), and 35% identity from ’ : amino acids 21-362 with a protein from Escherichia coli that is a sucrose-6 phosphate hydrolase (Accession Nos. NP_311270.1; NC_002695). )
A Gapped BlastP sequence alignment showed that SEQ ID NO:48 (368 amino : acids) has about 65% identity from amino acids 1-366 with a protein from ) . 25 Streptococcus mutans that is a multiple sugar-binding transport ATP-binding protein (msmK) (Accession No. sp|Q00752]MSMK_STRMU), about 65% identity from amino acids 1-366 with a protein from Streptococcus pyogenes that is a multiple sugar-binding ABC transport system (ATP-binding protein) (Accession Nos. :
NP_269942.1; NC_002737), about 66% identity from amino acids 1-367 with a . 30 protein from Streptococcus pneumoniae that is an ABC transporter ATP-binding protein - multiple sugar transport (Accession Nos. NP_359030.1; NC_003098), about - 65% identity from amino acids 1-366 with a protein from Streptococcus pyogenes that ~ is a multiple sugar-binding ABC transport system (ATP-binding protein) (Accession
Nos. NP_608016.1; NC_003485), and 66% identity from amino acids 1-367 with a .
protein from Streptococcus pneumoniae that is a sugar ABC transporter, ATP-binding protein (Accession Nos. NP_346026.1; NC_003028). :
A Gapped BlastP sequence alignment showed that SEQ ID NO:50 (490 amino ] acids) has about 63% identity from amino acids 11-489 with a protein from ]
Streptococcus mutans that is a gtfA protein (Accession No. pir BWSOGM), about - 63% identity from amino acids 1 1-490 with a protein from Streptococcus mutans that . is a sucrose phosphorylase (EC 2.4.1.7) (Accession No. pir||A27626), about 63% “ identity from amino acids 11-489 with a protein from Streptococcus mutans that is a sucrose phosphorylase (sucrose glucosyltransferase) (Accession No. sp|P10249]SUCP_STRMU), about 63% identity from amino acids 11-484 with a protein from Streptococcus pneumoniae that is a dextransucrase (sucrose 6- glucosyltransferase) (Accession Nos. NP_359301.1; NC_003098), and 63% identity : from amino acids 11-484 with a protein from Streptococcus pneumoniae thatisa : sucrose phosphorylase (Accession Nos. NP_346325.1; NC_003028).
A Gapped BlastP sequence alignment showed that SEQ ID NO:52 (328 amino acids) has about 55% identity from amino acids 47-316 with a protein from Bacillus subtilis that is a ribose ABC transporter (ribose-binding protein) (Accession Nos.
NP_391477.1; NC_000964), about 45% identity from amino acids 5-323 with a protein from Lactococcus lactis subsp. lactis that is a ribose ABC transporter substrate binding protein (Accession Nos. NP_267791.1; NC_002662), about 42%
SE identity from amino acids 4-278 with a protein from 7 etragenococcus halophilus that
Co 1s a ribose binding protein (Accession Nos. dbjBAA31869.1; AB009593), about 39% ~ identity from amino acids 15-316 with a protein from Bacillus halodurans that is a ; ribose ABC transporter (ribose-binding protein) (Accession Nos. NP_244599.1, -
NC_002570), and 42% identity from amino acids 4-315 with a protein from )
Pasteurella multocida that is an RbsB protein (Accession Nos. NP_245090.1;
NC_002663).
A Gapped BlastP sequence alignment showed that SEQ ID NO:54 (285 amino : acids) has about 60% identity from amino acids 1-277 with a protein from Bacillus ) 30 subtilis that is a ribose ABC transporter (permease) (Accession Nos. NP_391476.1;
NC_000964), about 59% identity from amino acids 1-277 with a protein from : . Bacillus subtilis that is a ribose transport system permease protein (rbcS) (Accession ~
No. sp[P36948|RBSC_BACSU), about 57% identity from amino acids 4-277 with a protein from Bacillus halodurans that is a ribose ABC transporter (permease)
(Accession Nos. NP_244598.1; NC_002570), about 58% identity from amino acids 4- 277 with a protein from Lactococcus lactis subsp. lactis that is a ribose ABC transporter permease protein (Accession Nos. NP_267792.1; NC_002662), and 54% . identity from amino acids 4-278 with a protein from Haemophilus influenzae that is a . 5 D-ribose ABC transporter, permease protein (rbsC) (Accession Nos. NP_438661.1; " NC_000907). : :
A Gapped BlastP sequence alignment showed that SEQ ID NO:56 (496 amino * acids) has about 59% identity from amino acids 5-496 with a protein from
Lactococcus lactis subsp. lactis that is a ribose ABC transporter ATP binding protein (Accession Nos. NP_267793.1; NC_002662), about 57% identity from amino acids 5- 496 with a protein from Bacillus subtilis that is a ribose ABC transporter (ATP- binding protein) (Accession Nos. NP_391475.1; NC_000964), about 51% identity from amino acids 5-496 with a protein from Bacillus subtilis that is an ATP binding protein (Accession No. pir{|[140465), about 49% identity from amino acids 5-495 with aprotein from Bacillus halodurans that is a ribose ABC transporter (ATP-binding protein) (Accession Nos. NP_244597.1; NC_002570), and 45% identity from amino acids 7-494 with a protein from Agrobacterium tumefaciens that is an ABC * transporter, nucleotide binding/ATPase protein [ribose] (Accession Nos.
NP_533484.1; NC_003304).
A Gapped BlastP sequence alignment showed that SEQ 1D NO:58 (134 amino ! acids) has about 58% identity from amino acids 4-134 with a protein from
Lactobacillus sakei that is a ribose permease (RbsD) (Accession Nos. gb|AAD34337.1; AF115391), about 51% identity from amino acids 4-134 with a - protein from Clostridium perfringens that is homologous to a ribose ABC transporter 3 (Accession Nos. NP_562547.1; NC_003366), about 50% identity from amino acids 4- 132 with a protein from Lactococcus lactis subsp. lactis that is a ribose ABC : transporter permease protein (Accession Nos. NP_267794.1; NC_002662), about 45% identity from amino acids 4-134 with a protein from Bacillus halodurans that is a 5 ribose ABC transporter (permease) (Accession Nos. NP_244596.1; NC_002570), and : . 30 51% identity from amino acids 4-134 with a protein from Staphylococcus aureus subsp. aureus that is a ribose permease (Accession Nos. NP_370793.1; NC_002758). - A Gapped BlastP sequence alignment showed that SEQ ID NO:60 (308 amino ~ : acids) has about 51% identity from amino acids 4-301 with a protein from . -
Lactobacillus sakei that is a ribokinase (RbsK) (Accession Nos. gb/AAD34338.1; :
AF115391), about 48% identity from amino acids 1-303 with a protein from :
Staphylococcus aureus subsp. aureus that is homologous to a ribokinase (Accession : Nos. NP_370792.1; NC_002758), about 45% identity from amino acids 3-305 with a } protein from Clostridium perfringens that is a ribokinase (Accession Nos. . : - 5 NP_562548.1; NC_003366), about 41% identity from amino acids 1-299 with a ) protein from Haemophilus influenzae that is a ribokinase (RbsK) (Accession Nos.
NP_438663.1; NC_000907), and 38% identity from amino acids 2-300 with a protein 2 from Yersinia pestis that is a ribokinase (Accession Nos. NP_403674.1; NC_003143).
A Gapped BlastP sequence alignment showed that SEQ ID NO:62 (285 amino acids) has about 63% identity from amino acids 1-285 with a protein from
Lactococcus lactis subsp. lactis that is a maltose ABC transporter permease protein : (Accession Nos. NP_267841.1; NC_002662), about 54% identity from amino acids 6- : 284 with a protein from Streptococcus pyogenes that is homologous to a ; maltose/maltodextrin ABC transport system protein (permease) (Accession Nos.
NP_269423.1; NC_002737), about 38% identity from amino acids 12-284 with a protein from Klebsiella oxytoca that is homologous to a malG protein (Accession No. pir||S63616), about 39% identity from amino acids 9-285 with a protein from Bacillus halodurans that is a maltose/maltodextrin transport system (permease) (Accession
Nos. NP_243790.1; NC_002570), and 36% identity from amino acids 7-285 with a protein from Bacillus subtilis that is homologous to a maltodextrin transport system permease (Accession Nos. NP_391294.1; NC_000964).
A Gapped BlastP sequence alignment showed that SEQ ID NO:64 (452 amino acids) has about 63% identity from amino acids 1-452 with a protein from :
Lactococcus lactis subsp. lactis that is a maltose ABC transporter permease protein ] (Accession Nos. NP_267840.1; NC_002662), about 52% identity from amino acids 3- B 452 with a protein from Streptococcus pyogenes that is homologous to a maltose/maltodextrin ABC transport system protein (permease) (Accession Nos.
NP_269422.1; NC_002737), about 52% identity from amino acids 3-452 with a ; protein from Streptococcus pyogenes that is homologous to a maltose/maltodextrin - 30 ABC transport system (permease) (Accession Nos. NP_607422.1; NC_003485), about 34% identity from amino acids 28-451 with a protein from Klebsiella oxytoca : i that is homologous to a malF protein (Accession No. pir]|S63615), and 33% identity ~ from amino acids 23-451 with a protein from Bacillus halodurans that is a : 106 _
maltose/maltodextrin transport system permease (Accession Nos. NP 243791 1;
NC_002570).
A Gapped BlastP sequence alignment showed that SEQ ID NO:66 (408 amino . acids) has about 49% identity from amino acids 1-407 with a protein from . - 5 Lactococcus lactis subsp. lactis that is a maltose ABC transporter substrate binding ) protein (Accession Nos. NP_267839.1; NC_002662), about 37% identity from amino acids 1-405 with a protein from Streptococcus pyogenes that is homologous to a : maltose/maltodextrin-binding protein (Accession Nos. NP_607421.1; NC 003485), about 36% identity from amino acids 1-405 with a protein from Streptococcus pyogenes that is homologous to a maltose/maltodextrin-binding protein (Accession :
Nos. NP_269421.1; NC_002737), about 27% identity from amino acids 1-393 with a protein from Listeria innocua that is homologous to a maltose/maltodextrin ABC- } transporter (binding protein) (Accession Nos. NP_471563.1; NC_003212), and 26% : identity from amino acids 1-403 with a protein from Bacillus subtilis that is homologous to a maltose/maltodextrin-binding protein (Accession Nos.
NP_391296.1; NC_000964). | -
A Gapped BlastP sequence alignment showed that SEQ ID NO:68 (368 amino acids) has about 64% identity from amino acids 1-366 with a protein from
Streptococcus mutans that is a multiple sugar-binding transport ATP-binding protein (msmK) (Accession No. splQ00752]MSMK_STRMU), about 64% identity from = n amino acids 1-366 with a protein from Streptococcus Jr— that is a multiple sugar-binding ABC transport system (ATP-binding) protein (Accession Nos.
NP_269942.1; NC _002737), about 64% identity from amino acids 1-366 witha : protein from Streptococcus pyogenes that is a multiple sugar-binding ABC transport N system (ATP-binding) protein (Accession Nos. NP_608016.1; NC 003485), about : 64% identity from amino acids 1-366 with a protein from Streptococcus pneumoniae that is an ABC transporter ATP-binding protein - multiple sugar transport (Accession
Nos. NP_359030.1; NC_003098), and 62% identity from amino acids 1-368 with a Fe protein from Lactococcus lactis subsp. lactis that is a multiple sugar ABC transporter i - 30 ATP-binding protein (Accession Nos. NP_266577.1; NC_002662).
A Gapped BlastP sequence alignment showed that SEQ ID NO:70 (512 amino i acids) has about 60% identity from amino acids 1-510 with a protein from ~
Streptococcus pyogenes that is homologous to a sugar ABC transporter (ATP-binding PE protein) (Accession Nos. NP_269365.1; NC_002737), about 60% identity from amino "
oo acids 1-510 with a protein from Streptococcus pyogenes that is homologous to a sugar
ABC transporter (ATP-binding protein) (Accession Nos. NP_607296.1; NC_003485), : about 59% identity from amino acids 5-503 with a protein from Lactococcus lactis . subsp. lactis that is a sugar ABC transporter ATP binding protein (Accession Nos. - 5 NP_267484.1; NC_002662), about 61% identity from amino acids 7-503 with a ) protein from Streptococcus pneumoniae that is a sugar ABC transporter, ATP-binding RE protein (Accession Nos. NP_345337.1; NC_003028), and 60% identity from amino - acids 7-503 with a protein from Streptococcus pneumoniae that is a ABC transporter
ATP-binding protein - ribose/galactose transport (Accession Nos. NP_358342.1;
NC _003098).
A Gapped BlastP sequence alignment showed that SEQ ID NO:72 (383 amino : acids) has about 49% identity from amino acids 7-351 with a protein from
Lactococcus lactis subsp. lactis that is a sugar ABC transporter permease protein (Accession Nos. NP_267485.1; NC 002662), about 47% identity from amino acids 4- 351 with a protein from Streptococcus pneumoniae that is an ABC transporter membrane-spanning permease (ribose/galactose transport) (Accession Nos.
NP_358343.1; NC_003098), about 47% identity from amino acids 4-351 with a protein from Streptococcus pneumoniae that is homologous to a sugar ABC transporter, permease protein (Accession Nos. NP_345338.1; NC_003028), about 49% identity from amino acids 4-342 with a protein from Streptococcus pyogenes that
Co is homologous to a sugar ABC transporter (permease protein) (Accession Nos.
NP_269364.1; NC_002737), and 49% identity from amino acids 4-342 with a protein ~~ from Streptococcus pyogenes that is homologous to a sugar ABC transporter (permease protein) (Accession Nos. NP_607295.1; NC_003485).
A Gapped BlastP sequence alignment showed that SEQ ID NO:74 (318 amino } acids) has about 67% identity from amino acids 1-318 with a protein from
Streptococcus pyogenes that is homologous to a sugar ABC transporter (permease protein) (Accession Nos. NP_607294.1; NC_003485), about 66% identity from amino : acids 1-318 with a protein from Streptococcus pyogenes that is homologous to a sugar ~~ - 30 ABC transporter (permease protein) (Accession Nos. NP_269363.1; NC_002737), ’ about 65% identity from amino acids 1-318 with a protein from Streptococcus : ) pneumoniae that is homologous to a sugar ABC transporter, permease protein ~ (Accession Nos. NP_345339.1; NC_003028), about 63% identity from amino acids 1- : 318 with a protein from Lactococcus lactis subsp. lactis that is a sugar ABC transporter permease protein (Accession Nos. NP_267486.1; NC_002662), and 61% identity from amino acids 6-318 with a protein from Listeria innocua that is : homologous to a sugar ABC transporter (permease protein) (Accession Nos. ) : NP_470764.1; NC_003212). i - 5 A Gapped BlastP sequence alignment showed that SEQ ID NO:76 (450 amino ) acids) has about 68% identity from amino acids 11-448 with a protein from Neisseria : meningitidis that 1s homologous to a sugar transporter (Accession Nos. NP_273437.1;
NC 003112), about 68% identity from amino acids 11-448 with a protein from ~ Neisseria meningitidis that is homologous to an integral membrane transport protein (Accession Nos. NP_284797.1; NC_003116), about 39% identity from amino acids 17-229 with a protein from Caulobacter crescentus that is homologous to a transporter (Accession Nos. NP_421086.1; NC_002696), about 21% identity from } amino acids 31-450 with a protein from Lycopersicon esculentum that is a sucrose i. transporter (Accession Nos. gb]AAG09270.1; AF176950), and 21% identity from amino acids 31-442 with a protein from Arabidopsis thaliana that is a sucrose transporter (Accession Nos. gb|]AAG09191.1; AF175321). :
A Gapped BlastP sequence alignment showed that SEQ ID NO:78 (495 amino acids) has about 32% identity from amino acids 8-482 with a protein from
Lactococcus lactis subsp. lactis that is a transporter protein (Accession Nos.
NP_266394.1; NC_002662), about 34% identity from amino acids 8-482 with a : : ~ protein from Listeria monocytogenes that is homologous to an efflux transporter oo (Accession Nos. NP_464506.1; NC_003210), about 34% identity from amino acids 8- 482 with a protein from Listeria innocua that is homologous to an efflux transporter . (Accession Nos. NP_470317.1; NC_003212), about 30% identity from amino acids 7- } 422 with a protein from Clostridium acetobutylicum that is an MDR related permease (Accession Nos. NP_149294.1; NC_001988), and 29% identity from amino acids 8- 425 with a protein from Streptomyces coelicolor that is homologous to a membrane transport protein (Accession Nos. emb|CAB89031.1; AL353870). x
A Gapped BlastP sequence alignment showed that SEQ ID NO:80 (471 amino - 30 acids) has about 32% identity from amino acids 1-440 with a protein from i Lactococcus lactis subsp. lactis that is a transporter protein (Accession Nos. ’ NP _266394.1; NC_002662), about 34% identity from amino acids 1-464 with a 3 protein from Listeria monocytogenes that is homologous to an efflux transporter | . (Accession Nos. NP_464506.1; NC_003270), about 34% identity from amino acids 1- :
464 with a protein from Listeria innocua that is homologous to an efflux transporter : (Accession Nos. NP_470317.1; NC_003212), about 29% identity from amino acids 1- 412 with a protein from Clostridium acetobutylicum that is an MDR related permease . . (Accession Nos. NP_149294.1; NC_001988), and 28% identity from amino acids 4- - 5 459 with a protein from Streptomyces coelicolor that is homologous to an exporter - (Accession No. pir||T36377). .
A Gapped BlastP sequence alignment showed that SEQ ID NO:82 (412 amino - acids) has about 49% identity from amino acids 18-400 with a protein from Listeria innocua that is homologous to a drug-efflux transporter (Accession Nos.
NP_472212.1; NC_003212), about 49% identity from amino acids 18-400 with a protein from Listeria monocytogenes that is homologous to a drug-efflux transporter (Accession Nos. NP_466263.1; NC_003210), about 48% identity from amino acids 18-397 with a protein from Escherichia coli that is homologous to a transport protein (Accession Nos. NP_415571.1; NC_000913), about 47% identity from amino acids 15-399 with a protein from Lactococcus lactis subsp. lactis that is a multidrug resistance efflux pump (Accession Nos. NP_266282.1; NC_002662), and 48% identity from amino acids 18-399 with a protein from Salmonella typhimurium that is homologous to an MFS family transport protein (Accession Nos. NP_460125.1; :
NC_003197). : 20 . A Gapped BlastP sequence alignment showed that SEQ ID NO:84 (462 amino acids) has about 38% identity from avin acids 9-413 with ORFC from Oenococcus oo oeni (Accession Nos. embJCAB61253.1; AJ250422), about 38% identity from amino acids 2-378 with a protein from Lactococcus lactis subsp. lactis that is a transporter | ) protein (Accession Nos. NP_267695.1; NC_002662), about 34% identity from amino acids 6-411 with a protein from Streptococcus pyogenes that is homologous to a drug resistance protein (Accession Nos. NP. 606824.1; NC 003485), about 33% identity from amino acids 6-411 with a protein from Streptococcus pyogenes that is : homologous to a drug resistance protein (Accession Nos. NP_268834.1 ; NC_002737), . and 34% identity from amino acids 2-454 with a protein from Lactococcus lactis : 30 subsp. lactis that is a drug-export protein (Accession Nos. NP_267504.1; . NC 002662). : _ A Gapped BlastP sequence alignment showed that SEQ ID NO:86 (490 amino ~ acids) has about 55% identity from amino acids 3-476 with a protein from Listeria : monocytogenes that is homologous to a drug-export protein (Accession Nos.
NP_466111.1; NC_003210), about 54% identity from amino acids 3-476 with a protein from Listeria innocua that is homologous to a drug-export protein (Accession
Nos. NP_472062.1; NC_003212), about 45% identity from amino acids 6-478 with a . protein from Lactococcus lactis subsp. lactis that is a multidrug resistance protein : . 5 (Accession Nos. NP_267065.1; NC_002662). about 49% identity from amino acids 8- _ R 484 with a protein from Bacillus subtilis that is homologous toa multidrug resistance - protein (Accession Nos. NP_388266.1; NC_000964), and 44% identity from amino » acids 18-425 with a protein from Bacillus subtilis that is homologous to a multidrug resistance protein (Accession Nos. NP_388782.1 ; NC_000964).
A Gapped BlastP sequence alignment showed that SEQ ID NO:88 (416 amino acids) has about 26% identity from amino acids 17-408 with a protein from
Desulfitobacterium hafniense (Accession Nos. gbl|AAL87781.1; AF403184), about : 25% identity from amino acids 26-408 with a protein from Streptococcus pneumoniae : that is transporter in the major facilitator superfamily (Accession Nos. NP_359046.1,
NC_003098), about 21% identity from amino acids 61-399 with a protein from
Campylobacter jejuni that is homologous to an efflux protein (Accession Nos.
NP_282813.1; NC_002163), about 19% identity from amino acids 25-368 with a protein from Agrobacterium tumefaciens that is homologous to an MFS permease (Accession Nos. NP_533033.1; NC_003304), and 25% identity from amino acids 19- 205 with a protein from Bacillus halodurans that is a multidrug resistance protein ' (Accession Nos. NP_244175.1; NC_002570).
