US20060251635A1 - Lactobacillus rhamnosus polynucleotides, polypeptides and methods for using them - Google Patents
Lactobacillus rhamnosus polynucleotides, polypeptides and methods for using them Download PDFInfo
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- US20060251635A1 US20060251635A1 US11/314,869 US31486905A US2006251635A1 US 20060251635 A1 US20060251635 A1 US 20060251635A1 US 31486905 A US31486905 A US 31486905A US 2006251635 A1 US2006251635 A1 US 2006251635A1
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- polynucleotide
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- protein
- polypeptide
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Abstract
Novel polynucleotides isolated from Lactobacillus rhamnosus, as well as probes and primers, genetic constructs comprising the polynucleotides, biological materials, including plants, microorganisms and multicellular organisms incorporating the polynucleotides, polypeptides expressed by the polynucleotides, and methods for using the polynucleotides and polypeptides are disclosed.
Description
- This application is a continuation of U.S. patent application Ser. No. 09/971,536, filed Oct. 2, 2001, which is a continuation-in-part of U.S. patent application Ser. No. 09/634,238 filed Aug. 8, 2000, now U.S. Pat. No. 6,544,772 and U.S. patent application Ser. No. 09/724,623, now U.S. Pat. No. 6,476,209, filed Nov. 28, 2000, and claims priority to PCT International Application No. PCT/NZ01/00160 filed Aug. 8, 2001.
- This application incorporates by reference in its entirety the Sequence Listing contained in the accompanying two compact discs, one of which is a duplicate copy. Each CD contains a single file, named “1043c4 SEQLIST.txt,” the size of which is 196 KB, and which was created on Dec. 20, 2005, in IBM-PC MS-Windows 2000 format pursuant to 37 CFR §1.52 (e).
- This invention relates to polynucleotides isolated from lactic acid bacteria, namely Lactobacillus rhamnosus, as well as probes and primers specific to the polynucleotides, genetic constructs comprising the polynucleotides, biological materials, including microorganisms and multicellular organisms, incorporating the polynucleotides, polypeptides encoded by the polynucleotides, and methods for using the polynucleotides and polypeptides.
- The present invention relates to polynucleotides and polypeptides isolated from a specific strain of lactic acid bacteria, namely Lactobacillus rhamnosus HN001 (L. rhamnosus HN001). Lactic acid bacteria and their enzymes are the major determinants of flavor and fermentation characteristics in fermented dairy products, such as cheese and yogurt. Flavors are produced through the action of bacteria and their enzymes on proteins, carbohydrates and lipids.
- Lactobacillus rhamnosus strain HN001 are heterofermentative bacteria that are Gram positive, non-motile, non-spore forming, catalase negative, facultative anaerobic rods exhibiting an optimal growth temperature of 37±1° C. and an optimum pH of 6.0-6.5. Experimental studies demonstrated that dietary supplementation with Lactobacillus rhamnosus strain HN001 induced a sustained enhancement in several aspects of both natural and acquired immunity (See PCT International Publication No. WO 99/10476).
- In addition, L. rhamnosus HN001 and certain other Gram-positive bacteria can specifically and directly modulate human and animal health (See, for example, Tannock et al., Applied Environ. Microbiol. 66:2578-2588, 2000; Gill et al., Brit. J Nutrition 83:167-176; Quan Shu et al., Food and Chem. Toxicol. 38:153-161, 2000; Quan Shu et al., Intl. J Food Microbiol. 56:87-96, 2000; Quan Shu et al., Intl. Dairy J. 9:831-836, 1999; Prasad et al., Intl. Dairy J. 8:993-1002, 1998; Sanders and Huis in't Veld, Antonie van Leeuwenhoek 76:293-315, 1999; Salminen et al., 1998. In: Lactic Acid Bacteria, Salminen S and von Wright A (eds)., Marcel Dekker Inc, New York, Basel, Hong Kong, pp. 203-253; Delcour et al., Antonie van Leeuwenhoek 76:159-184, 1999; Blum et al., Antonie van Leeuwenhoek 76:199-205, 1999; Yasui et al., Antonie van Leeuwenhoek 76:383-389, 1999; Hirayama and Rafter, Antonie van Leeuwenhoek 76:391-394, 1999; Ouwehand, 1998. In: Lactic Acid Bacteria, Salminen S and von Wright A (eds)., Marcel Dekker Inc, New York, Basel, Hong Kong, pp. 139-159; Isolauri et al., S 1998. In: Lactic Acid Bacteria, Salminen S and von Wright A (eds)., Marcel Dekker Inc, New York, Basel, Hong Kong, pp. 255-268; Lichtenstein and Goldin, 1998. In: Lactic Acid Bacteria, Salminen S and von Wright A (eds)., Marcel Dekker Inc, New York, Basel, Hong Kong, pp. 269-277; El-Nezami and Ahokas, 1998. In: Lactic Acid Bacteria, Salminen S and von Wright A (eds)., Marcel Dekker Inc, New York, Basel, Hong Kong, pp. 359-367; Nousianen et al., 1998. In: Lactic Acid Bacteria, Salminen S and von Wright A (eds)., Marcel Dekker Inc, New York, Basel, Hong Kong, pp. 437-473; Meisel and Bockelmann, Antonie van Leeuwenhoek 76:207-215, 1999; Christensen et al., Antonie van Leeuwenhoek 76:217-246, 1999; Dunne et al., Antonie van Leeuwenhoek 76:279-292, 1999).
- Beneficial health effects attributed to dietary supplementation with these bacteria include the following:
- Increased resistance to enteric pathogens and anti-infection activity, including treatment of rotavirus infection and infantile diarrhea—due to increases in antibody production caused by an adjuvant effect; increased resistance to pathogen colonization; alteration of intestinal conditions, such as pH; and the presence of specific antibacterial substances, such as bacteriocins and organic acids.
- Aid in lactose digestion—due to lactose degradation by bacterial lactase enzymes (such as beta-galactosidase) that act in the small intestine.
- Anti-cancer (in particular anti-colon cancer) and anti-mutagenesis activities—due to anti-mutagenic activity; alteration of procancerous enzymatic activity of colonic microbes; reduction of the carcinogenic enzymes azoreductase, beta-glucuronidase and nitroreductase in the gut and/or faeces; stimulation of immune function; positive influence on bile salt concentration; and antioxidant effects.
- Liver cancer reduction—due to aflatoxin detoxification and inhibition of mould growth.
- Reduction of small bowel bacterial overgrowth—due to antibacterial activity; and decrease in toxic metabolite production from overgrowth flora.
- Immune system modulation and treatment of autoimmune disorders and allergies—due to enhancement of non-specific and antigen-specific defence against infection and tumors; enhanced mucosal immunity; adjuvant effect in antigen-specific immune responses; and regulation of Th1/Th2 cells and production of cytokines.
- Treatment of allergic responses to foods—due to prevention of antigen translocation into blood stream and modulation of allergenic factors in food.
- Reduction of blood lipids and prevention of heart disease—due to assimilation of cholesterol by bacteria; hydrolysis of bile salts; and antioxidative effects.
- Antihypertensive effect—bacterial protease or peptidase action on milk peptides produces antihypertensive peptides. Cell wall components act as ACE inhibitors.
- Prevention and treatment of urogenital infections—due to adhesion to urinary and vaginal tract cells resulting in competitive exclusion; and production of antibacterial substances (acids, hydrogen peroxide and biosurfactants).
- Treatment of inflammatory bowel disorder and irritable bowel syndrome—due to immuno-modulation; increased resistance to pathogen colonization; alteration of intestinal conditions such as pH; production of specific antibacterial substances such as bacteriocins, organic acids, hydrogen peroxide and biosurfactants; and competitive exclusion.
- Modulation of infective endocarditis—due to fibronectin receptor-mediated platelet aggregation associated with Lactobacillus sepsis.
- Prevention and treatment of Helicobacter pylori infection—due to competitive colonization and antibacterial effect.
- Prevention and treatment of hepatic encephalopathy—due to inhibition and/or exclusion of urease-producing gut flora.
- Improved protein and carbohydrate utilization and conversion—due to production of beneficial products by bacterial action on proteins and carbohydrates.
- Other beneficial health effects associated with dietary supplementation with L. rhamnosus include: improved nutrition; regulation of colonocyte proliferation and differentiation; improved lignan and isoflavone metabolism; reduced mucosal permeability; detoxification of carcinogens and other harmful compounds; relief of constipation and diarrhea; and vitamin synthesis, in particular folate.
- Peptidases are enzymes that break the peptide bonds linking the amino group of one amino acid with the carboxy group (acid group) of an adjacent amino acid in a peptide chain. The bonds are broken in a hydrolytic reaction. There is a large family of peptidase enzymes that are defined by their specificity for the particular peptide bonds that they cleave (Barrett A J, Rawlings N D and Woessner J F (Eds.) 1998. Handbook of proteolytic enzymes, Academic Press, London, UK). The two main families are exopeptidases and endopeptidases.
- Exopeptidases cleave amino acids from the N- or C-terminus of a peptide chain, releasing free amino acids or short (di- and tri-) peptides. Different types of exopeptidases include:
-
- Aminopeptidases—release a free amino acid from the N-terminus of a peptide chain;
- dipeptidyl-peptidase (also known as dipeptidyl-aminopeptidases)—release a dipeptide from the N-terminus of a peptide chain;
- tripeptidyl-peptidases (also known as tripeptidyl-aminopeptidases)—release a tripeptide from the N-terminus of a peptide chain);
- carboxypeptidases—release a free amino acid from the C-terminus of a peptide chain;
- peptidyl-dipeptidases—release a dipeptide from the C-terminus of a peptide chain;
- dipeptidases—release two free amino acids from a dipeptide; and
- tripeptidases—release a free amino acid and a dipeptide from a tripeptide.
- Endopeptidases hydrolyze peptide bonds internally within a peptide and are classified on the basis of their mode of catalysis:
-
- serine-endopeptidases—depend on serine (or threonine) as the nucleophile in the catalytic reaction;
- cysteine-endopeptidases—depend on the sulphydryl group of cysteine as the nucleophile in the catalytic reaction;
- aspartic-endopeptidases—contain aspartate residues that act as ligands for an activated water molecule which acts as the nucleophile in the catalytic reaction; and
- metallo-endopeptidases—contain one or more divalent metal ions that activate the water molecule that acts as the nucleophile in the catalytic reaction.