A Gapped BlastP sequence alignment showed that SEQ ID NO:90 (548 amino acids) has about 38% identity from amino acids 17-546 with a protein from Listeria : innocua that is homologous to a transporter protein (Accession Nos. NP_471001.1; :
NC_003212), about 37% identity from amino acids 17-546 with a protein from
Listeria monocytogenes that is homologous to a transporter protein (Accession Nos.
NP_465149.1; NC_003210), about 36% identity from amino acids 1-534 with a protein from Streptococcus pneumoniae that is a polysaccharide transporter - (Accession Nos. NP_358976.1; NC_003098), about 36% identity from amino acids - ) 30 17-534 with a protein from Streptococcus pneumoniae that is homologous to a - polysaccharide biosynthesis protein (Accession Nos. NP_345978.1; NC_003028), and - 35% identity from amino acids 12-546 with a hypothetical protein from Lactococcus ~ lactis subsp. lactis (Accession Nos. NP_267962.1; NC_002662). I
A Gapped BlastP sequence alignment showed that SEQ ID NO:92 (485 amino acids) has about 44% identity from amino acids 1-484 with a protein from Listeria : monocytogenes that is homologous to an efflux transporter protein (Accession Nos. oe NP_464506.1; NC_003210), about 44% identity from amino acids 1-484 with a } : ~- 5 protein from Listeria innocua that is homologous to an efflux transporter protein Co (Accession Nos. NP_470317.1; NC_003212), about 34% identity from amino acids 9- . 420 with a protein from Clostridium acetobutylicum that 1s an MDR-related permease (Accession Nos. NP_149294.1; NC_001988), about 33% identity from amino acids ) 12-475 with a protein from Lactococcus lactis subsp. lactis that is a transporter protein (Accession Nos. NP_266394.1; NC_002662), and 34% identity from amino acids 1-457 with a hypothetical protein from Myxococcus xanthus (Accession Nos. B emb|CAB37973.1; X76640).
A Gapped BlastP sequence alignment showed that SEQ ID NO:94 (199 amino acids) has about 46% identity from amino acids 23-173 with a protein from Listeria innocua that is homologous to a drug-efflux transporter protein (Accession Nos.
NP_472212.1; NC_003212), about 45% identity from amino acids 23-173 witha protein from Listeria monocytogenes that is homologous to a drug-efflux transporter protein (Accession Nos. NP_466263.1; NC_003210), about 49% identity from amino : acids 23-173 with a protein from Lactococcus lactis subsp. lactis that is a multidrug resistance efflux pump (Accession Nos. NP_266282.1; NC_002662), about 46% ' identity from amino acids 23-173 with a protein from Salmonella enterica subsp. enterica serovar Typhi that is homologous to an efflux pump (Accession Nos.
NP_454977.1; NC_003198), and 46% identity from amino acids 23-173 with a : protein from Salmonella typhimurium that is homologous to a permease (Accession :
Nos. NP_459377.1; NC_003197).
A Gapped BlastP sequence alignment showed that SEQ ID NO:96 (538 amino acids) has about 32% identity from amino acids 4-525 with a protein from
Co Streptococcus pneumoniae that is a polysaccharide transporter (Accession Nos. 3
NP_358976.1; NC_003098), about 32% identity from amino acids 5-525 with a protein from Streptococcus pneumoniae that is homologous to a polysaccharide } biosynthesis protein (Accession Nos. NP_345978.1; NC_003028), about 33% identity : . | from amino acids 5-526 with a conserved hypothetical protein from Streptococcus oo pyogenes (Accession Nos. NP_606680.1; NC_003485), about 33% identity from amino acids 5-526 with a conserved hypothetical protein from Streptococcus pyogenes (Accession Nos. NP_268708.1; NC 002737), and 30% identity from amino : acids 4-526 with a hypothetical protein from Lactococcus lactis subsp. lactis (Accession Nos. NP_267962.1; NC_002662). a A Gapped BlastP Sequence ali gnment showed that SEQ ID NO:98 (328 amino c 5 acids) has about 57% identity from amino acids 1-323 with a protein from ) Pediococcus pentosaceus that is a sucrose operon regulatory protein (scrR) (Accession No: sp|P43472|SCRR_PEDPE), about 51% identity from amino acids 1- x 322 with a protein from Streptococcus pneumoniae that is a sucrose operon repressor (Accession Nos. NP_346162.1; NC_003028), about 49% identity from amino acids 1- 326 with a protein from Streptococcus mutans that is a sucrose operon regulatory - protein (scrR) (Accession No. sp|Q54430|SCRR_STRMU), about 49% identity from : amino acids 1-322 with a protein from Streptococcus pyogenes that is homologous to a sucrose operon repressor (Accession Nos. NP_607889.1; NC_003485), and 49% . identity from amino acids 1-322 with a protein from Streptococcus pyogenes that is homologous to a sucrose operon repressor (Accession Nos. NP_269821.1; oo NC_002737).
A Gapped BlastP sequence alignment showed that SEQ ID NO:100 (485 amino acids) has about 50% identity from amino acids 1-466 with a protein from
Streptococcus sobrinus that is a sucrose-6-phosphate hydrolase (ScrB) (Accession No. pir]|{S68598), about 49% identity from amino acids 1-461 with a protein from
Streptococcus pneumoniae that 5a sucrose-6-phosphate hydrolase (Accession Nos.
NP_359160.1; NC_003098), about 49% identity from amino acids 1-461 with a ~ protein from Streptococcus pneumoniae that is a sucrose-6-phosphate hydrolase g (Accession Nos. NP_346161.1; NC_003028), about 49% identity from amino acids 1- 466 with a protein from Streptococcus pyogenes that is homologous to a sucrose-6- phosphate hydrolase (Accession Nos. NP_607888.1; NC_003485), and 49% identity from amino acids 1-466 with a protein from Streptococcus pyogenes that is homologous to a sucrose-6-phosphate hydrolase (Accession Nos. NP_269820.1; 1
NC_002737). . 30 A Gapped BlastP sequence alignment showed that SEQ ID NO:102 (649 - amino acids) has about 65% identity from amino acids 1-645 with a protein from v Streptococcus mutans that is a phosphotransferase system enzyme II (EC 2.7.1.69), ~ sucrose-specific IIABC component (Accession No. sp|P12655[PTSA_STRMU), about : 56% identity from amino acids 1-647 with a protein from Pediococcus pentosaceus )
that is a phosphotransferase system enzyme II (EC 2.7.1.69), sucrose specific enzyme
IIABC (Accession No. sp[P43470|PTSA_PEDPE), about 52% identity from amino acids 1-643 with a protein from Lactococcus lactis that is an enzyme [I sucrose . protein (Accession Nos. emb|CAB09690.1; 297015), about 52% identity from amino } - 5 acids 114-647 with a protein from Lactobacillus sakei that is a sucrose-specific . - enzyme II of the PTS (Accession Nos. gb|AAK92528.1; AF401046), and 45% identity from amino acids 1-621 with a protein from Corynebacterium glutamicum - that is a phosphotransferase system IIB component (Accession Nos. NP _601842.1; - NC_003450).
A Gapped BlastP sequence alignment showed that SEQ ID NO:104 (667 amino acids) has about 42% identity from amino acids 192-661 with a protein from
Lactococcus lactis subsp. lactis that is a beta-glucoside-specific PTS system [IABC component (EC 2.7.1.69) (Accession Nos. NP_266583.1; NC_002662), about 39% identity from amino acids 191-652 with a protein from Listeria monocytogenes that is homologous to a phosphotransferase system (PTS) beta-glucoside-specific enzyme ~ IIABC (Accession Nos. NP_464560.1;"NC-_003210), about 37% identity from amino acids 191-662 with a protein from Clostridium longisporum that is a PTS-dependent enzyme II (Accession Nos. gb|]AAC05713.1; L49336), about 36% identity from amino acids 191-666 with a protein from Bacillus halodurans that is a PTS system, beta-glucoside-specific enzyme II, ABC component (Accession Nos. NP_241461.1;
SE NC_002570), and 36% identity from amino acids 191-650 with a protein from
Listeria innocua that is homologous to a PTS system, beta-glucosides specific enzyme [TABC (Accession Nos. NP_469373.1; NC_003212). : :
A Gapped BlastP sequence alignment showed that SEQ ID NO:106 (241 amino acids) has about 47% identity from amino acids 1-238 with a protein from
Bacillus subtilis that is a trehalose operon transcriptional repressor (Accession No. sp|[P39796]TRER_BACSU), about 41% identity from amino acids 4-238 with a protein from Bacillus halodurans that is a transcriptional repressor of the trehalose g operon (Accession Nos: NP_241 739.1; NC_002570), about 44% identity from amino i} 30 acids 9-237 with a protein from Listeria innocua that is homologous to a transcription
Pp regulator GntR family (Accession Nos. NP_470558.1; NC_003212), about 44% . . _ identity from amino acids 9-237 with a protein from Listeria monocytogenes that is ~ homologous to a transcription regulator GntR family (Accession Nos. NP_464778.1;
NC_003210), and 41% identity from amino acids 5-238 with a protein from .
Lactococcus lactis subsp. lactis that is a GntR family transcriptional regulator (Accession Nos. NP_266581.1; NC_002662).
A Gapped BlastP sequence alignment showed that SEQ ID NO:108 (570 . amino acids) has about 56% identity from amino acids 22-566 with a protein from : 2 5 Streptococcus pyogenes that is homologous to a dextran glucosidase (Accession Nos. no NP_608103.1; NC_003485), about 57% identity from amino acids 23-568 with a protein from Streptococcus pneumoniae that is a dextran glucosidase (Accession Nos. .
NP_359290.1; NC_003098), about 56% identity from amino acids 22-566 with a protein from Streptococcus pyogenes that is homologous to a dextran glucosidase (Accession Nos. NP_270026.1 ; NC_002737), about 57% identity from amino acids 23-568 with a protein from Streptococcus pneumoniae that is homologous to a dextran glucosidase DexS (Accession Nos. NP_346315.1; NC_003028), and 54% ~ identity from amino acids 17-570 with a protein from Clostridium perfringens that is an alpha-glucosidase (Accession Nos. NP_561478.1; NC_003366). :
A Gapped BlastP sequence alignment showed that SEQ ID NO:110 (370 amino acids) has about 67% identity from amino acids 1-368 with a protein from
Streptococcus pneumoniae that is an ABC transporter ATP-binding protein - multiple sugar transport (Accession Nos. NP_359030.1; NC _003098), about 67% identity from amino acids 1-368 with a protein from Streptococcus pneumoniae that is a sugar ABC transporter, ATP-binding protein (Accession Nos. NP_346026.1; NC_003028), about
Co 66% identity from amino acids 1-368 with a protein from Streptococcus mutans that is a multiple sugar-binding transport ATP-binding protein (msmK) (Accession No. sp|Q00752)MSMK_STRMU), about 68% identity from amino acids 1-365 with a oo protein from Listeria innocua that is homologous to a sugar ABC transporter, ATP- binding protein (Accession Nos. NP_469649.1; NC 003212), and 67% identity from amino acids 1-365 with a protein from Listeria monocytogenes that is homologous to : a sugar ABC transporter, ATP-binding protein (Accession Nos. NP_463809.1;
NC_003210). | :
A Gapped BlastP sequence alignment showed that SEQ ID NO:112 (278 : } 30 amino acids) has about 81% identity from amino acids 2-278 with a protein from p Streptococcus mutans that is a multiple sugar-binding transport system permease
SE protein (msmG) (Accession No. sp|Q00751|MSMG_STRMU), about 73% identity 3 : from amino acids 1-278 with a protein from Streptococcus pneumoniae that is a sugar .
ABC transporter, permease protein (Accession Nos. NP_346326.1; NC_003028), : 1s about 72% identity from amino acids 2-278 with a protein from Streptococcus pneumoniae that is a ABC transporter membrane spanning permease - multiple sugars (Accession Nos. NP_359302.1; NC_003098), about 85% identity from amino acids i 72-278 with a hypothetical protein fragment from Streptococcus mutans (Accession - 5 No. pirl{B27626), and 44% identity from amino acids 4-278 with a protein from ’ Clostridium acetobutylicum that is a sugar permease (Accession Nos. NP_350251.1;
NC_003030). | ;
A Gapped BlastP sequence alignment showed that SEQ ID NO:114 (291 : oo : amino acids) has about 73% identity from amino acids 4-290 with a protein from
Streptococcus pneumoniae that is an ABC transporter membrane-spanning permease - : multiple sugars (Accession Nos. NP_359303.1; NC_003098), about 73% identity from amino acids 4-290 with a protein from Streptococcus pneumoniae that is a sugar y
ABC transporter, permease protein (Accession Nos. NP_346327.1; NC_003028), about 73% identity from amino acids 1-290 with a protein from Streptococcus mutans : 15 that is a multiple sugar-binding transport system permease protein (msmF) (Accession
No. sp{Q00750MSMF_STRMU), about 53% identity from amino acids 6-291 with a protein from Clostridium acetobutylicum that is an ABC-type sugar transport system, permease component (Accession Nos. NP_350252.1; NC_003030), and 32% identity from amino acids 2-291 with a protein from Thermoanaerobacterium . thermosulfurigenes that is a potential starch degradation products transport system 0 oo permease protein (Accession No. sp|P37730|AMYD_THETU).
A Gapped BlastP sequence alignment showed that SEQ ID NO:116 (423 amino acids) has about 60% identity from amino acids 8-421 with a protein from :
Streptococcus mutans that is a multiple sugar-binding protein precursor (Accession :
No. sp|Q00749MSME_STRMU), about 56% identity from amino acids 9-421 with a : protein from Streptococcus pneumoniae that is a sugar ABC transporter, sugar- binding protein (Accession Nos. NP_346328.1; NC_003028), about 56% identity from amino acids 9-421 with a protein from Streptococcus pneumoniae that is an 3
ABC transporter substrate-binding protein - multiple sugars (Accession Nos. ) 30 NP_359304.1; NC_003098), about 29% identity from amino acids 9-420 with a . protein from Clostridium acetobutylicum that is an ABC-type sugar transport system, - periplasmic sugar-binding component (Accession Nos. NP_350253.1; NC_003030), ~ and 24% identity from amino acids 6-412 with a protein from Bacillus subtilis that is N homologous to a multiple sugar-binding protein (Accession Nos. NP_391140.1;
NC_000964).
A Gapped BlastP sequence alignment showed that SEQ ID NO:118 (279 . amino acids) has about 57% identity from amino acids 1-273 with a protein from = 5 Pediococcus pentosaceus that is a raffinose operon transcriptional regulatory protein (rafR) (Accession No. sp|P43465|RAFR_PEDPE), about 35% identity from amino acids 5-273 with a protein from Streptococcus mutans that is homologous to a i . transcription regulator (msmR) (Accession No. pir[|A42400), about 35% identity from amino acids 5-273 with a protein from Streptococcus mutans that is an msm operon regulatory protein (Accession No. sp|Q00753]MSMR_STRMU), about 36% identity from amino acids 19-273 with a protein from Streptococcus pneumoniae that is an msm operon regulatory protein (Accession Nos. NP_346330.1; NC_003028), and : 36% identity from amino acids 19-273 with a protein from Streptococcus pneumoniae - that is an msm (multiple sugar metabolism) operon regulatory protein (Accession
Nos. NP_359306.1; NC_003098).
A Gapped BlastP sequence alignment showed that SEQ IDNO:120 277 amino acids) has about 28% identity from amino acids 37-141 with a protein from
Treponema pallidum that is homologous to an rRNA methylase (Accession Nos.
NP 218549.1; NC_000919), about 32% identity from amino acids 74-141 with a protein from Guillardia theta that is a GTP-binding nuclear protein RAN (Accession
Nos. NP_113408.1; NC_002753), about 29% identity from amino acids 75-141 with a protein from Dictyostelium discoideum that is a GTP-binding nuclear protein
RAN/TC4 (Accession No. sp|P33519jRAN_DICDI), and about 25% identity from : amino acids 140-190 with a putative protein from Arabidopsis thaliana (Accession
Nos. NP_191798.1; NM_116104). :
A Gapped BlastP sequence alignment showed that SEQ ID NO:122 (530 amino acids) has about 26% identity from amino acids 8-524 with a protein from
Lactococcus lactis subsp. lactis that is an ABC transporter ATP binding and permease 7 protein (Accession Nos. NP_267678.1; NC_002662), about 25% identity from amino ) acids 49-518 with a protein from Streptococcus pneumoniae that is an ABC . transporter, ATP-binding protein (Accession Nos. NP_344680.1; NC_003028), about - 25% identity from amino acids 49-518 with a protein from Streptococcus pneumoniae + that is an ABC transporter ATP-binding/membrane spanning permease (Accession
Nos. NP_357731.1; NC _003098), about 24% identity from amino acids 47-511 with a protein from Synechocystis sp. PCC 6803 that is an ABC transporter (Accession Nos.
NP_440626.1; NC_000911), and 24% identity from amino acids 7-511 with a protein from Bacillus subtilis that is homologous to an ABC transporter (ATP-binding . protein) (Accession Nos. NP_388852.1; NC_000964). Co - 5 A Gapped BlastP sequence alignment showed that SEQ ID NO:124 (530 g amino acids) has about 24% identity from amino acids 4-524 with a protein from Co .
Lactococcus lactis subsp. lactis that is an ABC transporter ATP binding and permease E protein (Accession Nos. NP_267678.1; NC_002662), about 25% identity from amino acids 55-508 with a protein from Streptococcus pneumoniae that is an ABC transporter, ATP-binding protein (Accession Nos. NP_344680.1; NC_003028), about : 25% identity from amino acids 55-508 with a protein from Streptococcus pneumoniae that is an ABC transporter ATP-binding/membrane spanning permease (Accession :
EE Nos. NP_357731.1; NC_003098), about 24% identity from amino acids 1-511 with a : protein from Streptococcus pneumoniae that is a drug efflux ABC transporter, ATP- binding/permease (Accession Nos. NP_345800.1; NC_003028), and 24% identity from amino acids 1-511 with a protein from Streptococcus pneumoniae that is an
ABC transporter ATP-binding/membrane spanning protein (Accession Nos. NP_358796.1; NC_003098). E
A Gapped BlastP sequence alignment showed that SEQ ID NO:126 (527 amino-acids) has about 25% identity from amino acids 8-527 with a protein from
Lactococcus lactis subsp. lactis that is an ABC transporter ATP binding and permease protein (Accession Nos. NP_267678.1; NC_002662), about 24% identity from amino acids 13-520 with a protein from Streptococcus pneumoniae that is an ABC : “ transporter ATP-binding/membrane spanning permease protein (Accession Nos. -
NP_357731.1; NC_003098), about 24% identity from amino acids 13-520 with a : protein from Streptococcus pneumoniae that is an ABC transporter, ATP-binding protein (Accession Nos. NP_344680.1; NC_003028), about 22% identity from amino acids 22-511 with a protein from Streptococcus pneumoniae that is a drug efflux ABC : transporter, ATP-binding/permease protein (Accession Nos. NP_345800.1; :
NC_003028), and 22% identity from amino acids 22-511 with a protein from - Streptococcus pneumoniae that is an ABC transporter ATP-binding/membrane - spanning protein (Accession Nos. NP_358796.1; NC_003098). ~
A Gapped BlastP sequence alignment showed that SEQ ID NO:128 (534 amino acids) has about 23% identity from amino acids 14-512 with a protein from
Streptococcus pneumoniae that is a comA protein (Accession No. pir||A39203), about 26% identity from amino acids 3-512 with a protein from Lactococcus lactis that is a
Lactococcin A transport ATP-binding protein (IenC) (Accession No. . sp|Q00564|LCNC_LACLA), about 23% identity from amino acids 14-512 with a - 5 protein from Streptococcus pneumoniae that is a transport ATP-binding protein (ComA) (Accession Nos. NP_357637.1; NC_003098), about 25% identity from amino acids 113-509 with a protein from Streptococcus salivarius that is an ABC : transporter (Accession Nos. gb|AAC72026.1; AF043280), and 22% identity from - amino acids 14-512 with a protein from Streptococcus pneumoniae that is a competence factor transporting ATP-binding/permease protein (ComA) (Accession a Nos. NP_344591.1; NC_003028). oo
A Gapped BlastP sequence alignment showed that SEQ 1D NO:130 (527 : amino acids) has about 23% identity from amino acids 16-524 with a protein from
Lactococcus lactis subsp. lactis that is an ABC transporter ATP binding and permease protein (Accession Nos. NP _267678.1; NC 002662), about 25% identity from amino : acids 6-520 with a protein from Streptococcus pneumoniae that is an ABC transporter,
ATP-binding protein (Accession Nos. NP_344680.1; NC _003028), about 25% identity from amino acids 6-520 with a protein from Streptococcus pneumoniae that is an ABC transporter ATP-binding/membrane spanning permease (Accession Nos. :
NP_357731.1; NC_003098), about 24% identity from amino acids 105-51 1 with a oo protein from Streptococcus pneumoniae that is an ABC transporter ATP- binding/membrane spanning protein (Accession Nos. NP_358796.1; NC 003098), and 25% identity from amino acids 99-511 with a protein from Nostoc sp. PCC 7120 * that is an ABC transporter ATP-binding protein (Accession Nos. NP_490403.1; :
NC _003276). ”
A Gapped BlastP sequence alignment showed that SEQ ID NO:132 (529 amino acids) has about 25% identity from amino acids 10-526 with a protein from
Lactococcus lactis subsp. lactis that is an ABC transporter ATP binding and permease va oo protein (Accession Nos. NP_267678.1; NC_002662), about 26% identity from amino . 30 acids 112-525 with a protein from Streptococcus pneumoniae that is an ABC oo ” transporter ATP-binding/membrane spanning permease (Accession Nos. - NP_357731.1; NC_003098), about 26% identity from amino acids 112-525 with a ~ protein from Streptococcus pneumoniae that is an ABC transporter, ATP-binding : protein (Accession Nos. NP_344680.1; NC_003028), about 24% identity from amino :
acids 107-518 with a protein from Brevibacillus brevis that is homologous to an
ABC-transporter (TycD) (Accession No. pir][T31077), and 24% identity from amino acids 83-521 with a protein from Streptococcus pneumoniae that is a drug efflux ABC . } . transporter, ATP-binding/permease (Accession Nos. NP_345800.1; NC 003028). . 3 ! - 5 A Gapped BlastP sequence alignment showed that SEQ ID NO:134 (600 i ~ amino acids) has about 23% identity from amino acids 2-600 with a protein from -
Listeria innocua that is homologous to an ABC transporter (permease) (Accession :
Nos. NP_471553.1; NC_003212), about 23% identity from amino acids 1-598 with a protein from Listeria monocytogenes that is homologous to an ABC transporter oo 10 (permease) (Accession Nos. NP_465271.1; NC 003210), about 22% identity from amino acids 1-599 with a protein from Clostridium perfringens that is homologous to an ABC transporter (Accession Nos. NP_561767.1; NC_003366), about 22% identity ) from amino acids 1-564 with a protein from Clostridium perfringens that is : homologous to an ABC-transporter (Accession Nos. NP_561039.1; NC_003366), and 22% identity from amino acids 4-593 with a protein from Clostridium acetobutylicum that is homologous to a permease (Accession Nos. NP_346868.1; NC_003030). .