- Peptidases are important enzymes in the process of cheese ripening and the development of cheese flavor. The hydrolysis of milk caseins in cheese results in textural changes and the development of cheese flavors. The raft of proteolytic enzymes that cause this hydrolysis come from the lactic acid bacteria that are bound up in the cheese—either starter cultures that grow up during the manufacture of the cheese, or adventitious and adjunct non-starter lactic acid bacteria that grow in the cheese as it ripens (Law and Haandrikman, Int. Dairy J. 7:1-11, 1997).
- Many other enzymes can also influence dairy product flavor and functional and textural characteristics, as well as influencing the fermentation characteristics of the bacteria, such as speed of growth, acid production and survival. (Urbach, Int. Dairy J 5:877-890, 1995; Johnson and Somkuti, Biotech. Appl. Biochem. 13:196-204, 1991; El Soda and Pandian, J. Dairy Sci. 74:2317-2335, 1991; Fox et al., In Cheese: chemistry, physics and microbiology.
Volume 1, General aspects, 2nd edition, P Fox (ed) Chapman and Hall, London; Christensen et al., Antonie van Leeuwenhoek 76:217-246, 1999; Stingle et al., J. Bacteriol. 20:6354-6360, 1999; Stingle et al., Mol. Microbiol. 32:1287-1295, 1999; Lemoine et al., Appl. Environ. Microbiol. 63:1512-3518, 1997). Enzymes influencing the specific cellular and system characteristics and/or functions are examplified below: -
- Lysis of cells. These enzymes are mostly cell wall hydrolases, including amidases; muramidases; lysozymes, including N-acetyl muramidase; muramidase; N-acetylglucosaminidase; and N-acetylmuramoyl-L-alanine amidase. DEAD-box helicase proteins also influence autolysis.
- Carbohydrate utilization. Lactose, citrate and diacetyl metabolism, and alcohol metabolism are particularly important. The enzymes involved include beta-galactosidase, lactate dehydrogenase, citrate lyase, citrate permease, 2,3 butanediol dehydrogenase (acetoin reductase), acetolactate decaboxylase, acetolactate synthase, pyruvate decarboxylase, pyruvate formate lyase, diacetyl synthase, diacetyl reductase, alcohol decarboxylase, lactate dehydrogenase, pyruvate dehydrogenase, and aldehyde dehydrogenase.
- Lipid degradation, modification or synthesis. Enzymes involved include lipases, esterases, phospholipases, serine hydrolases, desaturases, and linoleate isomerase.
- Polysaccharide synthesis. Polysaccharides are important not only for potential immune enhancement and adhesion activity but are important for the texture of fermented dairy products. The enzymes involved are a series of glucosyl transferases, including beta-(1-3) glucosyl transferase, alpha-N acetylgalactosaminyl transferase, phosphogalactosyl transferase, alpha-glycosyl transferase, UDP-N-acetylglucosamine C4 epimerase and UDP-N-acetylglucosamine transferase.
- Amino acid degradation. Enzymes include glutamate dehydrogenase, aminotransferases, amino acid decarboxylases, and enzymes involved in sulphur amino acid degradation including cystothione beta-lyase.
- Sequencing of the genomes of numerous organisms, including humans, animals, microorganisms and various plant varieties, has been and is being carried out on a large scale. Polynucleotides identified using sequencing techniques may be partial or full-length genes, and may contain open reading frames, or portions of open reading frames, that encode polypeptides. Polypeptides may be identified based on polynucleotide sequences and further characterized. The sequencing data relating to polynucleotides thus represents valuable and useful information.
- Polynucleotides and polypeptides may be analyzed for varying degrees of novelty by comparing identified sequences to sequences published in various public domain databases, such as EMBL. Newly identified polynucleotides and corresponding polypeptides may also be compared to polynucleotides and polypeptides contained in public domain information to ascertain homology to known polynucleotides and polypeptides. In this way, the degree of similarity, identity or homology of polynucleotides and polypeptides having an unknown function may be determined relative to polynucleotides and polypeptides having known functions.
- Information relating to the sequences of isolated polynucleotides may be used in a variety of ways. Specified polynucleotides having a particular sequence may be isolated, or synthesized, for use in in vivo or in vitro experimentation as probes or primers. Alternatively, collections of sequences of isolated polynucleotides may be stored using magnetic or optical storage medium and analyzed or manipulated using computer hardware and software, as well as other types of tools.
- The present invention provides isolated polynucleotides comprising a sequence selected from the group consisting of: (a) SEQ ID NO: 1-33; (b) complements, reverse sequences and reverse complements of SEQ ID NO: 1-33 and fragments of SEQ ID NO: 1-33; (c) open reading frames contained in SEQ ID NO: 1-33 and their variants; (d) functional domains contained in SEQ ID NO: 1-33; and (e) sequences comprising at least a specified number of contiguous residues of a sequence of SEQ ID NO: 1-33 (x-mers). Oligonucleotide probes and primers corresponding to the sequences set out in SEQ ID NO: 1-33, and their variants are also provided. All of these polynucleotides and oligonucleotide probes and primers are collectively referred to herein, as “polynucleotides of the present invention.” RNA sequences, reverse sequences, complementary sequences, antisense sequences and the like, corresponding to the polynucleotides of the present invention, may be routinely ascertained and obtained using the polynucleotides identified as SEQ ID NO: 1-33 and are included in the term “polynucleotide.”
- The polynucleotide sequences identified as SEQ ID NO: 1-33 were derived from a microbial source, namely from fragmented genomic DNA of Lactobacillus rhamnosus strain HN001, described in PCT International Publication No. WO 99/10476. A biologically pure culture of Lactobacillus rhamnosus strain HN001 was deposited at the Australian Government Analytical Laboratories (AGAL), The New South Wales Regional Laboratory, 1 Suakin Street, Pymble, NSW 2073, Australia, as Deposit No. NM97/09514, dated 18 Aug. 1997.
- The polynucleotide sequences disclosed herein are primarily “full-length” sequences, in that they represent a full-length gene encoding a full-length polypeptide and contain an open reading frame.
- The present invention further provides isolated polypeptides, including polypeptides encoded by the polynucleotides disclosed herein. In certain specific embodiments, the polypeptides of the present invention comprise a sequence selected from the group consisting of SEQ ID NO: 42-75, and variants thereof. Polypeptides encoded by the polynucleotides of the present invention may be expressed and used in various assays to determine their biological activity. Such polypeptides may be used to raise antibodies, to isolate corresponding interacting proteins or other compounds, and to quantitatively determine levels of interacting proteins or other compounds. The polypeptides of the present invention may also be used as nutritional additives or supplements, and as additives in dairy processing and fermentation processing. Several polypeptides of the present invention also have human and animal health related benefits.
- Genetic constructs comprising the inventive polynucleotides are provided, together with transgenic host cells comprising such constructs and transgenic organisms, such as microbes, comprising such cells.
- The present invention also contemplates methods for modulating the polynucleotide and/or polypeptide content and composition of an organism, such methods optionally involving stably incorporating into the genome of the organism a genetic construct comprising a polynucleotide of the present invention. Such modulation may involve up regulating or down regulating expression from one or more polynucleotides of the present invention. Up regulation may be accomplished, for example, by providing multiple gene copies, modulating expression by modifying regulatory elements, promoting transcriptional or translational mechanisms, or the like. Similarly, down regulation may be accomplished using known antisense and gene silencing techniques, including RNAi. In one embodiment, the target organism is a microbe, preferably a microbe used in fermentation, more preferably a microbe of the genus Lactobacillus, and most preferably Lactobacillus rhamnosus, or another closely related microbial species used in the dairy industry.
- In a related aspect, methods for producing a microbe having an altered genotype and/or phenotype are provided, such methods comprising transforming a microbial cell with a genetic construct of the present invention to provide a transgenic cell, and cultivating the transgenic cell under conditions conducive to growth and multiplication. Organisms having an altered genotype or phenotype as a result of modulation of the level or content of a polynucleotide or polypeptide of the present invention compared to a wild-type organism, as well as components and progeny of such organisms, are contemplated by and encompassed within the present invention.
- The isolated polynucleotides of the present invention may be usefully employed for the detection of lactic acid bacteria, preferably L. rhamnosus, in a sample material, using techniques well known in the art, such as polymerase chain reaction (PCR) and DNA hybridization, as detailed below.
- The inventive polynucleotides and polypeptides may also be employed in methods for the selection and production of more effective probiotic bacteria; as “bioactive” (health-promoting) ingredients and health supplements; for immune function enhancement; for reduction of blood lipids such as cholesterol; for production of bioactive material from genetically modified bacteria; as adjuvants; for wound healing; in vaccine development, particularly mucosal vaccines; as animal probiotics for improved animal health and productivity; in selection and production of genetically modified rumen microorganisms for improved animal nutrition and productivity, improved flavor and improved milk composition; in methods for the selection and production of improved natural food bacteria for improved flavor, faster flavor development, improved fermentation characteristics, vitamin synthesis and improved textural characteristics; for the production of improved food bacteria through genetic modification; and for the identification of novel enzymes for the production of, for example, flavors or aroma concentrates.
- The isolated polynucleotides of the present invention also have utility in genome mapping, in physical mapping, and in positional cloning of genes of more or less related microbes. Additionally, the polynucleotide sequences identified as SEQ ID NO: 1-33, and their variants, may be used to design oligonucleotide probes and primers. Oligonucleotide probes and primers have sequences that are substantially complementary to the polynucleotide of interest over a certain portion of the polynucleotide. Oligonucleotide probes designed using the polynucleotides of the present invention may be used to detect the presence and examine the expression patterns of genes in any organism having sufficiently similar DNA and RNA sequences in their cells, using techniques that are well known in the art, such as slot blot DNA hybridization techniques. Oligonucleotide primers designed using the polynucleotides of the present invention may be used for PCR amplifications. Oligonucleotide probes and primers designed using the polynucleotides of the present invention may also be used in connection with various microarray technologies, including the microarray technology of Affymetrix (Santa Clara, Calif.).