A Gapped BlastP sequence alignment showed that SEQ ID NO:136 (249 amino acids) has about 58% identity from amino acids 1-242 witha protein from
Clostridium perfringens that is homologous to an ABC transporter (Accession Nos.
NP_561766.1; NC_003366), about 55% identity from amino acids 3-242 with a " protein from Clostridium perfringens that is homologous to an ABC transporter ) (Accession Nos. NP_561038.1; NC_003366), about 51% identity from amino acids 1- 242 with a protein from Listeria monocytogenes that is homologous to an ABC : transporter (ATP-binding protein) (Accession Nos. NP_465638.1, NC_003210), about 50% identity from amino acids 1-242 with a protein from Listeria innocua that 1s homologous to an ABC-transporter (ATP-binding protein) (Accession Nos.
NP_471552.1; NC_003212), and 54% identity from amino acids 3-242 with a protein from Clostridium acetobutylicum that is an ABC transporter, ATP-binding protein 3 (Accession Nos. NP_346867.1; NC_003030). ) 30 A Gapped BlastP sequence alignment showed that SEQ ID NO:138 (423 . amino acids) has about 21% identity from amino acids 2-391 with a hypothetical . protein from Streptococcus pyogenes (Accession Nos. NP_270004.1; NC_002737), ~ about 21% identity from amino acids 2-383 with a hypothetical protein from g
Streptococcus pyogenes (Accession Nos. NP_608080.1; NC 003485), about 26% :
: identity from amino acids 9-166 with a protein from Bacillus subtilis that is a yvbJ : protein (Accession Nos. NP_391268.1; NC_000964), about 25% identity from amino acids 92-281 with a protein from caprine arthritis-encephalitis virus that is an env . polyprotein precursor (Accession No. pir|{[VCLIC6), and 24% identity from amino - 5 acids 92-281 with a protein from Caprine arthritis-encephalitis virus that is an - envelope glycoprotein (Accession Nos. gb|AAD14661.1; AF105181).
A Gapped BlastP (version) sequence alignment showed that SEQ ID NO:140 : (438 amino acids) has about 27% identity from amino acids 86-216 with a protein from Brochothrix campestris that is a transport accessory protein (Accession Nos. gblJAAC95141.1; AF075600), about 26% identity from amino acids 107-219 with a protein from Streptococcus pneumoniae that is a bacterocin transport accessory ) protein (Accession Nos. NP_345950.1; NC_003028), about 26% identity from amino acids 107-219 with a protein from Streptococcus pneumoniae that is a Bta (Accession
Nos. gb|AADS56628.1; AF165218), 23% identity from amino acids 88-201 with a
I5 hypothetical protein from Bacillus anthracis (Accession Nos. NP_052783.1;
NC_001496), and 32% identity from amino acids 144-214 with a protein from Co
Neisseria meningitidis that 1s a thioredoxin (Accession Nos. NP_274384.1;
NC_003112).
A Gapped BlastP (version) sequence alignment showed that SEQ ID NO:142 (196 amino acids) has about 56% identity from amino acids 1-196 with a protein from
Lactobacillus gasseri (Accession Nos. dbj|BAA82351.1; AB029612), about 49% identity from amino acids 10-196 with a hypothetical protein from Lactobacillus sp. (Accession No. sp|P29470]YLA1_LACAC), about 28% identity from amino acids 41- : 196 with a protein from Lactobacillus casei that is an ABC-transporter accessory : factor (Accession Nos. NP_542220.1; NC_003320), 35% identity from amino acids Bb 90-196 with a protein from Lactobacillus plantarum that is an accessory factor for
ABC-transporter (PInH) (Accession Nos. emb]JCAA64190.1; X94434), and 30% identity from amino acids 41-196 with a protein from Lactobacillus sake that is C homologous to an ABC exporter accessory factor (SapE) (Accession No. ] 30 pirf]A56973). “ A Gapped BlastP (version) sequence alignment showed that SEQ ID NO:144 : . (720 amino acids) has about 62% identity from amino acids 9-720 with a protein from ~
Lactobacillus plantarum that is an ABC-transporter (PInG) (Accession Nos. : emb|CAA64189.1; X94434), about 62% identity from amino acids 6-720 with a -
protein from Lactobacillus sakei that is homologous to a translocation protein (sppT),
ATP-dependent (Accession No. pirl{S57913), about 62% identity from amino acids 2- . 720 with a protein from Lactobacillus sakei that 1s an ATP-dependent transport protein (SapT) (Accession No. pir|[156273), 62% identity from amino acids 9-720 | } - 5 with a protein from Lactobacillus casei that is an ABC transporter (Accession Nos. } NP_542219.1; NC_003320), and 57% identity from amino acids 25-718 with a . protein from Lactobacillus acidophilus that is an ABC transporter (Accession Nos. . © NP_604412.1;NC_003458).
A Gapped BlastP (version) sequence alignment showed that SEQ ID NO:146 .. (234 amino acids) has about 52% identity from amino acids 13-228 with a protein -from Staphylococcus aureus subsp. aureus that is homologous to an ABC transporter
ATP-binding protein (Accession Nos. NP_370833.1; NC_002758), about 50% identity from amino acids 11-234 with a protein from Streptococcus pyogenes that is : homologous to an ABC transporter (ATP-binding protein) (Accession Nos. :
NP_606994.1;, NC_003485), about 50% identity from amino acids 11-234 with a protein from Streptococcus pyogenes that is homologous to an ABC transporter (ATP-binding protein) (Accession Nos. NP_268993.1; NC_002737), 50% identity from amino acids 13-232 with a protein from Lactococcus lactis subsp. lactis that is an ABC transporter ATP-binding protein (Accession Nos. NP_266815.1;
NC_002662), and 53% identity from amino acids 11-233 with a protein from ’ Lactococcus lactis subsp. lactis that is an ABC transporter ATP-binding protein (Accession Nos. NP_268413.1; NC_002662). :
A Gapped BlastP (version) sequence alignment showed that SEQ ID NO:148 . (353 amino acids) has about 40% identity from amino acids 1-352 with a hypothetical protein from Lactococcus lactis subsp. lactis (Accession Nos. NP_268412.1;
NC_002662), about 38% identity from amino acids 1-352 with a conserved hypothetical protein from Staphylococcus aureus subsp. aureus (Accession Nos.
NP_370832.1; NC 002758), about 33% identity from amino acids 1-352 with a : conserved hypothetical protein from Streptococcus pyogenes (Accession Nos. : ) 30 NP_268992.1; NC_002737), 33% identity from amino acids 1-352 with a conserved . hypothetical protein from Streptococcus pyogenes (Accession Nos. NP_606993.1; : . NC_003485), and 34% identity from amino acids 1-352 with a protein from ~
Lactococcus lactis subsp. lactis that is an ABC transporter permease protein (Accession Nos. NP_266816.1; NC_002662).
A Gapped BlastP (version) sequence alignment showed that SEQ ID NO:150 (188 amino acids) has about 47% identity from amino acids 14-85 with a protein from
Lactococcus lactis subsp. lactis that is a transcriptional regulator (Accession Nos. . NP_266817.1; NC_002662), about 28% identity from amino acids 21-90 with a ’ - 5 protein from Aquifex aeolicus that is a transcriptional regulator in the TetR/AcrR . family (Accession Nos. NP_213195.1; NC_000918), about 30% identity from amino acids 14-75 with a protein from Clostridium acetobutylicum that is a transcriptional > : regulator in the AcrR family (Accession Nos. NP_348163.1; NC_003030), 29% identity from amino acids 25-109 with a protein from Streptomyces coelicolor that is homologous to a transcriptional regulator (Accession Nos. emb|CAB93030.1; .
AL357432), and 41% identity from amino acids 27-88 with a protein from g
Clostridium acetobutylicum that is a transcriptional regulator in the TetR/AcrR family . (Accession Nos. NP_347457.1; NC_003030). : oo
A Gapped BlastP (version) sequence alignment showed that SEQ ID NO:152 (236 amino acids) has about 65% identity from amino acids 3-236 with a protein from
Streptococcus pneumoniae that is an ABC transporter ATP-binding protein (Accession Nos. NP_359090.1; NC_003098), about 66% identity from amino acids 4- - : 236 with a protcin from Streptococcus pneumoniae that is an ABC transporter, ATP- binding protein (Accession Nos. NP_346092.1; NC_003028), about 65% identity : 20 from amino acids 4-236 with a protein from Streptococcus pyogenes that is homologous to an ABC transporter (ATP-binding protein) (Accession Nos.
NP_607321.1; NC_003485), 65% identity from amino acids 4-236 with a protein from Streptococcus pyogenes that is homologous to an ABC transporter (ATP-binding - protein) (Accession Nos. NP_269390.1; NC _002737), and 62% identity from amino acids 4-236 with a protein from Listeria monocytogenes that is homologous to a ABC : transporter, ATP-binding protein (Accession Nos. NP_464748.1; NC_003210).
A Gapped BlastP (version) sequence alignment showed that SEQ ID NO:154 (846 amino acids) has about 41% identity from amino acids 6-846 with a protein from a
Lactococcus lactis subsp. lactis that is an ABC transporter permease protein ] | 30 (Accession Nos. NP_267260.1; NC_002662), about 34% identity from amino acids 2- : . 846 with a hypothetical protein from Streptococcus pneumoniae (Accession Nos. - NP_359089.1; NC_003098), about 34% identity from amino acids 2-846 with a ~ hypothetical protein from Streptococcus pneumoniae (Accession Nos. NP_346091.1;
NC_003028), 33% identity from amino acids 4-846 with a hypothetical protein from Ce
Streptococcus pyogenes (Accession Nos. NP_269389.1; NC _002737), and 33% identity from amino acids 4-846 with a hypothetical protein from Streptococcus pyogenes (Accession Nos. NP_607320.1; NC_003485). | :
A Gapped BlastP (version) sequence alignment showed that SEQ ID NO:156 , - 5 (78 amino acids) has about 30% identity from amino acids 12-70 with a protein from } Arabidopsis thaliana (Accession Nos. gb|AAF19707.1; AC008047), about 30% ’ identity from amino acids 12-70 with a protein from Arabidopsis thaliana that is : - : homologous to an ATP dependent copper transporter (Accession Nos. NP_176533.1; )
NM_105023), about 32% identity from amino acids 1-65 with a hypothetical protein from Pyrococcus furiosus (Accession Nos. NP_579673.1; NC_003413), and 37% identity from amino acids 21-55 with a protein from Hepatitis TT virus (Accession : Nos. gb|AAK11712.1; AF345529).
A Gapped BlastP (version) sequence alignment showed that SEQ ID NO:158 (379 amino acids) has about 36% identity from amino acids 32-368 with a conserved hypothetical protein from Listeria innocua (Accession Nos. NP_470340.1;
NC_003212), about 37% identity from amino acids 32-353 with a conserved hypothetical protein from Listeria monocytogenes (Accession Nos. NP_464529.1;
NC_003210), about 36% identity from amino acids 87-370 with a protein from
Lactococcus lactis (Accession Nos. emb|CAA68042.1; X99710), 31% identity from amino acids 28-372 with a hypothetical protein from Lactococcus lactis subsp. lactis (Accession Nos. NP_267885.1; NC_002662), and 30% identity from amino acids 32- 348 with a protein from Actinosynnema pretiosum subsp. auranticum (Accession Nos. gb|AAC14002.1; U33059).
A Gapped BlastP (version) sequence alignment showed that SEQ 1D NO:160 (779 amino acids) has about 61% identity from amino acids 1-308 with a protein from ’
Streptococcus mutans that is an ABC transporter ATP binding subunit (Accession
Nos. gb]AAD09218.1; U73183), about 37% identity from amino acids 1-362 with a protein from Lactococcus lactis subsp. lactis that is an ABC transporter ATP-binding : and permease protein (Accession Nos. NP_266870.1; NC_002662), about 39% identity from amino acids 1-295 with a protein from Listeria monocytogenes that is . homologous to an ABC transporter, ATP-binding protein (Accession Nos. : - NP_464271.1; NC_003210), 47% identity from amino acids 1-221 with a protein ~ from Archaeoglobus fulgidus that is an ABC transporter, ATP-binding protein (Accession Nos. NP_070298.1; NC_000917), and 49% identity from amino acids 1-
218 with a protein from Archaeoglobus fulgidus that is an ABC transporter, ATP- binding protein (Accession Nos. NP_069851.1; NC_000917). :
A Gapped BlastP (version) sequence alignment showed that SEQ ID NO:162 , (38 amino acids) has about 66% identity from amino acids 1-27 with a protein from = 5 Clostridium acetobutylicum that is a mannose-specific phosphotransferase system component (Accession Nos. NP_149230.1; NC_001988), about 72% identity from ‘amino acids 3-27 with a protein from Listeria monocytogenes that is homologous to a . ~ PTS system mannose-specific factor [IAB (Accession Nos. NP_463629.1; :
NC_003210), about 72% identity from amino acids 3-27 with a protein from Listeria innocua that is homologous to a PTS system mannose-specific factor IAB (Accession Nos. NP_469488.1; NC_003212), 66% identity from amino acids 1-27 Co with a protein from Clostridium perfringens that is a PTS system protein (Accession )
Nos. NP_561737.1; NC_003366), and 65% identity from amino acids 2-27 with a ; protein from Streptococcus pyogenes that is a mannose-specific phosphotransferase - 15 system component [IAB (Accession Nos. NP_269761.1; NC_002737). - A Gapped BlastP (version) sequence alignment showed that SEQ ID NO:164 oo (105 amino acids) has about 60% identity from amino acids 1-103 with a protein from
Listeria monocytogenes that is homologous to a PTS system mannose-specific factor
IAB (Accession Nos. NP_463629.1; NC_003210), about 59% identity from amino acids 1-103 with a protein from Listeria innocua that is homologous to a PTS system ’ mannose-specific factor IIAB (Accession Nos. NP_469488.1; NC 003212), about "57% identity from amino acids 1-104 with a protein from Clostridium perfringens that is a PTS system protein (Accession Nos. NP_561737.1; NC_003366), 53% identity : from amino acids 1-104 with a protein from Clostridium acetobutylicum that is a 3 mannose-specific phosphotransferase system component [IAB (Accession Nos. N
NP_149230.1; NC_001988), and 54% identity from amino acids 1-96 with a protein from Streptococcus pyogenes that is a mannose-specific phosphotransferase system component IIAB (Accession Nos. NP_607831.1; NC_003485). :
A Gapped BlastP (version) sequence alignment showed that SEQ ID NO:166 : ) 30 (269 amino acids) has about 69% identity from amino acids 1-269 with a protein from | h . Listeria innocua that is homologous to a PTS system mannose-specific, factor [IC - (Accession Nos. NP_469489.1; NC_003212), about 69% identity from amino acids 1- 269 with a protein from Listeria monocytogenes that is homologous to a PTS system ) mannose-specific, factor IIC (Accession Nos. NP_463630.1; NC_003210), about 67% :
identity from amino acids 1-269 with a protein from Streptococcus pneumoniae that is a PTS system, mannose-specific [IC component (Accession Nos. NP_344821.1; :
NC_003028), 65% identity from amino acids 1-269 with a protein from Streptococcus . pyogenes that is homologous to a mannose-specific phosphotransferase system . - 5 component [IC (Accession Nos. NP_269762.1; NC_002737), and 64% identity from amino acids 1-269 with a protein from Clostridium acetobutylicum that is a - mannose/fructose-specific phosphotransferase system component [IC (Accession Nos. .
NP_149231.1; NC_001988).
A Gapped BlastP (version) sequence alignment showed that SEQ ID NO:168 (307 amino acids) has about 67% identity from amino acids 5-307 with a protein from
Listeria innocua that is homologous to a PTS system mannose-specific factor [ID : (Accession Nos. NP_469490.1; NC_003212), about 67% identity from amino acids 5- 307 with a protein from Listeria monocytogenes that is homologous to a PTS system : mannose-specific factor IID (Accession Nos. NP_463631.1; NC 003210), about 64% identity from amino acids 6-303 with a protein from Clostridium acetobutylicum that : is a mannose-specific phosphotransferase system component IID (Accession Nos.
NP_149232.1; NC_001988), 64% identity from amino acids 4-300 with a protein from Lactococcus lactis subsp. lactis that is a mannose-specific PTS system component IID (EC 2.7.1.69) (Accession Nos. NP_267864.1; NC_002662), and 64% identity from amino acids 5-307 with a protein from Streptococcus pneumoniae that is a PTS system, mannose-specific IID component (Accession Nos. NP_344820.1; :
NC_003028).
A Gapped BlastP (version) sequence alignment showed that SEQ ID NO:170 : . (111 aralne acids) has about 51% identity from amino acids 4-105 with a protein from
Streptococcus pyogenes that is homologous to a PTS system enzyme II protein : (Accession Nos. NP_269441.1; NC_002737), about 54% identity from amino acids 4- 110 with a protein from Listeria monocytogenes that is homologous to a cellobiose ‘phosphotransferase enzyme IIB component (Accession Nos. NP_466205.1; :
NC_003210), about 54% identity from amino acids 4-110 with a protein from Listeria innocua that is homologous to a cellobiose phosphotransferase enzyme IIB | N - component (Accession Nos. NP_472159.1; NC_003212), 50% identity from amino oo - acids 4-105 with a protein from Streptococcus pyogenes that is homologous to a PTS ~ system enzyme II (Accession Nos. NP_607438.1; NC_003485), and 50% identity from amino acids 1-109 with a protein from Lactococcus lactis subsp. lactis that is a cellobiose-specific PTS system IIB component (EC 2.7.1.69) (Accession Nos.
NP_266569.1; NC_002662).
A Gapped BlastP (version) sequence alignment showed that SEQ ID NO:172 (256 amino acids) has about 53% identity from amino acids 1-250 with a protein from . } 5 Streptococcus pneumoniae that is a phosphotransferase system sugar-specific Ell component (Accession Nos. NP_357876.1; NC_003098), about 53% identity from amino acids 1-250 with a protein from Streptococcus pneumoniae that is a PTS system I1C component (Accession Nos. NP_344847.1; NC_003028), about 43% : identity from amino acids 1-255 with a protein from Clostridium acetobutylicum that is a PTS cellobiose-specific component IIC (Accession Nos. NP_347026.1;
NC_003030), 38% identity from amino acids 1-249 with a protein from Lactococcus lactis subsp. lactis that is a cellobiose-specific PTS system 1IC component (EC 2.7.1.69) (Accession Nos. NP_266572.1; NC _002662), and 37% identity from amino . acids 1-255 with a protein from Listeria innocua that is homologous to a PTS system, celiobiose-specific IIC component (Accession Nos. NP_470241.1; NC_003212).
A Gapped BlastP (version) sequence alignment showed that SEQ 1D NO:174 (560 amino acids) has about 39% identity from amino acids 1-551 with a protein from
Bacillus halodurans that is a PTS system, beta-glucoside-specific enzyme II, ABC component (Accession Nos. NP_241162.1; NC_002570), about 39% identity from amino acids 1-551 with a protein from Listeria monocytogenes that is homologous to : a phosphotransferase system (PTS) beta-glucoside-specific enzyme ITABC component (Accession Nos. NP_464265.1; NC_003210), about 38% identity from amino acids 1-554 with a protein from Bacillus subtilis that is a phosphotransferase } : system (PTS) beta-glucoside-specific enzyme IIABC component (Accession Nos. : 25 NP_391806.1; NC_000964), 38% identity from amino acids 1-554 with a protein : from Bacillus subtilis that is a PTS system, beta-glucoside-specific [ABC component - (EIIABC-BGL) (beta-glucoside-permease IIABC component) (Accession No. splP40739PTBA_BACSU), and 37% identity from amino acids 1-554 with a protein } from Bacillus halodurans that is a PTS system, beta-glucoside-specific enzyme II,
ABC component (Accession Nos. NP_241461.1; NC_002570). ¢ . The top blast results for even SEQ ID NOS:176-364 is shown in Table 2.
Table 2. Top Blast result for SEQ ID NOS:176-308
SEQ Amino
Percent oo. :
ID ORF Acid Organism Description Accession No. .
Identity
NO: Range : ” Jto Lactobacillus emb|CADS55501. : 176 1463 83 . lactose permease - 639 helveticus 1 h Lactobacillus phosphocarrier “ 178 639 90 I to 88 ) . ref[NP_964671.1 johnsonii NCC 533 protein HPr ~ phosphoenolpyruva ’ ’ 1to Lactobacillus te-protein 180 640 83 : ref[NP_964672.1 576 johnsonii NCC 533 phosphotransferase (enzyme 1) . Co Lactobacillus h
Ito . emb|CAB76946. 182 431 77 133 delbrueckii subsp. pepR1 bulgaricus :
Ito Lactobacillus HPr(Ser) : : 184 676 71 ref[NP_964704.1 314 johnsonii NCC 533 kinase/phosphatase
Ito Lactobacillus fructose-1- i oo 186 1778 79 . 3 Tef]NP_965684.1 303 johnsonii NCC 533 phosphate kinase to Lactobacillus 188 1779 54 ref]NP_965685.1 251 johnsonit NCC 533 1to Lactobacillus 190 1433 77 glycerone kinase ~~ ref]lNP_784000.1 331 plantarum WCFS|1 oo ae . : dihydroxyacetone 3to Lactobacillus kinase, 192 1434 64 : reffNP_784001.1 194 plantarum WCFS| phosphatase domain dak2 ; to Lactobacillus glycerol uptake 194 1436 73 ] ref]NP_784003.1 231 plantarum WCFS| facilitator protein - i 1to Lactobacillus sucrose 196 1437 100 . gbl]AAO21868.1 480 acidophilus phosphorylase 1to * Lactobacillus 198 1438 100 ] alpha-galactosidase gbJAAO21867.1 732 acidophilus : lto Lactobacillus 200 1457 74 . aldose 1-epimerase refflNP_964716.1 327 johnsonii NCC 533 to - Ito Lactobacillus galactose-1-P- emb|CAA40526. 202 1458 84 - 486 helveticus uridyl transferase 1 ' Ito Lactobacillus emb|CAA40525. a . 204 1459 89 galactokinase 387 helveticus 1 .
SEQ Amino
Percent
ID ORF Acid Organism Description Accession No. :
Identity :
NO: Range ‘ 79 to Lactobacillus cell surface protein 206 1460 31 : refiINP_784891.1 ¢ : 305 plantarum WCFS| precursor 7 1 2to Lactobacillus .. 208 1461 27 ) reflNP_964254.1 201 Johnsonii NCC 533 to Lactobacillus “ 210 1462 74 . a beta-galactosidase ref]NP_964713.1 - 665 johnsonii NCC 533 1 to Lactobacillus 212 1467 99 beta-galactosidase ~~ dbj|BAA20536.1 628 acidophilus
BGAM_LACAC 1 to Lactobacillus . beta-galactosidase 214 1468 100 ) splO07685 316 acidophilus small subunit oo (LACTASE) :
Ito Lactobacillus UDP-galactose 4- emb|CADS55502. 216 1469 95 330 helveticus epimerase I
UTP--glucose-1- : 1 to Lactobacillus : = 218 1719 80 ] . phosphate ref[NP_965397.1 294 johnsonii NCC 533 uridylyltransferase
JE0395 phospho- 6 to Lactobacillus beta-galactosidase 220 874 87 : pir}JE0395 481 gasseri - Lactobacillus , ‘gasseri
COG0039: 3to Lactobacillus ref]ZP_00046547 222 910 66 Malate/lactate 308 gasseri 1 : : dehydrogenases :
COG2240: : 13 to Lactobacillus Pyridoxal/pyridoxi ref]ZP_00046499 : 224 1007 55 4 ) pr fzp_ ’ 279 gasseri ne/pyridoxamine 1 i kinase 3to Lactobacillus 226 1812 71 ; ~ alpha-glucosidase ref]lNP_965686.1 766 johnsonii NCC 533 ’ : : i : succinate- ’ : } 1 to Lactobacillus : 228 1632 69 . . semialdehyde ref]NP_965584.1 457 johnsonii NCC 533 ’ : : dehydrogenase : ’ COGO0446:
Ito Lactobacillus ref]ZP_00046159 - 230 1401 89 Uncharacterized 454 gasseri 1
NAD(FAD)- -
SEQ Amino
Percent
ID ORF Acid Organism Description Accession No. :
Identity ; : NO: Range dependent ¢ dehydrogenases ’ ~ : acctolactate synthase, pyruvate ’ : dehydrogenase C0G0028: *
Ito (cytochrome), Thiamine reflZP_00047198 232 1974 72 : 601 glyoxylate pyrophosphate- 1 : carboligase, requiring enzymes phosphonopyruvat Co . e decarboxylase 1 to Lactobacillus transmembrane emb|CAA05490. 234 1102 56 269 helveticus protein 1 : 1 to Lactobacillus ABC transporter 236 1783 68 . ref]NP_965688.1 ~ 298 johnsonii NCC 533 ATPase component
COGO0351:
Hydroxymethyl 9to Lactobacillus Y y yi ref]ZP_00046866 238 1879 72 imidine/phosphom - 268 gasseri . ethylpyrimidine
Kinase :
Streptococcus 8to , 240 680 56 633 agalactiae refiNP_735321.1
NEM316 :
COG1052: Lactate “8to Lactobacillus dehydrogenase and refiZP_00046778 3 242 55 96 ; 349 gasseri related 2 dehydrogenases . COGO0588: : to Lactobacillus ref]lZP_00047243 244 185 97 . Phosphoglycerate 230 gasseri Nl mutase 1to Lactobacillus lactate emb|CABO03618. . 246 271 91 } : 323 helveticus dehydrogenase 1 - Iyceraldehyde 3- . 1to Lactobacillus gl y . 248 698 92 . phosphate ref]NP_964727.1 ¥ 338 johnsonii NCC 533 dehydrogenase
Ito Lactobacillus phosphoglycerate -. 250 699 93 . refINP_964728.1 . 403 johnsonii NCC 533 kinase - 130 oo
/ So :
SEQ Amino i.
Percent :
ID ORF Acid Organism Description Accession No. :
Identity
NO: Range
COGO0166: . : 3to Lactobacillus Glucose-6- reflZP_00046229 : 252 752 83 i or 445 gasseri phosphate A oY isomerase 1to Lactobacillus reflZP_00046557 . 254 889 93 COGO0148: Enolase : z 428 gasseri A 6- to Lactobacillus 256 956 78 __ phosphofructokinas ref]NP_964935.1 319 johnsonii NCC 533 e 1to Lactobacillus COG0469: reflZP_00046514 . 258 957 88 - - 589 gasseri Pyruvate kinase 1 fructose- E 1 to Lactobacillus 260 1599 81 } bisphosphate ref]NP_964539.1 303 Jjohnsonii NCC 533 aldolase
COG1653: ABC- . type sugar
Ito Lactobacillus reflZP_00046816 262 1641 71 transport system, - 433 gasseri 2 periplasmic component phosphoenolpyruva to Lactobacillus te-dependent sugar : 264 452 69 ref]NP_965752.1 335 johnsonii NCC 533 phosphotransferase system ito Lactobacillus . ; 266 1479 71 . ref]NP_965117.1 ; 278 johnsonii NCC 533 - :
COG1263: . lto Lactobacillus Phosphotransferase ref|lZP_00046302 268 725 62 ] - 655 gasseri system HC 1 components, phosphoenolpyruva : 1to Lactobacillus te-dependent sugar a 270 1369 81 ) . ref]NP_964585.1 411 johnsonii NCC 533 phosphotransferase } . system EIIC,
E; 1 to Enterococcus PTS system, [IC ’ 272 227 52 . ref]NP_814084.1 e 436 faecalis V583 component 3 274 502 100 to Lactobacillus substrate-binding ~~ gb]AAO21856.1
SEQ Amino
Percent
ID ORF Acid Organism Description Accession No.
Identity : } .
NO: Range 431 acidophilus protein MsmE a y 1to Lactobacillus sucrose - . 276 507 100 . . gblAAO21861.1 ’ 480 acidophilus phosphorylase " | Streptococcus ’ . to 278 1483 59 49) agalactiae ref]NP_734585.1 .
NEM316 - high affinity ribose 1to Lactobacillus . . 280 1484 75 . transport protein refilNP_965069.1 131 johnsonii NCC 533 oT tbsD major facilitator : 1to Lactobacillus - 282 552 76 . superfamily ref]NP_964553.1 487 johnsonii NCC 533 ; permease : : : COG0477: ~ 3 to Lactobacillus Permeases of the ref]ZZP_00045998 284 567 79 - : : 400 gasseri major facilitator N : superfamily 79 to Lactobacillus 286 147 74 . refflNP_965113.1 405 johnsonii NCC 533
COG0477: 4to Lactobacillus Permeases of the reflZP_00046596 288 1853 80 -
R 163 gasseri major facilitator 1 superfamily phosphoenolpyruva 9to Lactobacillus te-dependent sugar 290 1012 77 ref]NP_964612.1 . 643 Johnsonii NCC 533 phosphotransferase system 1to Lactobacillus COGO0366: reflZP_00045981 292 1014 77 . . - 552 gasseri Glycosidases 1 1to Lactobacillus transmembrane 294 1440 100 gbl]AAO21865.1 277 acidophilus permease MsmG?2 " 1to Lactobacillus substrate-binding : 296 1442 100 . gbl]AAO21863.1 418 acidophilus protein MsmE2
COG1132: ABC- : ito Lactobacillus type multidrug reflZZP_00045932 \ 298 1132 62 . 525 gasseri transport system, B 3
ATPase and :
SEQ Amino :
Percent :
ID ORF Acid Organism Description Accession No. ;
Identity
NO: Range : } " ~ COGl132: ABC- £1 | . - type multidrug
I to Lactobacitlus reflZP_00045932 ’ 300 1358 37 transport system, 8) . 525 gasseri . } =
ATPase and permease
Ito Lactobacillus ABC transporter : 302 1838 71 . i ref]NP_965714.1 i 224 johnsonii NCC 533 ATPase component LL lto Lactobacillus : 304 1840 50 reffNP_965716.1 172 johnsonii NCC 533
COG1136: ABC- 2 1to Lactobacillus type antimicrobial ~~ ref]fZP_00045892 306 1913 72 } 233 gasseri peptide transport 1 : system, ATPase 19 to Lactobacillus 308 1938 59 reflNP_965786.1 364 johnsonii NCC 533
COG3590: 310 165 74 7 to Lactobacillus Predicted reflZP_00046938 : 650 gasseri metalloendopeptida 1 se 9 to Bacillus cereus Multidrug A ’ 312 251 43 184 ATCC 14579 resistance protein "Cf F-832933.1 ay 1to Lactococcus lactis multidrug NP 7 34252 39 117 subsp. lactis [11403 resistance protein reflNP_267065.1
Staphylococcus multidru > 316 253 35 1to 57 epidermidis ATCC . gE ref]NP_765487.1 12228 resistance protein 1to Lactobacillus ; 318 1062 86 173 johnsonii NCC 533 refiNP_965077.1 . ABC transporter - to Lactobacillus ATPase and 320 397 73 585 johnsonii NCC 533. permease reflNP_965013.1 components -
COG0477: i 322 1854 67 lto Lactobacillus Permeases of the reflZP_00046596 211 gasseri major facilitator 1 superfamily t . glucose-1- 324 681 66 flo Lactococcus lactis phosphate refj]NP_266853.1 = 380 subsp. lactis - adenylyltransferase
Required for 326 682 40 to Streptococcus glycogen refNP_358625.1 377 pneumoniae : . biosynthesis
SEQ Amino
Percent : ID ORF Acid Organism Description Accession No.
Identity
NO: Range ’ l1to Streptococcus - . 328 683 52 475 pneumoniae glycogen synthase reflNP_345595.1 . “a ~ Ito Lactococcus lactis glycogen 5 . 330 685 36 797 subsp. lactis phosphorylase refiNP_266256.1 : oy « } 5 to Lactococcus lactis - . 332 686 42 S48 subsp. lactis amylopullulanase ~~ ref]NP_266857.1 BN 334 1356 71 Ito Lactobacillus ref]NP_965359.1 : 118 johnsonii = . 336 1465 20 1 to Lactobacillus Co lactose operon ref]NP_064711.1 333 johnsonii =. . repressor . .. .. ABC-type sugar 338 1643 82 2t0 Lactobacillus transport system, reflZP_00046332 : 273 gasseri . permease Bl . component 1to Lactobacillus ABC transporter : 340 1645 “ 361 , johnsonii ATPase component refiNP_965601.1 ” 3to Lactobacillus : 342 1731 40 293 johnsonii ref[NP_964886.1 344 1732 75 Ito Lactobacillus © refjNP_964882.1 : : 257 Johnsonii -
COG2148: Sugar . transferases 346 1733 73 4 to Lactobacillus involved in ref|ZP_00045843 217 gasseri k . . 2 lipopolysaccharide . synthesis to Lactobacillus 348 1734 68 255 delbrueckii subsp. EpsD gblAAG44708.1 bulgaricus 350 1735 66 Sto Lactobacillus tyrosine-protein oNp 964879. 1 227 johnsonii kinase -
R Ito Lactobacillus : 352 1736 54 288 johnsonii ref]NP_964878.1 .
Sto Lactobacillus : 354 1737 52 340 delbrueckii subsp. EpsA gb|AAG44705.1 . bulgaricus .
I to Lactobacillus C0G2262: - reflZP_00046671 . 356 1738 75 417 gasseri GTPases 1 358 1739 42 23 Lactobacillus refiNP_964123.1 331 johnsonii - . ABC transporter p 360 1782 55 410 Lactobacillus permease ref]NP_965687.1 | : 405 Johnsonii component . beta- ; to Lactobacillus 362 1869 87 220 johnsonii phosphoglucomua ref]NP_964230.1 i COG 1554: - . -Trehalose and 364 1870 88 le Lactobacillus maltose hydrolases "*1- — 00 +7083 . } & (possible : : phosphorylaces) :
o o tn nN loll] om mle o . 3 vy ih pr I Rl Er MA hel s RTO FL FOR BR FR I > o o Oo |o o 1 ~ © a lo - wn Mp w © © Oo | N] © |m|© < . 3S
Z
£ { — in [ve <. | fey fo3) © I~ 0
[7] nn Nn ~ NM wn ~ [wv nN u ~ (30) [e¢] — {Ny on ™Mm | ™m §
Q ~N o oO Mm 1O o INE [=] o I=
Id o o o Oo {O oO oO {0 o 2 - v LL uw LL [TE FT rd ww Tl << a a a a |a a ja ja a : = u
Qo < . : and 5 [= . © © : a 2 oO oO oOo by! 5 = = Loa | <« .
C PT) uv — uv ft — Lom wv — @ a PE —t Fu] — wi w Fe — “ 2 a S iw c > S w - - S wi .
Ie] © v ° © I= [ao £ EE |o El =) wn 0 c E|E |c oo |e = o vo 2 om |o 8 Iain leo 8 . = o re} a wn Cc sila villi wn
Oo [©] le) > = a |¥|@ >|h (hb |@ > > << > = ££ | wn © un | © oT © : Zo = cle wo £ Sims 0818s o = c £ UO 3a wn [] Vow alm lo |[o wu [ = ~~ OO | © © Cl lo ow (= |@ @© = £ [1] + 3 I> nC “jojo |> = [TS nw nis ww [a] > 0 | | [5 ww
S < o E% | g |«|l€Elc selec 5 fa ° = a Z i= 12 c SIs i> c @a 14 © oo zc i>12 CIC |2 ©
[9] ue — Ie} Pg vw 15 Ll+ | [TO fy
[8] 0 + oO Fy oO [0] + & uo ~ c 9 Elle i228 © o = £ VF <Is|o lala £ =3 I) © a Oi= fo a l|lwm 7) o [al oo 0 . “ J 7) I | 0 o o i = wn .
Q & IT Q oo QQ oo |e le ja © > nn clgdial? < o 73] [eo] 0 [le] a oO [a¥ , 5 = n Ko c on 9 < un a a a
I) a o-
R= on .
Ef ™m — 3] ~ NN un ~ ~ : = 0 VU — < A 3 = [1] « [« J) ] ~— mM = © IN |Z oA D Ma “ 3 & Flea 2 NNGR|Y mE] vw :
Q + N Ss ~ ~ SE — =~ nv & 1 E = = © Q G6 oC] a [BN ~ 0 = <g 14) < 2 o & : gs a < ~— o ~— i c I (a) | OU {m
Ye £ ni! < ols|l & [22] « 8 £ 0 w o R= wi 1} w ot [2 Ie) { a = | I Jn jn | # = 0 o o wn <q |O wn = |= wn = i Ue] = [a |o o — a = Qa a el 2 © 2 © £ © CW ~ a on <r ~ ~ os wn
I o S185 ERIS RISA ~ 2 . o ai a < © @ © |o o~ oN | ev << a Fo ET : — =i fet — } £ o 2 2 5 = [7)] 5 .
a) nw i~N Om jlo jlo jo | ~ ®w [0 [mun |e] © 3 PTITIR TR TY oY T IPF ITIN A — ] [] ro w {Ww jw jw jw jw jw] ow Ww jw hw jw jw | w > ojojlojlojlojo jlo] o oO oOojloljlo lo} © ; ® [0 10 IN IN IN |= 9 I I a I w Mm | [0 |- Nom | — Mm NN Nn] ) .
Zz c 2 0 [NN |oo |O |o [0 | ~N © jo NJ lo] o v 4 No is = IN IN INT] [Vo JE A Ao J FS FO BE Vs n Mm [iM [© | (mm | mM Mm minim jm] om .
Q NON im NNN Oo oO |N Oo jiN [nN] © o olojlojlojolo jo} © Oo |o jo |o (o!] Oo 9 IVER EVAR VU FYE TOO SUVA [Ti ET [VERE EVER ITH RVI FTI ET 4 a joa |a ja la joa a | o a |a jo joo ja | a s . g . U
B 0. dd . “ — . [me © © v + 3 oO — oo ~N 9 Vimo |IVIVI]l a |3 O [m|2 0 |35 c SEEjlceElEEls 0 SsElB3lEd SE uw jw ww wow |w| wm |e = lw |w Ww ja i
Ss Sls jE Be (HE © —t .
So E|E|E{E |E |E|E E |v glEIEI= Elo E wn vo jo luo lolol | a |c vo ojo |e |e le je Of je (a gy) Oe (Ee |g ©
Oo vm {un {un I RR I Ic W = A I I = I P= 4 . = >a (CC |I>>1>] > wo [> |Y | uv [®] nln nijgtltnln (vw oo (Q >No lon (lo > g - > — T ©» 0 v On = jo jul, OU (©, @© 1 JO |O |x |} o - FO I IS SVL 2 2 J SR I RJ I: I wile 2 £ £ Plo mg |e |e B® lg G00 8 0 8 8 £ LILI G80 (0 8 |> 50 |8c|0|3 5
Ww wile laid viaje &@ |2 & 16 ijn l0 |v |2 < clc |e |x |c |e lc Cc > vic lcs |c |= © © |©o | © © |@ |@© © A € |olo |E |o ja ©
LISI IES Isis 5 |= © ly [ow je |= © » ooo |wiojo of © Jg ££ ijo|loj~|Ooig = pd clic alec] © Jo ic |€ iE |S |g © = alajald |alafal a |g £ila|ajg |a|g =<
I wlu lu Fiolo ol oo |g 20 vlog . 4] o|o |o o lo |o la} a Ve (o|vwije |g © ‘ oC Llc lc nic jc |e) ££ 1&5 9c ic |>lc|P © a jajajajaja) a |g Sa |o|”ia | S } . = a. c 9) £ << [% |= o u- o a 0 |2 = =| © oO lon RI 7o Wl I Vo IE FN nS ;
SIS ELE InwIBwwidIBl7lo @f mm (Hiro jo] o -
Flo IS nS LT Do TT a SO ATE A A I AL LA Lo I £ 8 ¥ IN| Is clon |i A] = - |g im . : = g [NINIMmI™"lMINI® jin | M Mm [= | |= wun :
E & I~ LV | uv |H —- {tn . < n — gS oN : c Uimnmjuilc|lolL]|U]| © IU jo |Z |u — — r= J PO = f= Je | gC f= f= | = wow fw wojw fw | ow — jw jw ip Jw] =
E Ha lod 1 Lead 1nd [A I w tld l= | Ww 2 EEREREEIL |» PELE]
Q a (a fo |Uija joo laa | a Eo|a ja | ja | = a K o : < in lin jin iN INS
F SRI ISIRIS] 8 o |oloiI~N|InN| ©
NN JO |p jo Ho) © 2 RIRININT WN 1 lo} EA = ol Lo Ll LT ET EI fa) T (¢ [v jojo jo |v] wo + (sls jo jo | Ww ’ ee [j= NIN MM mM mM Mmmm in| mM gz o o Jo |=~ o on No] m © Mn ™ ) 3 EE EE EEE AN —-— [] J t - r] w w |w w uw w jw Ww Ww ww w > o o |o = o o |o o o |o © ! ©° vn Mm <Q LN I < ~ ™ © w No {HH | « -“ |vla}) wo © |< tn 0 > . c 2 oN wo I~ oe) @® — jun ™M nN < 0 n ™ in |< ~ ol = tn o fo ~N 0 wn ™M jn nn Ln ~N lO Yo) oO ~N nn y o oO | o Oo o lo IN o o Oo : . 9 oO oO |o o Oo o |o oO o o o .