- The polynucleotides of the present invention may also incorporate regulatory elements such as promoters, gene regulators, origins of DNA replication, secretion signals, cell wall or membrane anchors for genetic tools (such as expression or integration vectors).
- The polynucleotide sequences, encoded polypeptides and genetic constructs of this invention are useful for improving the properties of microbes that are used in the manufacture of milk-derived products, such as cheeses, yogurt, fermented milk products, sour milks, and buttermilk. Microbial metabolism during fermentation, which results in the breakdown of proteins, lipids and lactose in milk, influences the speed of ripening, the texture and consistency of fermented milk products, and the development of flavors and aromas during ripening. Undesirable flavors in milk products are produced, for example, by the food of milk-producing animals, microbial action and enzymatic activity during fermentation, and require removal. The present invention provides polynucleotides and polypeptides and methods for their use in modifying the flavor, aroma, texture and health-related benefits of milk-derived products.
- Methods are described for modulating the polynucleotide content or composition of microbes used in the dairy industry by transforming the microbes with one or more polynucleotide sequences of Lactobacillus rhamnosus strain HN001. Certain of the inventive polynucleotides encode polypeptides that increase the survivability of microbes during industrial fermentation processes, wherein exposure to osmotic, temperature and other stresses can lead to reduced microbial viability, impaired metabolic activity and suboptimal fermentation conditions. While the present invention is described with particular reference to milk-derived products, it will be recognized that microbes such as Lactobacillus, which are used in the dairy industry, are also used in the production of other foods and beverages (e.g., fermented vegetables, beer, wines, juices and sourdough breads). The polynucleotides and polypeptides described herein and their methods of use may also be employed in the processing of these foods and beverages. In addition, the inventive polypeptides and polynucleotides may be employed to provide food products, including food products prepared from milk, that have modified properties such as, but not limited to, improved flavor, aroma, texture, nutritional benefits, immune system modulation properties and health benefits.
- This invention also provides transgenic microbial populations comprising expressible polynucleotide sequences of Lactobacillus rhamnosus strain HN001 which provide health-related benefits. For example, the polypeptides encoded by the inventive sequences include enzymes that detoxify carcinogens, degrade allergenic proteins and lactose, and produce bioactive peptides and biogenic amines. Microbes transformed with these polynucleotide sequences can be taken internally as a probiotic composition or alternatively, the microbes or their encoded polypeptides can be added to products to provide health-related benefits. Nonpathogenic bacteria, preferably lactic-acid producing species of Bacillus, Lactobacillus, Sporolactobacillus or Bifidiobacterium, that are able to colonize the gastrointestinal tract, preferably the gastrointestinal tract of a mammal, are useful for preventing or reducing pathogen colonization of the gastrointestinal mucosa, and for replacing normal flora that are depleted, for example, by drug therapy. The polynucleotide sequences of this invention can be used to transform microbes for use in a therapeutic composition that is effective for treating or preventing a gastrointestinal condition or disorder caused by the presence of pathogenic microbes in the gastrointestinal tract or by the absence of normal intestinal microbes in the intestinal tract. Such probiotic compositions can be administered alone or in combination with another pharmaceutical agent, depending on the condition that is to be treated.
- All references cited herein, including patent references and non-patent publications, are hereby incorporated by reference in their entireties.
-
FIG. 1 shows the nucleotide sequence of L. rhamnosus strain HN001 esterase gene AA7 (SEQ ID NO: 3) showing ATG initiation and translation stop codons (boxed). -
FIG. 2 shows the amino acid sequence of HN001 esterase AA7 (SEQ ID NO: 44). -
FIG. 3 demonstrates the esterase activity of the AA7 fusion protein. Production of ethyl butyrate from para-nitrophenyl butyrate substrate was measured by change in OD at 410 nm. While buffer only (♦) and the HN001 non-esterase fusion protein (●) showed minimal esterase activity, the ST1 esterase from Streptococcus thermophilus (σ) and the AA7 esterase fusion protein (ν) showed strong activity. -