S iw (ri rt Ts Te TH ITS re a w {a a a ja a a o |a a o a a = < w [+8 £1E E E E :
L 2132 3 3 3 2 5. ElelE |2 g 2 : @ 2s |al|a o & 0 |& 3 vs Elg|2lE 2lg 2d] |t PR o |.E o z c £ o |S | a pd a ps > a £ a
Q Oo |= |e |® 52 & IT nw Cc xX |x © : wn w B2luls|5 als alec is 2 | vc 8
EE Sic | © £ © £8 ju |x © [ © S e [¥] o Ole lL Oo 9 |n Ro) S TO |(« 8 g 0 J |g Oleg © [8 FE o oO > | i—r} Oo I= aQ Q lu a > 1c o = a cls vl lo wiv ig|g © a jo w :
El E |o®|2|l|E SE El8lSlcals |B]
E ® c close |g 2212 3 @ |t|g © y= a [3} [+H] U celzigis EIB E128 88 [2]8 6 “= o Sloe lc Elec Elxi<|s g!| < [Ele E wn = QO | | oo |u ow? © Aa 5 = [oJ Rui PT} Oo © 3 E22 |x| 28 20 5S - “5 2 w = gp |O ud pus > om Co Cc o a © |© a ££ la £5 . a £ - a « . © t = [ — {QO 2 Fru 1 = « < 5 (2c |? 2 < ale 5 o O lo t= R= [v] |e : <C a a 1c |= a Cc Ee]
Ww o |E £ © R= a mn (od m fie} e1] mM . pod a 2 |S 0 2 - oN oo lo |~ n> NN o : so |< on m [Blo] o N ol=| & 123K! :
Fle eg . ™ ) |< ~~ ~N Tin R ~ ig ~N
EE not o [Do] « o It [2] a Qilsl © £ an 0 ~ © @D Nm @ ~ :
E® 8 on & < n — oN — A = I ™ ~ ~ } c ao! Bz o o o |e o c a 5 & s|2 | 2 [5g] ge |¢g 2 — £ i I= Ey © a] > 1% © + 2 © [i © — = = = | rey i“ i oS a |Z 4 lala] 2 FS A §
RS o ; } a Oo a a > 0 a w od 2) in < 3 Bla] 38 |3 B82 |2 [23 ro) tn in in in in th {0 AA 3 AA x a @ © |o ~ < 0 | < te) o oN =. ™ mM |< < < < |< un nN ve) 16] o @) X w Z . nn o Mm Noo < ™ < o | vs) [Ro 3 - ~ [un va < << —~ |o — — NN . — J 1 ] ] 3 J [1 [] t [J © w ww 19} w tw uw w wg, | w > o Oo |o o o Oo Oo |1O o oO is | © : 1 o << N < < AS I © nls |< - w ~N ~ [00 [ea] it fe, | —t [+03 I) oN » fo) : . > . c 2 © ~N {un Ln mn ™ Mm |m ~ © |= | © . 0 oN + {Oo o 7) nn ow |< m nn in [tn 0 nn un Io Oo 0 O oO | wn a INE Ea)
Qo o ~~ |O o ~N oN © |— oO oO Jo | oOo 2 hot oO © |Oo o o oO [wile o oO |O | O ; -Q Ww uw jw Le w Ww Wwojuo ww wow | Ww - << a a ja a a o a {io a a ja | a = : << ™ [« 8
E 1.£ CTE E : i |g i |g 5 [2] |B a >. uy > > Clo o |E o
Oo [7,] a wn [7] = ‘o 3 = © a . c a oO Fe Prd LB “— iN 9 SEIS | BE |g_12EleSleln|z 3 a @ 5 a «+ lo «|g |x| «|g |> FT]
S a s2] 2512 518lals 5 |0lE|S [9)) 5 alo |o “ CT CC |@ CC a wn bi 4 on E =
Fed £ 1 Q Q - 0 fat Ia) Q = c Q @ © fram) Q +“ al al0 |e D = |= a = oOo | gud £ hl I] oo @ LY oO 2 95106 6 [8 EB E|*|E = 18 | © < => S o lo ie a CT 60 oll |2 Eloi ; . o — © clo |2 A © O|® O |g Qa QQ w |gjc | QO c E C © |v |g © 0 olo ol |€ lo ©|, lo|® £ [] v = jo |o hg Cc wlc wn | |O Cc cc |C |x > ww a oO |= |+ = = @les ©1091, = =lo |v | 5 < vo £12 |o 8) E 0 |E oo oS |2 BE [>t E - 5 © | |m oa 0 FF | E c |= 5 E {0c Py © o | |= | & w= EESTI < |e gle |S ly Si2inlo 1) [TR J UT am aif [5 |= v 13 c = rank] c £ io lo |E | |8 io > o c 0 ~~ 10 oO © E |= o pul Q 3 y [9] © > wn [a% oy o . oO 3 [1)] C TT — —_ Ka
Q ER ° bH nwil>|13 £15101 a o oO — oo ° |2 3519 [7 c pu ££ ££ ful J le) = @ oO u} ao 0 , z 5 c = & © &
Z c og | © © mn — “ a om [aa] om [en] ™
T o °@ ‘0 l=} — — . — . : re) QQ «= ~N | ~ ow ~ a wn [e)) ; os |< an 2 IN[2IR2| o NINN ois | D :
Flee sl 5 [RN Xfm | § 9: | on (oie |2 ££ 8 t A IC ET Pl al co [Biv] o |m[S|h
E ¥ ~ I PEN wn — ~ ve) ™ | {n apd [4 1} . ~ ~ o~ 4 ~ ™ d Tle l| ¢ a PU HT 3 2 gigs | 2 | 2 [B98 gs [i+ Lom] £ s [31% “i © © © |O ! Q |= | © . fae — © QC lc | “ ° ro} 1 {OU [@) o Per} >| © — fir] | 1
I |& lm] & | | = a olw a] (a) Q ig << (a) 0 wn [> fo Oo | E a 0} a a +) 4 > la . ry) o © a ao G ; w nn Oo |~ < in 0s) ¥o) o 0 |v jo | % + + 18-1 & S183 ; © |o|o| oa a o I8le| & 51212 (o] Lo) a | +4 —{ Rae | =i Ln i Mm 0 <r a <t O [© o oN < Oo jo los] B= —- ts oO |O ~ ~ ~ w | a a |S 9 : o 8 w = 172]
0 0 [« tn ™ |v {in |G |o jo ~ a — |o 3 WO |= <t <t |v —- | oO oN oN O |M ad J t 1 ) ' ] ’ | [] ) ] J © Ww jw w wou jw fg jw jw w w wu
S o io oO Oo |o |o |g {9 |9 o o oOo |{o
Cn] So Nils IFINI@ 6 | © [Nd w < [un < — Ne |g | | ~N — — 0 o . r4 =
S 0 jI~N © 0 |~N |nfo n [on 0 0 ~ |w » ~ lwo 7p) ~ |v oN JO [in ~ ~ t+ ® Mm 1m ™ Mm (in [HO [3 wn tn wn lm hd NO o ~N Oo jlo Jo |o |m oO oO fe = : Q Oo {oo o oO {lo [0 jo [jo |o Oo o oO |o ; v Ww. |i LL [VER [VERN ITH Fra JY IT IT uw wou << a ja a |a la ja (a ja {a a a a (a = : < u. a : E E £ . > a [T] fT] 0 E » @ ° wn . i Oo ov \ 3 QO mo |3 O | lm | } o > SN c I mele SEs o T 2 oly vlols g MI Ll il = Al Cl = l = S 5/2 §|E|E : < [IG IP 4] £ |EIE|B g|E|E |=] a 5a 5218 wn ov lo |c o lo |< c 3 | = [J] og Qe en [Of © © 2 |0 Ig . © 4 wn wn a [7] [24] c pros Q c fa E — E ¢ Cc = uw > | > uv [>= [>= |S je lS |= = o [CE (w] > nln |Q@ lon l|lo lo (= oc lm|+ Sle 9 (¥ iT : 23 > 0) oT © = c Vie O32 es = vo lo | vo |o |e alES bn £
SN ‘= d Bilge 31g (gl5|8|2losiu Elo 218 |° = E © © © © © © © R 5 © ic © Cc © - |T
E © 5 [Cle sie iz|lg Slug 5/8 5 (5c << = nin |g «lv ju |o|w oa o S | © . 0 CIC |x 2c |c 21810 |0 (J £ < E |= & ISIE SEER ze FIR 2% gg |= 5 s £ £ 2 2 ££ el |c |v |© Els 51s |= = < ajalig £ |aial|- (© Oo £10 ¢ |X 0 a nin le ale jo |= (co Lt ET o (VU
Hf a °ol12|5 ie |o|R |e a ta t£ = |G - | 2 : £ [£82 c|& |8]a |< @ =) = |< s [oY Oo [=% -~ c c QO £ 0 = = » << [3 . mo © oO © w m £ E © a. mo m om
I) 0 | 2 by = uv 0 ~ | fe) wn , a v0 +o ™ on iN o — lon coc |< on Sle vw RIFlo|n|=|@2f o a |= {in = °c | ~- < "No («jn |) a ~ < cE 8 & nll INS IE PR LN PP Pel Nl Bo © lo |o —- — o jon Q Vo)
E (0 4 5 - < A |= (a La I = ve) < | : << 0 .
Q — — ~ pA | |S 2 c O |mo | QO jo 8 1c a oO BL |= ~ = = |= < = 18 le [218 jw 4 u ov IR pi wf | = w |w (5S |E is |; 5 p= 3c
E oo YE fa 062d |e] & 5 Is [9 0 a Fle wo [BRP |elalF ES) ) ’ o ja. ~ o |o L£ I< 0 a) | | m . a oa a. a 8 a } [a9] [a1] - < n : w PI ~ NN me lo jo Qo ~- oN mM oo o lo — all Ea Ea Ra Tu IR Ia] << < < |< o IF IF o oO jo |o |o |«¢ |< < <t < |< 4 i i ~~ ~~ i ~~ i i Lan} -{ fa) IN jy | IT it © |® [Oo {lo ~ < wo © - - oe. Oo |O o OQ OO |O {rm |v — — — o 9 i i i Lan 4 i v4 v4 «i vi i ri Lo | Bl w = 1]
P) o OW nn [in jo [Vv |Mm jn Jo |O jo |T | [vw |v [I~
E ol Rl Rl ol CR Ce Co El a El hl
Ir w woof qu Www wo jw fu wo lw jw jw jw fw
S o o|lo|o|jojoio |oljo jo |o |o jo |o |o jo » : SE Ll I CH CT I ON I a CN bo I Ce EO ET : - : uw © AVI Fe J ES SI VS FCO (Si pi DSI YS FyoYE PEO PI [SY : - I . ’ : 4 . c - 8 wn nn (in fg fn jv (un [Nn + [8 jn (vb |[~ |(n . » 0 oO © (0 |v jojo (jo |v |(o|jv |n |o jo |v jo 7 — Oo Oo [0 |v |o|o lo |v |0ilv |t | lo |v jo @ o oo |o jo |o|o (on (00 lm |C |O in |O +
S oO oO lo |O |O |o lo |o jo |o|o jo |lo |o jo |o - rd [TERY VEG VEG J VI STIG VIG FV § Tl § TH VW Evil Eyal Fru Fru Fr <g o o ja [a (a ja | |a ja |a ja ja ja |ad (a ja = [4 i a -
S & 0 x 2 : 0°
Q = oH . [o)] a : = : lo [0 . Cc le jul ul . g = Q [\}) . oOo f- Cc . Cc h] 22 ° Q o |= Q . [17] OR =S IR IES [SO CU [Soy [ES pr ols [2 |E =e {VO o X glee (Ele 2 |9|8 ie |E lo i® a & > cc oul2i81g |g fee |e o a |l© (9 |E |o 2 alala alalalg [alE lalaly |a o — > OD joj iu [vl |jn |g 2lojo Mv a alv c E 5 « |c |g |c g clc |e c |g (VY Is c c.
E © Ss J IBIEi PBIB IC ICID (SIC lo ||P PTO : < 3S S00 Q | QV] lg io |U(u le fu ° @ oi I< |< {5 |<< |< |2 |< |5 SE FR — 2 T Oo a eo a lo a 3 = Cc c . in 0 © © & ks] far iS . © O QO z | 2 2 w a ™ vo | 2 a —= (7) O 0 ~ os fs 0 IN oy o © ~
Sl 8S NEE ERR EERE EEE SE Ea ERE Ei oa
Flo ec . JR rE rt rt LO De tN RT EN SS ER BC LY BC EY - cn le) 0 < INYO oss ltisiN [oY lo . - ~ <T || 4 (SF |< to} i lo}
E = La Fo ST TN Fo TN A Fo WN FW FS Cl AA =i a IVS BB ol << wn v : ov : c c |B 8) © © c cleceyje |= 1c |e |c civ |O |e | C - El ER FR = ER ER EC OR El = MR El 4 £ < ZEEE EEEE EE RIE5E u [41] .-
Ss T [VIO Ig UO IOI]E [Ug |S |V |u| ju a jot m (0 ja { [© Od [sd] | |S (@ | om
TI < |< LC [IC |X |< <u |a |< |< < [a] o (& : << << ww ™ ~ f |N IN joo jo [lO | jo |o jo [ov [Mm o <t + (nm jm jin ln iN jo joo jo ov [mm + : o <t Lan a I BO I TO Ha TO a TR KV TE Vo TE TS NO IF SNUG oo J I o J 0) =i ort ft let yd [ed fed (= | et eH |e eH
Qa © NT Jo Jo {oo IN |g |o [¢ | [Tj foo i ” —- -- — NIN IN NIN mm mim (te [| n o © i Ra BEE BE EE BE EE IE IE I I I I I RS I RI I I RE I I w = : wn
/ S . S ~ o 0 ~ un | 3 — Mm lo | © ~ 0 ~ ~
E EEE EE EE a EE ER i © w us we w ' > oO oloild| ¥ o © |e] o |[& S S] > © eg 8 | TR [Ela RIQR o pd - . c © ~ wv oN (oO ™ oN - © a [a] oO |o |O i o nn 2 a po a MN a O Oo |v [© 0 a ™m © MN Mm | [In hd ~N oO {Nm ™ oN o o o oO | [in a 9 =) o |o lo o o oO o o oO {oo lo 7 & id [TH [TH fT il [Tl LC i [YE FYE fT : ’ : oa a [a |a a a a ja a. a ja ja og - [78 . a. ”
S I) = - I. Qc 0 clan I © © 2 I's 10) o |© = [SI oO ~ |» bu] : a |o = 3 QO |3 = |E |= j ony — [¥) wn < — on < Ie) [3] . bl £ bey @ — | — o c y br a 4 — LJ a pro) © pad o SI » gs YI-Il5 Ye |g |E
QQ + - ry pe. - © i) o fu c 1S 3 = & Pelee slg IREEREIZIEIS © © 2 clo o 9 | a | 9 : o o v vo |D 2 2 jo |Y oe |e e cle |218 ola a 4 l|l% la %|8 <1. b>; o £ co |E SIE 210 UV >| | >a |= |. fon als — = = & |T wn © [2] Oo Ir o oO — [)] wn [\7} DISS © c ] [4] 1 at — £ . e| % 5 2lala|2 8852 alae gla|s|k — o © ' lo} D8 © = |6 © N £ > O js bt ic Ov JF |> lo |> = c £E |c < + wo vc = 2 nw | Q hr = CA TR I = Lv [© IR 2 [40] > | + 5S wn Past ~ [73 c N oO 5 os (2lg |v a Sle la clo |E |e [hwy Q < Q oO hd © —_— © — = © Q. QQ [= o ud E12 Oo = |< © N © a a lvIlE 5 | g ££ (02 5 IE |o|= = E18 «IE o 2 |€ ig 9 loc |S 2 s |@ 5 © a|2|e &|ZIE|? o o> a c ald lc & a I= a . oc . Qo 1% a o |e (a o < jw > wv un x 3 n ln on o 0 ] a by = o 2 s a = n Pad £ wn : x — |® 2 a a Ea a | a a ™ o To a . —_— [5] [e] Es) ~ . rey U oo | te} iH © 3 :
Flo ec [RINT] Q i no a9 |e nN - cm y © EA oN ™ — y IS > ~ so . = t old lo |~] wn SHR Co = EN FA PS hang
E 8 NO tn < Tin ~ : oO |Z — — o <( wn £ < 18x18] 9 © < J < |& pr £ — — pt £ 4 Sw? = ls ME GIES — pre = ps o Qc |=] a dd I (wn I FL R= = 5 a w fom wn —- wn - 3 3 oe : < i a i o SN PE a £ SS
TT] oa a
Ww <t oO |e v AA PAS PAR IT, WY [Ye 0) 2213 [ooo
S oa on lov [0 7 ~ a in 4 m I< ix oo pd a bl <t © pd pull A ow |O |© a <t o |o |v leo) = << < te) © |O |o - =. wn © |© © O ~ ~ ~ ~ ~N |o | - o (o} -{ 4 4 ~~ Lon} i 4 ~— ~—4 + ee on 2 n : :
[3 Ve}
Pn nd Ei br= AT- J 1S E° b-G VR Fe SN ™ nn {Oo
S Mm [=o IN mo [SIN |m jo [Qo ]| 2 oa |n r= wow |b (wo fw fd | fd fl fds [gu : Ww w r= : s Slololololo|l8lololo|8|lol Y o |o o ei AA et A CEI LAC BE Pl Ao J CT LO PS Ci BN Bt m |e <I {MN He PCT i EI a TE PON on oN oN o - 2 . £ : 0 ~ [0 fn |m|¢ [bb |m |v jo |o [in . a oO (nN On mmm [Nw © 3 ® ® : 0 Omit on |g NINN [=o in] N o oN bed oO 10 IN NN JO JO IN [NN |O JO [IN |m o~ — o El
Y Slelelololoolo|oio oo | © o |o gl b rif | | | | {a | | © ww i oa ad laja ld a |a ja lad {a |a |la| a a a : = ’ q tw . a > ~ ~ £ c |Z = & bs a 9 = |E = o © 3 c EC |unlnld |S k= 2 |e a w "lo |= 3 | |@m |X © [7] 0 oO 3 © jvijec lc jE |O £ Cc c c c vo IPIEIE IZ |Y c |o 2 ° 5 on ACRE RERERS EH BY a2 (2 gl o ole |S [2 |8 | |clc|O |S cg gg gf 9 Eliz |v|o md (a|=le & tT 51S 5B > g|Clele(Z|8lElE|L|R|al|E|2 82 0 . fe) o— a lw wig ls |0o]0|w |® 2 ia Q — 9 = £ = E ~|2igle lo (elY(2 |? |s ® (a ®|> © >IieElE le Ian IE lg lhl El £5
N (3) rae) w So |X |= |m jC — QL I a. < c OG |[¥ |X |c |v |= E EIB
Oo wl |o om | |= | © 218 = lg cs
L Llc lc |>|m 10 0 |= |= S18 a2 13 [e) iS [= {= |= |= Oo > Oo lc ole oo a < clog la (E23 © |E 21202 zy 9 REAR ERES s |® < 14 - <9 — —_ _— [14] [] [) 2 ZS |® la {a |e |= c 0 o ©
O lc iT |T |X |® a a 3 i; @ a |e a5 < g 18 a 73) o + 9 © a = a |g 0 fo o = ' < [10] [1] [33] ww Mm om I a 0 Oo 0 2 |2 = = [3] Oo I~ ™M o ‘ — a < 4 br NH eNfeN je - RLM iG [0 a o wn ~ m |m jo fm Bn | |o [VN] « Nm ~ lo ££ . Lie | | <+ [2 |m ~N 13 € nv Wi iglolZT VY VN glo INlgslmis] « SN = 5 TIPE INICGIXI~I8 oR] ~ in — £ 8 |~ — ~ ~ — — < n i
Ue . wv te | | © (S fo © wn = |g |g |g Slala|s | [8 : = Igle ole |2|¥ a |E|8 Y%] a ol |S
SInjE EIR lo|m|m|2|n(2 || © = ; s 3 “3% ooo 2 a = 21 2 S |e a wi 8 |& STB od |= a = | w® = .