FIG. 4 shows the dose-response of the AA7 fusion protein. While buffer-only (λ) showed no esterase activity, increasing amounts of His-patch/Thio/AA7 fusion protein; 5 μl (υ), 10 μl (σ) and 20 μl (ν) purified protein showed increasing rates of substrate hydrolysis. The increase in substrate hydrolysis was proportional to the amount of AA7 fusion protein added. -
FIG. 5 shows the effect of the serine esterase inhibitor PMSF on esterase AA7 activity. Esterase activity of the His-patch/Thio/AA7 fusion protein was assessed in the absence (ν) and presence (σ) of 10 mM PMSF. A buffer-only reaction (●) was used as a negative control. The presence of PMSF reduced HN001 esterase AA7 enzyme activity. -
FIG. 6 shows the nucleotide sequence of L. rhamnosus strain HN001 autoaggregation gene AG5 (SEQ ID NO: 10) showing ATG initiation and translation stop codons (boxed). -
FIG. 7 shows the amino acid sequence of HN001 autoaggregation protein AG5 (SEQ ID NO: 52). -
FIGS. 8A and 8B are images of phase contrast photomicrographs.FIG. 8A illustrates an image of a phase-contrast photomicrograph (exposure ⅛ sec, final magnification×240) showing obvious clumping of washed L. rhamnosus strain HN001 cells in the presence of AG5 autoaggregation protein tagged with GST.FIG. 8B illustrates an image of a phase-contrast photomicrograph (exposure ⅛ sec, final magnification×240) showing no clumping of washed L. rhamnosus strain HN001 cells in the presence of an irrelevant (non-adhesion) HN001 protein tagged with GST, as a negative control. -
FIG. 9 shows the nucleotide sequence of L. rhamnosus strain HN001 malic enzyme gene AA5 (SEQ ID NO: 2) showing ATG initiation and translation stop codons (boxed). -
FIG. 10 shows the amino acid sequence of HN001 malic enzyme AA5 (SEQ ID NO: 43). -
FIG. 11 demonstrates malate enzyme activity measured experimentally as rate of pyruvate reduction by crude lysate preparations of EJ1321 cell transformants. ν PBS buffer-only; σ 3.5 μg wild-type EJ1321 cell lysate; υ 3.5 μg cell lysate of EJ1321 transformed with pGEX-6P-3 construct encoding an irrelevant HN001 protein (AD5); λ 3.5 μg cell lysate of EJ1321 transformed with pGEX-6P-3 construct encoding HN001 malic enzyme AA5. -
FIG. 12 shows experimental data illustrating the effect of increasing amounts of EJ1321 crude lysate on malic enzyme activity. ν 5 μl wild-type EJ1321 cell lysate; σ 5 μl cell lysate of EJ1321 transformed with pGex-6P-3 encoding AA5; υ 50 μl cell lysate of EJ1321 transformed with pGex-6P-3 encoding AA5;λ 200 μl cell lysate of EJ1321 transformed with pGex-6P-3 encoding AA5. -
FIG. 13 shows the nucleotide sequence of L. rhamnosus strain HN001 malate dehydrogenase gene AG3 (SEQ ID NO: 9) showing TTG initiation and translation stop codons (boxed). -
FIG. 14 shows the amino acid sequence of HN001 malate dehydrogenase AG3 (SEQ ID NO: 51). -
FIG. 15 shows the nucleotide sequence of L. rhamnosus strain HN001 dihydrodipicolinate synthase gene AI2 (SEQ ID NO: 13) showing ATG initiation and translation stop codons (boxed). -
FIG. 16 shows the amino acid sequence of HN001 dihydrodipicolinate synthase AI2 (SEQ ID NO: 55). -
FIG. 17 shows the nucleotide sequence of L. rhamnosus strain aspartate aminotransferase gene AH9 (SEQ ID NO: 12) showing GTG initiation and translation stop codons (boxed). -
FIG. 18 shows the amino acid sequence of HN001 aspartate aminotransferase AH9 (SEQ ID NO: 54). -
FIG. 19 shows the nucleotide sequence of L. rhamnosus strain HN001 serine dehydratase subunits α (AF8) and β (AF7). ATG translation initiation codons and termination codons are shown, boxed for AF8, and shaded for AF7. -
FIG. 20 shows the experimentally measured percentage serine utilisation by HN001 strain in liquid culture with 5 mM initial serine concentration. ν HN001 transformed with vector only; υ pTRKH2 construct containing HN001 serine dehydratase. -
FIG. 21 shows the experimentally determined percentage serine utilization by HN001 strain in liquid culture with 12 mM initial serine concentration, ν HN001 transformed with vector only, and υ pTRKH2 construct containing HN001 serine dehydratase. -
FIG. 22 shows the amino acid sequence of L. rhamnosus strain HN001 serine dehydratase subunit α (AF8; SEQ ID NO: 49), and the amino acid sequence of L. rhamnosus strain HN001 serine dehydratase subunit β (AF7; SEQ ID NO: 48). -
FIG. 23 shows the nucleotide sequence of L. rhamnosus strain HN001 histidinol-phosphate aminotransferase gene AG2 (SEQ ID NO: 8) showing ATG initiation and translation stop codons (boxed). -
FIG. 24 shows the amino acid sequence of HN001 histidinol-phosphate aminotransferase AG2 (SEQ ID NO: 50). -
FIG. 25 shows the nucleotide sequence of L. rhamnosus strain HN001 malY-aminotransferase gene AJ6 (SEQ ID NO: 17) showing ATG initiation and translation stop codons (boxed). -
FIG. 26 shows the amino acid sequence of HN001 malY-aminotransferase AJ6 (SEQ ID NO: 59). -
FIG. 27 shows the nucleotide sequence of L. rhamnosus strain HN001 malY-aminotransferase gene AJ7 (SEQ ID NO: 18) showing ATG initiation and translation stop codons (boxed). -
FIG. 28 shows the amino acid sequence of HN001 malY-aminotransferase AJ7 (SEQ ID NO: 60). -
FIG. 29 shows the nucleotide sequence of L. rhamnosus strain HN001 cystathione β-lyase gene AC8 (SEQ ID NO: 5) showing ATG initiation and translation stop codons (boxed). -
FIG. 30 shows the amino acid sequence of HN001 cystathione β-lyase AC8 (SEQ ID NO: 46). -
FIG. 31 shows experimental results demonstrating cystathione β-lyase activity measured as rate of mercaptide formation.υ 10 μl purified HN001 cystathione β-lyase AC8 fusion protein;ν 10 μl purified CAT fusion protein;ν 10 μl H2O only;λ 10 μl elution buffer only. -
FIG. 32 shows the experimentally determined dose-response of the AC8 fusion protein. Cystathione β-lyase activity of increasing amounts of His-patch/Thio/AC8 fusion protein; 10 μl (υ), 25 μl (ν) and 50 μl (σ) purified protein showed increasing rates of mercaptide formation. The increase in mercaptide formation was proportional to the amount of AC8 fusion protein added. -
FIG. 33 shows the nucleotide sequence of L. rhamnosus strain HN001 phosphoenolpyruvate hydratase AK4 (SEQ ID NO: 20) showing ATG initiation and translation stop codons (boxed). -
FIG. 34 shows the amino acid sequence of L. rhamnosus strain HN001 phosphoenolpyruvate hydratase AK4 (SEQ ID NO: 62). -
FIG. 35 shows the nucleotide sequence of L. rhamnosus strain HN001 tagatose bisphosphate aldolase AK1 (SEQ ID NO: 19) showing ATG initiation and translation stop codons (boxed). -
FIG. 36 shows the amino acid sequence of L. rhamnosus strain HN001 tagatose bisphosphate aldolase AK1 (SEQ ID NO: 61). -
FIG. 37 shows the nucleotide sequence of L. rhamnosus strain HN001 phosphoglycerate kinase AK6 (SEQ ID NO: 22) showing TTG initiation and translation stop codons (boxed). -
FIG. 38 shows the amino acid sequence of L. rhamnosus strain HN001 phosphoglycerate kinase AK6 (SEQ ID NO: 64). -
FIG. 39 shows the nucleotide sequence of L. rhamnosus strain HN001 triosephosphate isomerase AK5 (SEQ ID NO: 21) showing ATG initiation and translation stop codons (boxed). -
FIG. 40 shows the amino acid sequence of L. rhamnosus strain HN001 triosephosphate isomerase AK5 (SEQ ID NO: 63). -
FIG. 41 shows the nucleotide sequence of L. rhamnosus strain HN001 phosphoryl carrier protein HPR AA9 (SEQ ID NO: 4) showing ATG initiation and translation stop codons (boxed). -
FIG. 42 shows the amino acid sequence of L. rhamnosus strain HN001 phosphoryl carrier protein HPR AA9 (SEQ ID NO: 45). -
FIG. 43 shows the nucleotide sequence of L. rhamnosus strain HN001 glyceraldehyde-3-phosphate dehydrogenase AK7 (SEQ ID NO: 23) showing ATG initiation and translation stop codons (boxed). -
FIG. 44 shows the amino acid sequence of L. rhamnosus strain HN001 glyceraldehyde-3-phosphate dehydrogenase AK7 (SEQ ID NO: 65). -
FIG. 45 shows the nucleotide sequence of L. rhamnosus strain HN001 sorR transcription regulator AL3 (SEQ ID NO: 24) showing ATG initiation and translation stop codons (boxed). -
FIG. 46 shows the amino acid sequence of L. rhamnosus strain HN001 sorR transcription regulator AL3 (SEQ ID NO: 66). -
FIG. 47 shows the nucleotide sequence of L. rhamnosus strain fpg gene AL4 (SEQ ID NO: 25) showing ATG initiation and translation stop codons (boxed). -
FIG. 48 shows the amino acid sequence of HN001 fpg AL4 (SEQ ID NO: 67). -
FIG. 49 shows the nucleotide sequence of the L. rhamnosus strain HN001 acetoin dehydrogenase gene AP1 (SEQ ID NO: 32) showing ATG initiation and translation stop codons (boxed). -
FIG. 50 shows the amino acid sequence of HN001 acetoin dehydrogenase AP1 (SEQ ID NO: 74). -
FIG. 51 illustrates the experimental results of an acetoin reductase assay as measured by oxidation of NADH co-factor by OD at 340 nm in the presence of acetoin substrate. λ, elution buffer only; ν, purified irrelevant GST-fusion protein; ν, purified GST protein; υ, purified AP1-GST fusion protein. -
FIG. 52 shows the nucleotide sequence of the L. rhamnosus strain HN001 aflatoxin B1 aldehyde reductase gene AI7 (SEQ ID NO: 15) showing ATG initiation and translation stop codons (boxed). -
FIG. 53 shows the amino acid sequence of HN001 aflatoxin B1 aldehyde reductase AI7 (SEQ ID NO: 57). -