IT |T << < U] 8 |O w 0 [00 [|e oN Jo IN| © os) fo) 7; filmi inINm@|m mm [vb] ©» nn ‘ 0 Oo frlo Jo TIN IRITIEIEISISS RANA oN Ig [or |o [oo [ne |v jo jlo] ~ oN < - ae © {0 | [© |© {© |ov joo |v [ov ov [©] © o |o o Oo ord vet fet fe fed fet [et fed [et (e- N oN oN oN : : uw = : (F2]
0] [084411 PCT/US20605/007594 o Ve)
Q nn [Ve] =i <t i ™~N ™~ 0 3 dB EA © ~ a © 3 Ss |N™
ER IT A A I EA BE I A i
S =H = o o o o & So |o|Y : IS) ro a < = a 2 re DO ) ? } Oo it 0 . w : - ~ ~ ™ © . " 5 1 — ~ ~ A LJ oS 3 . . N =) : . 2 ol wo ~ ™ on o
[4] < mM ™ o Nm R ~ > ps 9 < © oe} ~ a on ™ o a [(
Ni ~ o~ o ~N ~ — — o |o =
Q oO o oO o © o o
O o e he Q oO oO |O : < w [VIN Ww uw. [SW uw [798 : a a a a a a a aja < lL mw o 2 = |m ® — = © £ © : 0 ud TT g £ Lo 2
U = Pd . £ ©
S 8 |3 3 | 5 E IE > c ~ 2 = g s 5 952 |B Ig El 2 x o> > 3 = © Z oo flo [> . O 3 = ~ - o wn I= e gla @ E 3 |S c ° oS 3m |E ° : 3S [> oN ol BE 2 @ $ Oo (® < > 2 |E = FE nt © ggg c l= cc -— = > c 2 gS |E E£ £13 s8l= |= |x |x EG o £ 5 |® © iE ® (0 BF e @ e © £ Z|o 2 |5 |B = [i] M lo «Oa » UV [0] © Hin £ “— £ o a E br E|E E TT ~~ I= Ix : = B o | ” 5 w o ca mls Ti>|o < oO|nw T |g Vis Xjuo Dileo © | Y= oO i> : wn om |S = nw S| wv wn) oo ao 909 ° o° o ale o = 91318
ES re +
S Oo Pr} Oo ta) j= 19) = Y o|0 0 ° [2 |> > = - = on |B 8 o > oO & 8 Iz |° 2|s [8 |° 9 1) [=] : = S 0 2 lwo © a © x 0} 2 [i U pd : ou G} E |= @ : a) } vo | 2 2 = [v} OC iin ©
Kal << QU 4 ~ hn) wn 3 ah lo ~N N ™ ~ — < < ~ lo fd oc . |Q] wv o~ ~” o ~ o oN ~NOIS £ a8 ¢ [Nf m or on — Ire) m Mm g o s o © a IN < 2 I) ~ nN g [1)] ) ~ | © z ~ 3 lo ~ i = O ra [eo] _ | [\)] a} i c o — o = c| © o 5 = © a LAN RRS © ° = = 2 © io © TT I'v |Z
E > oO ° E £ IT} | > : > > = c o 5 £ £ o ) A E 8 Is 5 ‘ a S Io) o Oo © ‘© a a [T]8 > Oo 0 > J =3 tw ™ a | >
AEE ERE E |G 0 o = w a ~ ~ ~ © o o| © © © © > > > 2x o < < < < < < ~ + I~ =i 4 i ~— =i i 4 -—t ~— © oO oN o~ ~ ” a |g <2 > = M) AJ a A bl o 9 o~ od ~ ~ ~ o~ ~ oN Ny :
S wn } )
v o oN ~ IQ lo] in < NE RA EEN un ™ o oo © 0 he) 4 | ©
IE 1] ' 1 — 0 ' ' An I 1] .
EE EEN CAE EA CRE
:- o o 7 © | |= [81% = |S |= [8]8|w w - ~ ™ ~N | ~ < — - jn
G .
Z c } 2 O O <tr wn [— oO 0 wn oO (in |v . 7) tn O oO wn ~~ ~ ~ o Oo |o|o “a o 0 oN oO | o CIN ~N 0 {0 |N hd : o oN o - |O o oN oO O |O]O 2 bd : oO o oO oO o o Oo oO jo | oO . o LL tw [TE IT w we ow ju | Ww gq a a a a a ao a a a lo | a = g w a o : £ hic © wn 2 a : =
Ii) £ ~ ’ =z ft
Q 0n © be > - [1] hd & o |a 8 |= |E |S gc 9 © 3 < ir] ££ = © |w £ o Zz © x |Z[&|a Xd E|E = = « y Cc = c © n o a =] 2 EIR(3 o|0 © 9 S| | 2 o 0 © © J lo |= brs Cc | [LI : >] c g O BO 0 lod ole E - 60] 8 : <C = vo CC > Q Q [ >= = Oo = |9@ © Fa nw [Dio ic Ble S15 || 2 o E lo iL oc [mils gr ER ®|al2l0 c £t Eo 2 0 cloi|e o|8 og Elo|sg lo £ [\] VT oO {> = fe husk > {0 LC o a nn © C ag fo
S > 0 le E © OV lz {O o |E - oc |+ [1] < re o = J >lo |3 © |S = o Slo | © 3 CT © > cole {2 © a Tila > 8 ° @ 5 E |Zlgle oa 28 © |< | E mn c uo 3 © © o |> |= vo = |& 3 ©
P= fo © u“ o |l& |= c E 0 hy 3 © f= oo > lo |= = o i fen] o T 9 a < 2 E a \ + fis] : © : % BG EE 3 |S i mM
R=) o .
CC 3]% 0 8 © [N53 o o
Si< 88 ~ © |o IT |vlo| « Ne TT BIS ~ << [] — NIN |1n [)) ~ 0 —
Fle cg - ~ - ~~ - RRA ££ © ¢ ~ < o ~~ |W < ~ - ™m Oo IN g 5 ™ < Q ~|o|m IT) < o pull PAV IH - £ — ~N | — = rr p= i . vt “ < % H x LW) 0) c = = Sls | 8 E E |Z |p : ] - —- fos] BT (oO hb > > © |S | © £ | I frant bd N Fo 1g ° fe = a [512 1 c c 10 | & : ao 3 S| ol<| = X NR ERE g a a o o |< . > a & g . 0 wn w ~ ~N |e a <t < Nn | on : o © S o — {m o ~ ~ o lo | ~ : o on on o © |W < oO oO — |N | © i ~—{ — ri i i —t i i a o~ ~ < © | o o~ oN < [© | © } - a o~ ~ ~ Nl a ™m el (mm o o o~ ~ nN ~N {eN ol ~ ~ NNN ; w = :
[7]
o ~ © : v oO | S ~ pi Yo) nn © oN hor pu 3 ON ED Sl YR EO A BA :
S$ ool 4 o |¥] o oO o oc 4 i © Im Ln << Oo ~ ~— o > 1M ~ ) O . ) 2 ! ~N ow |M — —t < [¢)) a} (5) — —- 1} ~ : z c
L ~N jo | © oo |lo|. wv el o «|e 7) ~N |[N ol © o nn 0 o < 0 w oO | © ™m ™ o 0 [os] oO —
Q NO oN o Oo Oo ol o~ Oo o a o Oo |Oo o Oo o Oo oO o oO o 5 o wf u wu. [TS LL Th LL wo w < a |a a a a a a o a a =< q } [in . a (=)} : £ 8 :
[7] - od clo Blo £|>|a © © py : c Sls lc Elo ®m |= re vo £lo E v ©lc|o 8s E|E|2 a |= 58 © : 3 £ > CP |> 5 |e << ro 5 3|® © : oo oll IX olX “— |Z £ £ 59 © © |o o © lo 5 a —= [a © wn © - CC > Cy © |p £|c 0) = vino lo 0h = = |.E
Tis. 8% S| oln ® 0 £ | Tv I|T : hel . 0 [£5 > 1® £ Fa : 3] Lille z= Zoe a a = & Slr ©l7 ® oo o|% Ef 53
Co = JER [nv oN RIE|Y Slo Bm gm ale : : o E 3{S|lv gv Olalo | slo Tle S |e £ hel re = Ji= lc E io 5 LU |o oO £ © Clo |T | R(T 6lx E13 “(S22 |> [' Z a 9 gO nig {> © lc ££ o|& ~ |S < 0 [8a ia © |o (5 0 Cc |o vile 2s
C nw |< |v wn C | >| T wiv © |v 9ic o > © vo [= |o 28 cB wo
Lo Ble ls cis oO u vile glm ci > Q QO Q ° 0 @ 20 |e = I] 0
Co» E|®|e ole Llc |B |= g 218 92 lo io Tio © > 51> 2 |x 3 Ole lg Slog >lo|o £ = TO |Z A
[72] O a |— |= oO = IG) > (© he} [+3] wn |Z ] > [] Qo |= ~. ed > = Q a“ © oO QQ : © © + © » Ld ° (9) & ® = “
R= eX . 213 0 |o in a ~ © 3 2 ag 9 bry < 3 fe) =} m 0 ~ ~— — O ™ : o ~N < ™ ~ < 0 To) ra o = oO cc I A IS ~ —- ~ — N ~ i < cC 68 ¥ | wn | a Xe} ~ 5 A Py y —y ; < 7) By
Z v © = c vo l|3| = = z Ss) ®) = -— 7} © | s 1 ] ! © B® |E 1 © < + — iy ££ Xx . £ > | © ped [9] Nb] | | hd A oO = 8 Slee | 2 5 |8 |&8|& i
J ge] = & © © — |g ~N . uw o lo in| ~ - © © |o : [4 © |® in th © ~ ~ Fer) Io a } (o] O |W i o~ o~ Va] Ve] le] fo) == o oN < re) 0 e) © o oe < |< < < < < < < <t Ln o o ~N jN ~ ~ ~ ~ ~ o~ ~N ~ ; hf 2 BS un
WO {VO <
BY PO I =< TO IN el BS ol a I Xs ~N fon) ie} o
EO Rl ER I I Cl EN ER A BC nl rr ! ow wow jw jw us w w [18] we :
S Lilo 8lcojlojlolo] © |o oo |of © : 1 © Mm [T= joa [Nn [Tr N90 © Ap hp w FC PSI Ro EL PE VI FV TN Eo © ~ ™ ™m on o . ’ 2 c 2 SE La IR Lo IR IT NE EE SS EN [TN |S o o + 2 0 . v < [~ [1 |W [IN JO [OV | |x [ap] — ~ ~ wn : BEREEIRIBEL| 8 BE B| 8 : — | mM o ha) N o Oo |lojo |o lo lo io lo |o oO le) Oo o o 5
Y wwe joe jue jw fue ju jue | u. Te ww. u ww << ao {a joa |a la ja |a ja o o a a a . = : < : uw . c i
T wn — fs [= [ [=] + © . © LL |= —
Q £ cle |= 8 (a |® c 4
C ’ o SZ jE |E |m |g |— c no I [ oO IT ££ © o Oo |G Cc > fe) = 3 wi - l'c | Elo |T |e is |= a c |e w : o Cle |e Q vw lw [BIE E ® |g v 0 |e dio |B (2s |E is o £12 E n E © 3 ao |l— lo |a lg |© |< (¥] a) S
Ee) al lcci |oalo Hole re
Oo — “ © [\}] 3 c T | nv . 0 © Ix | 15 Elm |e pa = FY) . 2 > L2ITIc(dd|o|® @ 5 {vw cig © ola ol : sl = lelFIEiglP|aldl2(a|8 2le| E |S o c| E Sls |S |Z |o|®|E|m|2]0c 2181 8S |B) © = © Of le En sS(s ls 10 2 3 o £ io ©
E w SEX elm lo |N [2B |5 EO a [23 © = £ ic clu ils | 3 2 we <C oO |E |Z clu o|l=2 ln O | oo << | » = o = N a |g [{0] [+7] = 5 OU |e o = > c o lola lg Elolald |E Zz |& © & slv|a|2i|8 =a |e |E s = 2 QvlRiad |e le |R|a |5 |B 2 FE lc ©
Ee fe) > 2 |= | > [7 < |w- £~ < |g 5 |e 5 |e | > JU lo - : n CE SEU ERR A a £ &
[7] © >1317 10 |g tn a o oo —_ a fu. a +t © — wn wn c
Oo > uw uv “in = © a = [on Qa 1a c Q o. =
I's Z us [VIN O o
[10] a lia} 13) v | 2 a ro v 9 o ~ wn {on (03 -~ ND o < < o I NIN jn NS fo INS INS (nln fH oY ~ iq ~ ’
Pu o o EAE REINER ASHEN ™M ~ © ~ 3 © : ca © [LTT VN Ulolg|N|M| ao [ZT Sg] © - 5 ~ jm in jon je [Oo (oy [is O ~N [© © — . E - — ™M |< — MN tn ly c u |= g 4 — 2 |e al = 0 lo o|Nle lu a © Ie) < ® lala |x DIE IBI® IE] 0 |x| = :
Eg Q © lo |L |& wv = © “2, [a8] [ ox | wl : 5 sie lel 8 121% [5] ! o. o n a. a. . w ~N jo lo jo In IN INDI NN a on un o wn |@ [eo ln jin (nn jn [DE in |in T ON ~ le) ~ jo [oo joo Joo lov Jo |) 1S < < 3 3 ~ o NN | | (© 0 | © ]|]O |x < < Yo} 0 lve} - = in (in in {1b ln jn In {© [© O 0. | WO 0 © o o ARNEL EL EERE EN ~~ ~ ~ ~N ~ :
Ww ra ’ - ES
[1]
o m |v |o {nN jin © oO | wn o jv [NY ~ tn = Mm [— [© lo jm @ in |O <t T = (TD ~N <r -— 1) i |) [] 1 1 1 |] , 4 ] 1 1 r=] Ww |W jw jw jw w ww w wow w w > oO |O |O |o |o o oO |o o © |o|8 o o
TemiRm| 3 loo R [HI] % |o w — |e [= [A in © in | © n(n 5 ~ ~ [=] pr - = 2 © [I~ Joo joo |~ wn wn lm 0 © IN joo © ~ wn ~N [ON IN oO oN | un ~ (WO iN o~N < 0 mM | jm [mn [= oO |O ™m Mm Mm |e wn tn : o NO IN | | Oo un |o o ~N |o |o o — w=
Q oO |o |o {oo jo o Q {Oo o oO |o |o o o : u [YU ETI I ri ru IT rl (I Tt THE Fri fr nd IT . << oa ja ja |a |a a a |o a Oo ja ja a {a = : [18 o : £ E £ ’ [TI N FT) L © > © Ir ° un = = oO bd a OU lo jo (U |a = 5 OU lace | a od — fe |= |= E I nn LC {= |= |= . jot — = ee oo © {0 min nEEE IEE oO :
[0] w (Ww jw |w c w“— + LenS 1 VE I 1 78) £ Fo) Cc Cc . 3 ele le 5 = 515 Ble |cl518 25
E|E |E |E Cc ‘0 {| [Oo E E |E © |? ogo |E wn ) Vv | lu |v |'F oo {nC [J] o jo a ls we lo lo (O br] Oo c @ VV | |e | U fie] Ea) oO Ov in lun ' )) lola 2 |v vis ls E ' : 2 > > {>[>1e + oO |x 2 II 15” 5 l|2 (w} wv lo jn jun [Y ° + 2 bry 0a |= le 8S < > ole ole ig a |= || v lo lo 5 o lS . o - no luin in 2 wn Slo le iw |ulwe oS hd : c E © (Co (© c Q «+ ulm (@wfo c = [od IRCA I wa a © n) < © © ju es _ {Cc © fo
EOE HIBIBBIEIE BEBE alzl|nlE Et <« c lc lc ic |= O S52 (XX 9 |c |c|we 3 E |= = © (0 i{o o |© om Cla Clo (oP 0 |O® oO — i pat - 0 < 2 © [= © find “ = ou ww Fa EI a er] © OU |~— |o = jer 42 £ = E © wn © {ojo |o |C Jw |e Bll lm io o [= = £ lc |£ |€ {© * mfg © |C€ |< 2 go = aio jaja |X oo Saja |m|o £ IX
Ir nw ivnialn |© a = = EO EO = Cc ||P o © [CO |O |O |= olga 812 ]oja|8 = |= oc LL | | |C |© 7 on 2 l= |< =z | © a (aa (a |T O co £.ia |a fod :
[7] Qa c [43] p ed ox c 5 - . , < O Qa ce Q uw © R= © - a o [48] [¢0] ™ vw | 2 a —= (¥] 0 Jo -~ AIS -
Sl 8 2 lo oll vw Olam | © Jolla? © [I~ © 20 SPIN I8It ln smc IRIRIZ|I NN [= : + © ct - “|TT iIm ™M ™m |N NE I Aa ~ ~ | oN ™ -
SEE iH ele Raley RIS] @ |o : . £ 5 5 len |eN N ~N ~ AV Bb 0
L- 4 wn - wn . i — | — c olololo 7] e 13] lola l®] & —_— = |e |= [ie] + — — > 1] po (TVR ITER STURN [TYR I IR TS PU on Ww |w |E S o
E HREEREE flo | wm 1 1|® PL Io . o nin juin ld] © alo I 5 ; a FEEREE | 2 [Bal EIEIR] & |% a 0 o I al & |» <C [a3] \ uw win INlan] ® Tia] Nn | 0 (nN : : & INRINIRIE Rw 232] I II (lo) ~ ~ Lan] ~ tn i ~—t wn ™~ ri La —{ Lan} Ln] a 0 | [jo {Nn | fos} oO |v o o |o | <t [Ye] I. ee OO IN IN IN ~ © jo on oO (on [on o oa o [SR Ta I FaVI FoI Fat o~ ~eN ~N NNN ~N oN - (od wu = 74}
o nn 0 IN jn | Yon|o]s|inlnjol~|~]~
El ll A ES Ee Ea Ee EE Re Ea ES BER Ee Ea EA = wow ww jw www w|wlojujdio] ou ldddbd]d
S sig 12 [2 |e |8]|elelo|olololo|o|s]| o |olala]|s & 22 T 3T |ofR|e|rn Non] 1» fom
CAI Ea a I EA EE IE F< 7 E oV RS ONY [Ro] IER NY IL Jy BE IPRS IPO 5 . 4 “ c s . 8 Vv |v fn jo |[o|a|lunltinlmls|olio @ oo |o|o |a |+|m|o|o|o|o|m|~|3l] J (8lRIRS ’ s 1218181218 [3IZISI8IEIE 2 le El 2 (5]2]818
Mop Oo | : - uv © 10 lo |o |o |olololalelalalalaldl 8 [21818]|Y — wou {uC |u| CIR b-4 rE EE EE Fr he Fd bd Fl a Fr 1g I pve 1 ry rg fre : : oljajala|a|aloial a (&jalala b= . u [+8 £ c > oe a 2 HE 0 a EE S| = = NEI = [2 > 2 El 9185 alo : o vlo|l 3 oO i
S 22 |elalalels| |Hes (else n sls is ls le IZI= 512155 ole! 1906 of «| 81Llel . O giggles 5 Sl EjEI& wl Els Ely] 2w
S uy £1 > > ol wl wnlof>>0 o|ltle c bd > 2 1o 19 lo |E [EES elsiclvls|=]l5le of ola] 8 = 2 Jala (a|a lc |E|ElojE| sola EICS ofl] E{=]E
ST IE I Ce Bed BE EC RE ESE Be fe _— . =|=|% = rg J £ £888 1% lolol BCI G| 50] 25 8 5{= os] & TS LA EN EE PA slave BB Gl al | olaal == 2° 5 a 0 (aa (2 |5|S(@ 2] 832] F TET ele] SEs N « < |< | | [QO ol ofl <| [SIG = E alc Eli XT)
IT Vi © al [7,] [
Ju] ala 3|3S{o|™ vl OU Diol w
E 2lZ[=| 9s [Z]e & 2c a © > = ols E nlc 2 | ole [ges iE [+4 g) < Yio ) = Q Es < < HEE » ] w ol c
Ww E 0 ™ ov |B a —= oO O Im [in — NTIS <n
SIT E28 lala |®is|m lS5tvd—=lo|n|lolo SoA ed INT id YY o TP El ET PA oinfalolofsini— IN © |DIN] Zo - 0 - ) lee fey LO IA cS RS Had Pg cd I Bal I ov] BEA aN] Rand Hal Bt
E 8 £ [FIT |v [0 IN [N]BRIN]O| SI 2ILIN lal © |e] a] os
ET 8 SSA CA POR FOR CE Ea] B=Ad BN Fol Rad ad Bod 1 Rall © [mS g g 7] : =
J] ™m
IE Slalal— 17
Cc |c|c lc AIS] | ©] ©] — £ s|o |e (ss |® [=[Ts|s]l5] 519s ! ‘a © Fa BEC IA A al © ele Sl < [aX Cc . £ =, = = = ~ 0 hr “ nl nl] ©] 24 wn © ‘%
Ww |nle ViciclSlE Cc ic|ls . ° OOO IU |S o|olY| Elica] ls oc || & i mn | (0 |] = 10 wl = = - IN gle <|E[EIZIZI E58] & (2/2 8|8 . Z|1Z2{|0j<<|<|2] © |<] w NN [0 {© |m | On 1 nln Mm jin] fm] o Mn me je [JIBS HNO sol + |[|m|m|= - o =m jo jo lo ISIS DBIZ RB Bm|<t]| © |olN|NI~
La ET Eo I RR pe NOOO O| JTS = lt] ® ON lu Jo |Oolo|lojo a. 213s Is 12 |alalRlRl lel als el 2 |g] o © I I le a Fo I EY FI RE RY I EE RE EI IE EI EN Es . oa 2 .
[7]
™ i} } ~ (e)] 0 o [Fp}
CE EAE
S ololo|¥ | o 7 wn joo} oo = — w ! ] } : 1 o rd . a = 0 wo |n| wv oN ™
[7] —- [Oo] m mM ™M ’ . wn ow IN] WO Yo) O . ‘ v Mm [oo] m Mm mM i. v o |ol © o oo x 0 ow jul @© Til Til
L- 4 ao ja] ao a oa = < hu .
Qo. © © . 0) I ir HE EES g c [s|E [5 |E - 2 ° lolx c =
Cc ror | QQ
Q b= Pho] Q ori - 3 a 19a Sa IK oo = lz tn |S) [¥} = . LH] br = ~ 0 ~ .
CW c =| olin fad 75)
S 5 EIT > £ E>
OQ Stulz c = << > Dol ulg =|8 8g = . : : . fe) = L. im @*= OS © } : 2 E |=mS|g|CElgslCE £ .’ Oooo lo Yeo } [TH L 3lolwnT sn ®T : : < o Slee [2 slo :
S -|elgle [|e ° oO = o
In) o Plo |= > = > ol= > ~ = c = 0 —_ oO I} S1> o > [}] — [74] [8] [74] [4 To |82lo [> |e . ©| OV w= Oo . : = UO jcl> © I> 1]
I U] O a ™ - a
L 10s ro) oN © oN © ie) < QU in [nN 5 22) < . ’ © on No 0 ~
Fle c nl | Sl of vo : £ © ¢ ola] "| & | &
E © — A ve 3 Ce) dd < 0» z| El oO wn wn tn fu Ww O OO c a ON I —- o o o © | — ful ful <lgl ol © i=] . £ gla >1 > > oe ” °o alg] © < ed i : o 1121 o o o « |g| © ] O oo
Pix 221 =
J |x] O (U) © w ~ jo] © oo o ’ . « m jo| ~ ~ ~ . o ~ |o] © © 0 : — 4 i =i ~~ a |p | | - re in lo] © 0 © m |ml m ™ mM . o 9 : w = ky wv
Example 3: Sugar metabolism genes
Lactobacillus acidophilus has the ability to utilize a variety of carbohydrates, : including mono-, di- and poly-saccharides, as shown by its API50 sugar fermentation RR : 5 pattern. In particular, complex dietary carbohydrates that escape digestion in the upper Gl-tract, such as raffinose and fructooligosaccharides (Gibson et al. (1995) J. ] - Nutr. 125:1401-1412; Barrangou et al. (2003) Proc. Natl. Acad. Sci. U. S. A : 100:8957-8962) can be utilized. The NCFM genome encodes a large variety of genes related to carbohydrate utilization, including 20 phosphoenolpyruvate sugar- transferase systems (PTS) and 5 ATP binding cassette (ABC) families of transporters. * Putative PTS transporters were identified for trehalose (ORF 1012)(SEQ ID NOS: 103 and 289), fructose (ORF 1777) (SEQ ID NO:35), sucrose (ORF 401) (SEQ ID ;
NO:101), glucose and mannose (ORF 452 (SEQ ID NOS:1 and 263), ORF 453 (SEQ
ID NO:161), ORF 454 (SEQ D NO:163), ORF 455 (SEQ ID NO:165) and ORF 456 : 15 (SEQ ID NO:167)), melibiose (ORF 1705)(SEQ ID NO:33), gentiobiose and cellobiose (ORF 1369) (SEQ ID NOS:17 and 269), salicin (ORF 876) (SEQ ID
NO:169), ORF 877 (SEQ ID NO:3), ORF 879 (SEQ ID NO:171)), arbutin (ORF 884) (SEQ ID NO:27), and N-acetyl glucosamine (ORF 146) (SEQ ID NO:21). Putative
ABC transporters were identified for FOS (ORF 502 (SEQ ID NOS:39 and 273) ORF , 20 504 (SEQ ID NO:43), ORF 506 (SEQ ID NO:47)), raffinose (ORF 1439 (SEQ ID
NO:109), ORF 1440 (SEQ ID NOS:111 and 293), ORF 1441 (SEQ ID NO:113), ORF 1442 (SEQ ID NOS:115 and 295), and maltose (ORF 1854-ORF 1857). A putative oo : lactose-galactose permease was also identified (ORF 1463) (SEQ ID NO:175). Most A of these transporters share a genetic locus with a glycosidase and a transcriptional : regulator, allowing localized transcriptional control.
In silico analyses of the genome revealed the presence of genes representing the complete glycolysis pathway. Additionally, members of the general carbohydrate utilization regulation network were identified, namely HPr (ORF 639 (SEQ ID
NO:177), ptsH), EI (ORF 640 (SEQ ID NO:179), ptsI), CcpA (ORF 431 (SEQ ID 7
NO:181), ccpA), and HPrK/P (ORF 676 (SEQ ID NO:183), ptsK), indicating an active carbon catabolite repression network based on sugar availability.
Example 4: Differentially expressed genes
Global gene expression patterns obtained from growth on eight different carbohydrates were visualized by cluster analysis (Eisen et al. (1998) Proc. Natl. :
Acad. Sci. USA 95:14863-14868) using Ward's hierarchical clustering method, volcano plots and contour plots. Overall, between 23 and 379 genes were differentially expressed between paired treatment conditions (with p-values below the
Bonferroni correction), representing between 1% and 20% of the genome, : respectively. All possible treatment comparisons were considered, and a gene was considered induced above a particular level if it showed induction in at least one 10 . treatment comparison. For genes that showed induction in more than one instance, : the highest induction level was selected. Although 342 genes (18% of the genome) - oo showed induction levels above two fold, only 63 genes (3% of the genome) showed : induction above 4 fold, indicating a relatively small number of genes were highly induced. Although overall expression levels of the majority of the genes remained : 15 consistent regardless of the growth substrate (80% of the genome), select clusters showed differential transcription of genes and operons. Nevertheless, for each sugar, a : limited number of genes showed specific induction.
In the presence of glucose, ORF 1679 (SEQ ID NO:133) and ORF 1680 (SEQ oo ID NO:135) were highly induced when compared to other monosaccharides (fructose, . 20 galactose) and di-saccharides (sucrose, lactose, trehalose). The induction levels compared to other sugars varied between 3.5 and 6.3 for ORF 1679 (SEQ ID NO:133) and between 3.7 and 4.7 for ORF 1680 (SEQ ID NO:135). ORF 1679 (SEQ'ID )
NO:133) encodes an ABC nucleotide binding protein, including commonly found : nucleotide binding domain motifs, namely WalkerA, WalkerB, ABC signature * sequence and Linton and Higgins motif. ORF 1680 (SEQ ID NO:135) encodes an
ABC permease, with 10 predicted membrane spanning domains. No solute binding protein is encoded in their vicinity, suggesting a possible role as an exporter rather . ol ] than an importer. Several genes and operons were specifically repressed by glucose, 5 including ORFs 680 (SEQ ID NO:239)-ORF 686, which are involved in glycogen metabolism. Since glycogen is metabolized by the cell in order to store energy, in the presence of the preferred carbon source such as glucose, energy storage is not "necessary. Other genes repressed in the presence of glucose included proteins -
involved in uptake of alternative carbohydrate sources, and enzymes involved in hydrolysis of such carbohydrates.
The three genes of the putative fructose locus, ORF 1777 (SEQ ID NO:35) (FruA, fructose PTS transporter EIIABC™), ORF 1778 (SEQ ID NO:185) (Fruk, . phosphofructokinase EC 2.7.1.56) and ORF 1779 (SEQ ID NO:187) (FruR, transcription regulator) were differentially expressed. Induction levels were up to 3.9, : 4.3 and 4.6 for frud, fruK and fruR, respectively. These results suggest fructose is transported into the cell via a PTS transporter, into fructose-6-phosphate, which the phosphofructokinase FruK phosphorylates into fructose-1,6 bi-phosphate, a glycolysis © 10 intermediate.
In the presence of sucrose, the three genes of the sucrose locus were : differentially expressed, namely ORF 399 (SEQ ID NO:97) (ScrR, transcription regulator), ORF 400 (SEQ ID NO:99) (ScrB, sucrose-6-phosphate hydrolase EC 3.2.1.26), and ORF 401 (SEQ ID NO:101) (ScrA, sucrose PTS transporter
EIIBCAS™). When compared to glucose, induction levels were upto 3.1,2.8 and 17.2 : for scr. scrB and scrA, respectively. ORF 401 (SEQ ID NO: 101) in particular showed high induction levels, between 8.0 and 17.2 when compared to mono- and di- saccharides. These results indicate that sucrose is transported into the cell via a PTS transporter, into sucrose-6-phosphate, which is subsequently hydrolyzed into glucose- 6-phosphate and fructose by ScrB. :
The six genes of the FOS operon were differentially expressed, namely ORF 502 (SEQ ID NOS:39 and 273), ORF 503 (SEQ ID NO:41), ORF 504 (SEQ ID
NO:43), ORF 506 (SEQ ID NO:47) (MsmEFGK ABC transporter), ORF 505 (SEQ :
So ID NO:45) (BfrA, B-fructosidase EC 3.2.1.26) and ORF 507 (SEQ ID NOS:49 and 275) (GtfA, sucrose phosphorylase EC 2.7.1.4). Induction levels varied between 15.1 and 40.6 when compared to mono- and di-saccharides, and between 5.5 and 8.9 when compared to raffinose. These results suggest FOS is transported into the cell via an
ABC transporter and subsequently hydrolyzed into fructose and sucrose by the fructosidase. Sucrose is likely subsequently hydrolyzed into fructose and glucose-1-P : by the sucrose phosphorylase. In addition to the FOS operon, FOS also induced the fructose operon, the sucrose PTS transporter, the trehalose operon and an ABC ) transporter (ORF 1679-ORF 1680) (SEQ ID NOS:133 and 135, respectively). - ~
In the presence of raffinose, the six genes of the raffinose operon were specifically induced. The raffinose locus consists of ORF 1442 (SEQ ID NOS:115 and 295), ORF 1441 (SEQ ID NO:113), ORF 1440 (SEQ ID NOS:111 and 293), ORF . - 1439 (SEQ ID NO:109) (MsmEFGK; ABC transporter), ORF 1438 (SEQ ID
NO:197) (MelA o-galactosidase EC 3.2.1.22), and ORF 1437 (SEQ ID NO:195) (GtfA,, sucrose phosphorylase EC 2.7.1.4). Induction levels varied between 15.1 and 45.6, when compared to all other conditions. Additionally, ORFs 1433 (SEQ ID : : NO:189), 1434 (SEQ ID NO:191) (di-hydroxyacetone kinase EC 2.7.1.29), and ORF 1436 (SEQ ID NO:193) (glycerol uptake facilitator) were induced between 1.9 and 24.7 fold when compared to other conditions. }
In the presence of lactose and galactose, ten genes distributed in two loci were : differentially expressed, namely ORF 1463 (SEQ ID NO:175) (LacS permease of the
GPH translocator family), ORF 1462 (SEQ ID NO:209) (LacZ, B-galactosidase EC 3.2.1.23), ORF 1461 (SEQ ID NO:207), ORF 1460 (SEQ ID NO:205)(surface protein), ORF 1459 (SEQ ID NO:203) (GalK, galactokinase EC 2.7.1.6), ORF 1458 : : oo (SEQ ID NO:201) (GalT, galactose-1 phosphate uridylyl transferase EC 2.7.7.10),
ORF 1457 (SEQ ID NO:199) (GalM, galactose epimerase EC 5.1.3.3), ORFs 1467 (SEQ ID NO:211), 1468 (SEQ ID NO:213) (LacLM, B-galactosidase EC 3.2.1.23 : large and small subunits), and 1469 (SEQ ID NO:215) (GalE, UDP-glucose , 20 epimerase EC 5.1.3.2). LacS (SEQ ID NO:175) is similar to GPH permeases previously identified in lactic acid bacteria. Although LacS (SEQ ID NO:175) contains an EIIA at the carboxy-terminus, it is not a PTS transporter. Also, LacS . (SEQ ID NO:175) includes a His at position 553, which might be involved in - interaction with HPr, as shown in S. salivarius (Lessard et al. (2003) J. Bacteriol. ~ 185:6764-6772). In the presence of lactose and galactose, galKTM (SEQ ID :
NOS:199, 201, and 203) were induced between 3.7 and 17.6 fold; lacSZ (SEQ ID
NOS:175 and 209) were induced between 2.8 and 17.6 fold; lacL (SEQ ID NO:213) . and galE (SEQ ID NO:215) were induced between 2.7 and 29.5, when compared to : ¥ other carbohydrates not containing galactose, i.e., glucose, fructose, sucrose, trehalose and FOS. These results suggest lactose is transported into the cell via the LacS permease of the galactoside-pentose hexuronide translocator family. Inside the cell, lactose is hydrolyzed into glucose and galactose by LacZ. Galactose is then - phosphorylated by GalK into galactose-1 phosphate, further transformed into UDP- ]
galactose by GalT. UDP-galactose is subsequently epimerized to UDP-glucose by
GalE. UDP-glucose is likely turned into glucose-1P by ORF 1719 (SEQ ID NO:217), which encodes a UDP-glucose phosphorylase EC 2.7.7.9, consistently highly g expressed. Finally, the phosphoglucomutase EC 5.4.2.2 likely acts on glucose-1P to . yield glucose-6P, a glycolysis substrate.
The three genes of the putative trehalose locus were also differentially : expressed. The trehalose locus consists of ORF 1012 (SEQ ID NOS:103 and 289)(encoding the TreB trehalose PTS transporter EIABC™ EC 2.7.1.69), ORF 1013 : (SEQ ID NO:105) (TreR, trehalose regulator) and ORF 1014 (SEQ ID NOS:107 and 291) (TreC, trehalose-6 phosphate hydrolase EC 3.2.1.93). Induction levels were between 4.3 and 18.6 for treB (SEQ ID NOS:103 and 289), between 2.3 and 7.3 for treR (SEQ ID NO:105), and between 2.7 and 18.5 for treC (SEQ ID NOS:107 and ] 291), when compared to glucose, sucrose, raffinose and galactose. These results suggest trehalose is transported into the cell via a PTS transporter, phosphorylated to trehalose-6 phosphate and hydrolyzed into glucose and glucose-6 phosphate by TreC.
In addition, genes showing differential expression included sugar- and energy- : related genes ORF 874 (SEQ ID NO:219) (beta galactosidase EC 3.2.1.86), ORF 910 (SEQ ID NO:221) (L-LDH EC 1.1.1.27), ORF 1007 (SEQ ID NO:223 (pyridoxal kinase 2.7.1.35), ORF 1812 (SEQ ID NO:225) (alpha glucosidase EC 3.2.1.3), ORF , 20 1632 (SEQ ID NO:227) (aldehyde dehydrogenase EC 1.2.1.16), ORF 1401 (SEQ ID
NO:229) (NADH peroxidase EC 1.11.1.1), ORF 1974 (SEQ ID NO:231) (pyruvate oxidase EC 1.2.3.3), adherence genes ORF 555, ORF 649, ORF 1019; aminopeptidase ORF 911, ORF 1086; amino acid permease, ORF 1102 (SEQ ID
NO:233) (membrane protein), ORF 1783 (SEQ ID NO:235) (ABC transporter), and
ORF 1879 (SEQ ID NO:237) (pyrimidine kinase EC 2.7.4.7).
Example 5: Real time RT-PCR
Five genes that were differentially expressed in microarray experiments were : selected for real-time quantitative RT-PCR experiments, in order to validate induction } levels measured by microarrays. These genes were selected for both their broad expression range (LSM between -1.52 and +3.87), and induction levels between sugars (fold induction up to 34). All selected genes showed an induction level above 6 ~~ fold in at least one instance. Also, the annotations of the selected genes were correlated functionally with carbohydrate utilization. The five selected genes were: beta-fructosidase (ORF 505) (SEQ ID NO:45), trehalose PTS (ORF 1012) (SEQ ID
NOS:103 and 289), glycerol uptake facilitator (ORF 1436) (SEQ ID NO:193), beta- galactosidase (ORF 1467) (SEQ ID NO:211), and ABC transporter (ORF 1679) (SEQ
IDNO:133).
For the five selected genes, induction levels were compared between six : different treatments, resulting in 15 induction levels for each gene. The induction : levels measured by microarrays were plotted against induction levels measured by Q-
PCR, in order to validate microarray data. Individual R-square values ranged between 0.642 and 0.883 for each of the tested genes (between 0.652 and 0.978 using data in a log, scale). When the data were combined, the global R-square value was 0.78 (0.88 : using data in a log; scale). A correlation analysis was run in SAS (Cary, NC), and showed a correlation between the two methods with P-values less than 0.001, for : Spearman, Hoeffding and Kendall tests. Additionally, a regression analysis was run in excel (Microsoft, CA), and showed a statistically highly significant (p < 1.02x1 0%) correlation between microarray data and Q-PCR results. Nevertheless, Q-PCR measurements revealed larger induction levels, which is likely due to the smaller dynamic range of the microarray scanner, compared to that of the Q-PCR cycler.
Similar results have been reported previously (Wagner et al. (2003) J. Bacteriol. 185:2080-2095). -
Example 6: Comparative analysis
Comparative analyses of global transcription profiles determined for growth on eight carbohydrates identified the basis for carbohydrate transport and catabolism . in L acidophilus. Specifically, three different types of carbohydrate transporters were differentially expressed, namely phosphoenolpyruvate: sugar phosphotransferase system (PTS), ATP binding cassette (ABC) and galactoside-pentose hexuronide . (GPH) translocator, illustrating the diversity of carbohydrate transporters used by :
Lactobacillus acidophilus. Transcription profiles suggested that galactosides were transported by a GPH translocator, while mono- and di- saccharides were transported by members of the PTS, and polysaccharides were transported by members of the
ABC family. C
Microarray results indicated fructose, sucrose and trehalose are transported by
PTS transporters EHABC™ (ORF 1777) (SEQ ID NO:35), EIIBCAS* (ORF 401) (SEQ ID NO:101) and EITABC™™ (ORF 1012) (SEQ ID NOS:103 and 289), respectively. Those genes are encoded on typical PTS loci (Figure 1), along with . oo 5 regulators and enzymes that have been well characterized in other organisms. In contrast, FOS and raffinose are transported by ABC transporters of the MsmEFGK : family, ORFs 502 (SEQ ID NOS:39 and 273), 503 (SEQ ID NO:41), 504 (SEQ ID :
NO:43), and 505 (SEQ ID NO:45); and ORFs 1437(SEQ ID NO: 195, ORF 1438 (SEQ ID NO:197), 1439 (SEQ ID NO:109), ORF 1440 (SEQ ID NOS:111 and 293),
ORF 1441 (SEQ ID NO:113), and ORF 1442 (SEQ ID NO:115 and 295), respectively. In the case of trehalose and FOS, microarray results correlate well with functional studies in which targeted knock out of carbohydrate transporters and : : hydrolases modified the saccharolytic potential of Lactobacillus acidophilus NCFM. :
Differential expression of the EIIABCT™ is consistent with recent work in
Lactobacillus acidophilus indicating ORF 1012 (SEQ ID NOS: 103 and 289) is. involved in trehalose uptake. Similarly, differential expression of the fos operon is consistent with previous work in Lactobacillus acidophilus indicating those genes are involved in uptake and catabolism of FOS, and induced in the presence of FOS and " repressed in the presence of glucose (Barrangou et al. (2003) Proc. Natl. Acad. Sci. , 20 USA 100: 8957-8962). Additionally, induction of the raffinose msm locus is consistent with previous work in Streptococcus mutans (Russell et al. (1992) J. Biol. :
Chem. 267: 4631-4637) and Streptococcus pneumoniae (Rosenow et al. (1999)
Genome Res. 9:1189-1197).
A number of lactic acid bacteria take up glucose via a PTS transporter. The
EIM PTS transporter has the ability to import both mannose and glucose (Cochu et al. 2003). The Lactobacillus acidophilus mannose PTS system is similar to that of
Streptococcus thermophilus, with proteins sharing 53-65% identity and 72-79% similarity. Specifically, the EITM is composed of three proteins IABM*, ICY" and :
IID", encoded by ORF 452 (SEQ ID NOS:1 and 263) (manL), ORF 455 (SEQ ID .
NO:165) (manM) and ORF 456 (SEQ ID NO:167) (manN), respectively (Figure 1).
Most of the carbohydrates examined here specifically induced genes involved in their ‘ own transport and hydrolysis, but glucose did not. Analysis of the mannose PTS - revealed that the genes encoding the EIIABCD™*" were consistently highly expressed, regardless of the carbohydrate source. This expression profile suggests glucose is a preferred carbohydrate, and Lactobacillus acidophilus is also designed for efficient utilization of different carbohydrate sources, as suggested previously for :
Lactobacillus plantarum (Kleerebezem et al, (2003) Proc. Natl. Acad. Sci. USA 100:1990-1995).
The genes differentially expressed in the presence of galactose and lactose } included a permease (LacS), and the enzymatic machinery of the Leloir pathway.
Members of the LacS subfamily of galactoside-pentose-hexuronide (GPH) : translocators have been described in a variety of lactic acid bacteria, including
Leuconostoc lactis (Vaughan etal. (1996) Appl. Environ. Microbiol. 62:1574—1582), 10S. thermophilus (van den Bogaard et al. (2000) J. Bacteriol. 182:5982-5989),
Streptococcus salivarius (Lessard et al. (2003) J. Bacteriol. 185:6764-6772) and
Lactobacillus delbrueckii (Lapierre et al. (2002) J. Bacteriol. 184:928-935). Although
LacS contains a PTS EIIA at the carboxy terminus, it is not a member of the PTS family of transporters. LacS has been reported to have the ability to import both galactose and lactose in select organisms (Vaughan et al. (1996) Appl. Environ. :
SE ‘Microbiol. 62:1574-1582; van den Bogaard et al. (2000) J. Bacteriol. 182:5982— 5989). Although the combination of a LacS lactose permease with two §- galactosidase subunits LacL and LacM has been described in Lactobacillus plantarum (Kleerebezem et al. 2003) and Leuconostoc lactis (Vaughan et al. (1996) Appl. : , 20 Environ. Microbiol. 62:1574-1582), it has never been reported in Lactobacillus acidophilus. Even though constitutive expression of lacS and lacLM has been reported previously (Vaughan et al. (1996) Appl. Environ. Microbiol. 62:1574—1582), these results indicate specific induction of the genes involved in uptake and catabolism of . both galactose and lactose. Operon organization for galactoside utilization is variable and unstable among Gram-positive bacteria (Lapierre et al. (2002) J. Bacteriol. 184:928-935; Vaillancourt et al. (2002) J. Bacteriol. 184:785-793; Boucher et al. (2003) Appl. Environ. Microbiol. 69:4149-4156; Fortina et al. (2003) Appl. Environ.
Microbiol. 69:3238-3243; Grossiord et al. (2003) J. Bacteriol. 185:870—878). Even a amongst closely related Lactobacillus species, namely Lactobacillus johnsonii,
Lactobacillus gasseri and Lactobacillus acidophilus, the lactose-galactose locus is not well conserved (Pridmore et al. (2004) Proc. Natl. Acad. Sci. USA 101:2512-2517).
Although it was previously suggested that the phosphoenolpyruvate: -- ’ phosphotransferase system is the primary sugar transport system of Gram-positive bacteria (Ajdic et al. (2002) Proc. Natl. Acad. Sci. USA 99:14434-14439; Warner and : Lolkema (2003) Microbiol. Mol. Rev. 67:475-490), current microarray data indicate : that ABC transport systems are also important. While PTS transporters are involved in uptake of mono- and di-saccharides, those carbohydrates are digested in the upper . : 5 GIT. In contrast, oligosaccharides reach the lower intestine whereby commensals are likely to compete for more complex and scarce nutrients. Perhaps under such : conditions ABC transporters are even more crucial than the PTS, given their apparent : roles in transport of oligosaccharides like FOS and raffinose. In this regard, the ability to utilize nutrients that has been are non digestible by the host has been associated : 10 with competitiveness and persistence of beneficial intestinal flora in the colon (Schell et al. (2002) Proc. Natl. Acad. Sci. USA 99:14422-14427).
Transcription profiles of genes differentially expressed in conditions tested indicated that all carbohydrate uptake systems and their respective sugar hydrolases were specifically induced by their substrate, except for glucose. Moreover, genes within those inducible loci were repressed in the presence of glucose, and cre : sequences were identified in their promoter-operator regions. The promoter-operator : regions of differentially expressed genes and operons were searched for putative catabolite response elements according to consensus sequences :
TGNNWNCGNNWNCA (SEQ ID NO:365) (Miwa et al. (2000) Nucleic Acids Res. , 20 28:1206-1210) and TGWAANCGNTNWCA (SEQ ID NO:366) (Weickert and
Chambliss (1990) Proc. Natl. Acad. Sci. USA 87:6238-6242). Together, these results indicate regulation of carbohydrate uptake and metabolism at the transcription level, : and implicate the involvement of a global regulatory system compatible with carbon catabolite repression. Carbon catabolite repression (CCR) controls transcription of proteins involved in transport and catabolism of carbohydrates (Miwa et al. (2000)
Nucleic Acids Res. 28:1206—1210). Catabolite repression is a mechanism widely distributed amongst Gram-positive bacteria, mediated in cis by catabolite responsive elements (Miwa et al. (2000) Nucleic Acids Res. 28:1206-1210; Wickert and :
Chambliss (1990) Proc. Natl. Acad. Sci. USA 87:6238-6242), and in trans by . repressors of the Lacl family, which is responsible for transcriptional repression of genes encoding unnecessary saccharolytic components in the presence of preferred i substrates (Wickert and Chambliss (1990) Proc. Natl. Acad. Sci. USA 87:6238-6242; -
Viana et al. (2000) Mol. Microbiol. 36:570-584; Muscariello et al. (2001) Appl. ) _ Environ. Microbiol. 67:2903-2907; Warner and Lolkema (2003) Microbiol. Mol. Rev. “ 158 oo
Loe 3 :
67:475-490). This regulatory mechanism allows cells to coordinate the utilization of diverse carbohydrates, to focus primarily on preferred energy sources. CCR is based upon several key enzymes, namely HPr (ORF 639 (SEQ ID NO:177), ptsH), El (ORF 640 (SEQ ID NO:179), ptsl), CcpA (ORF 431 (SEQ ID NO:181), ccpA), and HPrK/P (ORF 676 (SEQ ID NO:183), ptsK), all of which are encoded within the Lactobacillus acidophilus chromosome. : ~ Carbon catabolite repression has already been described in lactobacilli (Mahr - ~ “etal. 2000). The PTS is characterized by a phosphate transfer cascade involving PEP,
EL, HPr, EIIABC, whereby a phosphate is ultimately transferred to the carbohydrate substrate (Saier, 2000; Warner and Lolkema, 2003). HPr is an important component oo : of CCR, which is regulated via phosphorylation by enzyme I and HPrK/P. When HPr is phosphorylated at His15, the PTS is active, and carbohydrates transported via the . PTS are phosphorylated via EIIABCs. In contrast, when HPr is phosphorylated at
Ser46, the PTS machinery is not functional (Mijakovic et al. (2002) Proc. Natl. Acad.
Sci. USA 99:13442-13447). - Although the phosphorylation cascade suggests regulation at the protein level, several studies report transcriptional modulation of ¢ccpd and pisHL In S. thermophilus, CcpA production is induced by glucose (van den Bogaart et al. 2000).
In several bacteria, the carbohydrate source modulates prsHI transcription levels (Luesink et al. 1999). In contrast, expression levels of ccpA, pst, pisl and ptsK did not vary in the presence of different carbohydrates in Lactobacillus acidophilus. - These results are consistent with regulation via phosphorylation at the protein level.
Similar results have been reported for ccpd expression levels in Lactobacillus . pentosus (Mahr et al. (2000) Appl. Environ. Microbiol. 66:277-283), and ptsHI : transcription in S. thermophilus (Cochu et al. (2003) Appl. Environ. Microbiol. 69:5423-5432).
Globally, microarray results allowed reconstruction of carbohydrate transport and catabolism pathways (Figure 2). Although transcription of carbohydrate | i transporters and hydrolases was specifically induced by their respective substrates, these glycolysis genes were consistently highly expressed: D-lactate dehydrogenase : ~ (D-LDH, ORF 55 (SEQ ID NO:241)), phosphoglycerate mutase (PGM, ORF 185 (SEQ ID NO:243)), L-lactate dehydrogenase (L-LDH, ORF 271 (SEQ ID NO:245)), ~ glyceraldehyde 3-phosphate dehydrogenase (GPDH, ORF 698 (SEQ ID N0:247)), phosphoglycerate kinase (PGK ORF 699 (SEQ ID NO:249)), glucose 6-phosphate : 159 i) .
isomerase (GPI, ORF 752 (SEQ ID NO:251)), 2-phosphoglycerate dehydratase (PGDH, ORF 889 (SEQ ID NO:253)), phosphofructokinase (PFK, ORF 956 (SEQ ID
NO:255)), pyruvate kinase (PK, ORF 957 (SEQ ID NO:257)) and fructose ~ biphosphate aldolase (FBPA, ORF 1599 (SEQ ID NO:259)). A glycerol-3-phosphate .
ABC transporter (ORF 1641 (SEQ ID NO:261)) was also among the genes that were consistently highly expressed. Orchestrated carbohydrate uptake likely withdraws T energy sources from the intestinal environment and deprives other bacteria of access i to such resources. Consequently, Lactobacillus acidophilus may compete well against other commensals for nutrients. : : 10 In summary, a variety of carbohydrate uptake systems were identified and . characterized, with respect to expression profiles in the presence of different . carbohydrates, including PTS, ABC and GHP transporters. The uptake and catabolic 3 machinery is highly regulated at the transcription level, suggesting the Lactobacillus acidophilus transcriptome 1s flexible, dynamic and designed for efficient carbohydrate utilization. Differential gene expression indicated the presence of a global carbon catabolite repression regulatory network. Regulatory proteins were consistently highly expressed, suggesting regulation at the protein level, rather than the transcriptional level. Collectively, Lactobacillus acidophilus appears to be able to efficiently adapt its metabolic machinery to fluctuating carbohydrate sources available in the - 20 nutritional complex environment of the small intestine. In particular, ABC . transporters of the MsmEFG family involved in uptake of FOS and raffinose likely play an important role in the ability of Lactobacillus acidophilus to compete with intestinal commensals for complex sugars that are not digested by the human host. :
Ultimately, this information provides new insights into how undigested dietary compounds influence the intestinal microbial balance. This study is a model for comparative transcriptional analysis of a bacterium exposed to varying growth substrates.
Example 7: Multidrug Transporters :
Microorganisms such as Lactobacillus acidophilus have developed various methods in which to resist the toxic effect of antibiotics and other deleterious compounds. One such method involves transporters that promote the active efflux of Co drugs, by which drug resistance may be affected for a particular microorganism.
There are two major classes of multidrug transporters: secondary multidrug transporters that utilize the transmembrane electrochemical gradient of protons or sodium ions to drive the extrusion of drugs from a cell; and ATP-binding cassette (ABC)-type multidrug transporters that utilize the free energy of ATP hydrolysis to pump drugs out of the cell. :
Secondary multidrug transporters are subdivided into several distinct families of transport proteins: the major facilitator superfamily (MFS, Pao et al. (1998) : :
Microbiol. Mol. Biol. Rev. 62:1-34), the small multidrug resistance (SMR) family (Paulsen et al. (1996) Mol. Microbiol. 19:1167-1175), the resistance-nodulation-cell division (RND) family (Saier et al. (1994) Mol. Microbiol. 11:841-847), and the multidrug and toxic compound extrusion (MATE) family (Brown et al. (1999) Mol.
Microbiol. 31:394-395. These families are not solely associated with multidrug export, and include proteins involved in other-proton motive force-dependent transport processes or other functions. :
MES membrane transport proteins are involved in synport, antiport, or uniport of various substrates, among which are antibiotics (Marger and Saier (1993) Trends oo "Biochem. Sci 18:1 3-20). Analysis and alignment of conserved motifs of the resistance-conferring drug efflux proteins revealed that these proteins can be divided into two separate clusters, with either 12 or 14 transmembrane segments (Paulsen and i 20 Skurry (1993) Gene 124:1-11). The NCFM genome contains several genes that encode MF transporters attributed to multidrug transport. Included among these are the transporters encoded in ORFs 552 (SEQ ID NO:77), 566 (SEQ ID NO:79), 567 (SEQ ID NO:81), 1446 (SEQ ID NO:85), 1471 (SEQ ID NO:87), 1621 (SEQID - E
NO:91), 1853 (SEQ ID NO:93), 1854 (SEQ ID NO:321), 164 (SEQ ID NO:309), - 251-253 (SEQID NOs:311, 313, 315) and 1062 (SEQ ID NO:317).
ABC transporters require four distinct domains: two hydrophobic membrane domains, which usually consist of six putative transmembrane a-helices each, and two hydrophilic nucleotide binding domains (NBDs), containing Walker A and B motifs = (Walker et al. (1982) EMBO J. 1:945-951) and the ABC signature (Hyde et al. (1990)
Nature 346:362-365). The individual domains can be expressed as separate proteins or they may be fused into multidomain polypeptides in several ways (Faith and Kolter (1993) Microbiol. Rev. 57:995-1017; Higgens (1992) Annu. Rev. Cell Bio. 8:67-113; ~
Hyde et al. (1990) Nature 346:362-365). A multidrug ABC transporter in the NCFM genome similar to the ABC multidrug transporter /mrA4 from Lactococcus lactis and Ce Co : horA from Lactobacillus brevis is encoded by ORF 597 (SEQ ID NO:320). : All publications, patents and patent applications mentioned in the specification ‘are indicative of the level of those skilled in the art to which this invention pertains. oo
All publications, patents and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was ’ specifically and individually indicated to be incorporated by reference. ’
Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the | Lo appended claims. 162 oo
Claims (19)
1. An isolated nucleic acid selected from the group consisting of: a) a nucleic acid comprising a nucleotide sequence as set forth in SEQ ID NO: 289,1, 3,5,7,9,11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39,41, 43, 45,47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71,73, 75,77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223,225, 227, 229, 231, 233, 235, 237, 239,241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361 or 363 or a full length complement thereof; b) a nucleic acid comprising a nucleotide sequence having at least 90% sequence identity to a nucleotide sequence as set forth in SEQ ID NO: 289,1, 3,5,7,9,11,13,15,17,19, 21, 23, 25,27, 29, 31, 33, 35, 37,39, 41, 43, 45, 47,49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73,75, 77, 79, 81, 83, 85, 87, 89,91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115,117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215,217,219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245,247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275,277,279, 281, 283, 285, 287, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361 or 363, wherein said nucleic acid encodes a polypeptide having carbohydrate utilization related activity or multi-drug transporter activity; ¢) a nucleic acid that encodes a polypeptide comprising an amino acid sequence as set forth in SEQ ID NO:290, 2, 4, 6, §, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64,
66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228,230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272,274, 276, 278, 280, 282, 284, 286, 288,292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362 or 364, wherein said nucleic acid encodes a polypeptide having carbohydrate utilization related activity or multi-drug transporter activity; and, d) a nucleic acid comprising a nucleotide sequence encoding a polypeptide having at least 90% amino acid sequence identity to the amino acid sequence as set forth in SEQ ID NO:290, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22,24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228,230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362 or 364, wherein said polypeptide has carbohydrate utilization activity or multi-drug transporter activity.
2. A vector comprising the nucleic acid of claim 1.
3. The vector of claim 2, further comprising a nucleic acid encoding a heterologous polypeptide.
4. A cell comprising the vector of claim 2.
5. An isolated polypeptide selected from the group consisting of: a) a polypeptide comprising an amino acid sequence as set forth in SEQ ID NO:290, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274,276, 278, 280, 282, 284, 286, 288, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362 or 364 ;
b) a polypeptide comprising an amino acid sequence having at least 90% sequence identity with an amino acid sequence as set forth in SEQ ID NO:290, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74,76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362 or 364, wherein said polypeptide has carbohydrate utilization related activity or multi-drug transporter activity; and,
d) a polypeptide encoded by a nucleotide sequence having at least 90% sequence identity to a nucleotide sequence as set forth in SEQ ID NO: 289,1, 3,5,7,9,11,13,15,17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47,49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87,
89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115,117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245,247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275,271,279, 281, 283, 285, 287, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361 or 363, wherein said polypeptide has carbohydrate utilization related activity or multi- drug transporter activity.
6. The polypeptide of claim S$ further comprising a heterologous amino acid sequence.
7. An antibody that selectively binds to a polypeptide selected from the group consisting of: a) a polypeptide consisting of an amino acid sequence as set forth in SEQ ID NO:290, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174,178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362 or 364 ; b) a polypeptide consisting of an amino acid sequence having at least 90% sequence identity with an amino acid sequence as set forth in SEQ ID NO:290, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42,44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74,76, 78, 80, 82,
84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210,212,214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362 or 364, wherein said polypeptide has carbohydrate utilization related activity or multi-drug transporter activity; and, ¢) a polypeptide encoded by a polynucleotide consisting of a nucleotide sequence having at least 90% sequence identity to a nucleotide sequence as set forth in SEQ ID NO: 289,1, 3,5,7,9, 11, 13, 15, 17, 19, 21, 23,25,27,29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65,67, 69,71, 73,75,77,79, 81, 83, 85, 87, 89, 91, 93, 95, 97,99, 101, 103, 105,107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137,139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165,167,169, 171, 173,177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227,229, 231, 233,235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361 or 363, wherein said polypeptide has carbohydrate utilization related activity or multi-drug transporter activity
8. A method for producing a polypeptide, comprising culturing the cell of claim 4 under conditions in which a nucleic acid encoding the polypeptide is expressed, said polypeptide being selected from the group consisting of: a) a polypeptide comprising the amino acid sequence as set forth in SEQ ID NO:290, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112,
114,116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142,
144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172,
174, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204,
206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234,
236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264,
266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 292, 294, 296,
298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328,330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356,
358, 360, 362 or 364,
b) a polypeptide comprising an amino acid sequence having at least 90% sequence identity with an amino acid sequence as set forth in SEQ ID NO:290, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74,76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210,212,214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270,272,274, 276, 278, 280, 282, 284, 286, 288, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362 or 364, wherein said polypeptide has carbohydrate utilization related activity or multi-drug transporter activity; and,
c) a polypeptide encoded by a nucleotide sequence having at least 90% sequence identity to a nucleotide sequence as set forth in SEQ ID NO: 289,1, 3,5,7,9,11,13,15,17, 19,21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107,109, 111, 113,115,117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173,177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215,217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245,247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273,
275,277,279, 281, 283, 285, 287, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361 or 363, wherein said polypeptide has carbohydrate utilization related activity or multi- drug transporter activity.
9. A method for modifying the ability of an organism to transport a carbohydrate into or out of a cell, comprising introducing into said organism the nucleic acid of claim 1.
10. A method for modifying the ability of an organism to accumulate a carbohydrate, comprising introducing into said organism the nucleic acid of claim 1.
11. A method for modifying the ability of an organism to utilize a carbohydrate as an energy source, comprising introducing into said organism the nucleic acid of claim 1.
12. A method for modifying the flavor of a food product fermented by a microorganism, comprising introducing into said microorganism the nucleic acid of claim 1. ©
13. A method for modifying the texture of a food product fermented by a microorganism, comprising introducing into said microorganism the nucleic acid of claim 1.
14. A method for modifying the ability of an organism to produce a modified carbohydrate, comprising introducing into said organism the nucleic acid o claim 1.
15. A method for modifying the ability of an organism to survive food processing and storage conditions, comprising introducing into said organism the nucleic acid of claim 1.
16. A method for modifying the ability of an organism to survive in a GI tract, comprising introducing into said microorganism the nucleic acid of claim 1.
17. A method for modifying the ability of an organism to produce a carbohydrate, comprising introducing into said organism the nucleic acid of claim 1.
18. A method for modifying the ability of an organism to transport a drug into or out of a cell, comprising introducing into said organism the nucleic acid of claim 1.
19. A plant cell comprising a nucleic acid construct comprising a nucleic acid of claim 1. DATED THIS 19™ DAY OF OCTOBER 2007
S$. TIRY SPOOR & FISHER APPLICANT’S PATENT ATTORNEYS
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US55112104P | 2004-03-08 | 2004-03-08 |
Publications (1)
Publication Number | Publication Date |
---|---|
ZA200708939B true ZA200708939B (en) | 2008-09-25 |
Family
ID=38019338
Family Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
ZA200607432A ZA200607432B (en) | 2004-03-08 | 2005-03-08 | Lactobacillus acidophilus nucleic acid sequences encoding carbohydrate utilization-related protein and uses therefor |
ZA200708939A ZA200708939B (en) | 2004-03-08 | 2007-10-16 | Lactobacillus acidophilus nucleic acid sequences encoding carbohydrate utilization-related proteins and uses therefor |
Family Applications Before (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
ZA200607432A ZA200607432B (en) | 2004-03-08 | 2005-03-08 | Lactobacillus acidophilus nucleic acid sequences encoding carbohydrate utilization-related protein and uses therefor |
Country Status (2)
Country | Link |
---|---|
CN (1) | CN1950392B (en) |
ZA (2) | ZA200607432B (en) |
Families Citing this family (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN108018254B (en) * | 2018-01-26 | 2021-05-07 | 遵义市第一人民医院 | LytR function-deficient streptococcus pneumoniae, vaccine, and preparation methods and applications thereof |
CN112391331B (en) * | 2020-11-12 | 2022-09-27 | 江南大学 | Recombinant escherichia coli for overexpression of GatA gene and application thereof |
CN114990140B (en) * | 2022-04-28 | 2024-04-19 | 广西大学 | Cassava pyridoxal kinase gene and application thereof in improving salt tolerance of plants |
-
2005
- 2005-03-08 ZA ZA200607432A patent/ZA200607432B/en unknown
- 2005-03-08 CN CN2005800146731A patent/CN1950392B/en not_active Expired - Fee Related
-
2007
- 2007-10-16 ZA ZA200708939A patent/ZA200708939B/en unknown
Also Published As
Publication number | Publication date |
---|---|
ZA200607432B (en) | 2008-05-28 |
CN1950392B (en) | 2011-06-15 |
CN1950392A (en) | 2007-04-18 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
AU2005218666B2 (en) | Lactobacillus acidophilus nucleic acid sequences encoding carbohydrate utilization-related proteins and uses therefor | |
US7608700B2 (en) | Lactobacillus acidophilus nucleic acid sequences encoding stress-related proteins and uses therefor | |
US7824894B2 (en) | Lactobacillus acidophilus nucleic acids encoding fructo-oligosaccharide utilization compounds and uses thereof | |
US7754868B2 (en) | Nucleic acid encoding two-component sensing and regulatory proteins, antimicrobial proteins and uses therefor | |
ZA200708939B (en) | Lactobacillus acidophilus nucleic acid sequences encoding carbohydrate utilization-related proteins and uses therefor | |
MXPA06010198A (en) | Lactobacillus acidophilus nucleic acid sequences encoding carbohydrate utilization-related proteins and uses therefor. |