FIG. 54 shows the experimental results of aflatoxin B1 aldehyde reductase assay according to oxidation of the NADPH co-factor in the presence of acetoin substrate. X, water only; +, Sepharose column elution buffer only; λ, irrelevant GST-fusion protein; ν, 10 μl purified AP4-GST fusion protein;σ 20 μl purified AP4-GST fusion protein. -
FIG. 55 shows the experimental determination of 6-Phospho-β-galactosidase enzyme activity as measured by substrate utilization using crude lysates of strains transformed with pGex-6P-3 encoding A05 (υ), pGex-6P-3 encoding an irrelevant protein (ν), or using lysis buffer only (X). -
FIG. 56 shows the experimentally determined 6-Phospho-β-galactosidase enzyme activity as measured experimentally by substrate utilization using increasing amounts of crude lysate from strains transformed with pGex-6P-3 encoding A05-GST fusion protein. υ, 50 μl lysate; ν, 100 μl lysate; σ, 200 μl lysate; λ, 200 μl lysis buffer only. -
FIG. 57 shows the nucleotide sequence of the L. rhamnosus strain HN001 aromatic aminotransferase gene AH7 (SEQ ID NO: 11) showing ATG initiation and translation stop codons (boxed). -
FIG. 58 shows the amino acid sequence of HN001 aromatic aminotransferase AH7 (SEQ ID NO: 53). -
FIG. 59 shows the nucleotide sequence of the L. rhamnosus strain HN001 acetate kinase gene AP5 (SEQ ID NO: 33) showing ATG initiation and translation stop codons (boxed). -
FIG. 60 shows the amino acid sequence of HN001 acetate kinase AP5 (SEQ ID NO: 75). -
FIG. 61 shows the nucleotide sequence of the L. rhamnosus strain HN001 basic surface protein gene AC9 (SEQ ID NO: 6) showing ATG initiation and translation stop codons (boxed). -
FIG. 62 shows the amino acid sequence of HN001 basic surface protein AC9 (SEQ ID NO: 47). - FIGS. 63A-C show the nucleotide sequence of the L. rhamnosus strain HN001 aromatic outer membrane protein A AL8 (SEQ ID NO: 27) showing ATG initiation and translation stop codons (boxed).
-
FIG. 64 shows the amino acid sequence of HN001 outer membrane protein AL8 (SEQ ID NO: 69). - FIGS. 65A-D show the nucleotide sequence of the L. rhamnosus strain HN001 aromatic extracellular matrix binding protein AM4 (SEQ ID NO: 28) showing ATG initiation and translation stop codons (boxed).
- FIGS. 66A-B show the amino acid sequence of HN001 extracellular matrix binding protein AM4 (SEQ ID NO: 70).
- FIGS. 67A-D show the nucleotide sequence of the L. rhamnosus strain HN001 aromatic high-molecular-weight adhesion protein AL7 (SEQ ID NO: 26) showing ATG initiation and translation stop codons (boxed).
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FIG. 68 shows the amino acid sequence of HN001 high-molecular-weight adhesion protein AL7 (SEQ ID NO: 68). -
FIG. 69 shows the nucleotide sequence of the L. rhamnosus strain HN001 aromatic PEB1 AJ4 (SEQ ID NO: 16) showing ATG initiation and translation stop codons (boxed). -
FIG. 70 shows the amino acid sequence of HN001 PEB1 AJ4 (SEQ ID NO: 58). -
FIG. 71 shows the experimentally determined relative density of autoradiographic signals from AJ4 protein (grey bars) to dot blots of intestinal proteins, compared to a positive control (mapA, white bars) and negative control (irrelevant HN001 protein, black bars). Results for each dot (duplicates) are shown. -
FIG. 72 shows the nucleotide sequence of the L. rhamnosus strain HN001 dihydrodipicolinate reductase AI3 (SEQ ID NO: 14) showing ATG initiation and translation stop codons (boxed). -
FIG. 73 shows the amino acid sequence of HN001 dihydrodipicolinate reductase AI3 (SEQ ID NO: 56). -
FIG. 74 shows the nucleotide sequence of the L. rhamnosus strain HN001 Fructose-bisphosphate aldolase AM8 (SEQ ID NO: 29) showing ATG initiation and translation stop codons (boxed). -
FIG. 75 shows the amino acid sequence of HN001 Fructose-bisphosphate aldolase AM8 (SEQ ID NO: 71). -
FIG. 76 shows the nucleotide sequence of the L. rhamnosus strain HN001 chaperone protein dnaK AM9 (SEQ ID NO: 30) showing ATG initiation and translation stop codons (boxed). -
FIG. 77 shows the amino acid sequence of HN001 chaperone protein dnaK AM9 (SEQ ID NO: 72). -
FIG. 78 shows the nucleotide sequence of the L. rhamnosus strain HN001 6-phospho-β-galactosidase gene AO5 (SEQ ID NO: 31) showing translation stop codon (boxed). -
FIG. 79 shows the amino acid sequence of HN001 6-phospho-β-galactosidase AO5 (SEQ ID NO: 73). - FIGS. 80A-B show the nucleotide sequence of the L. rhamnosus strain HN001 peptidase pepO (SEQ ID NO: 1) showing ATG initiation and translation stop codons (boxed).
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FIG. 81 shows the amino acid sequence of HN001 peptidase pepO (SEQ ID NO: 42). - The polynucleotides disclosed herein were isolated by high throughput sequencing of DNA libraries from the lactic acid bacteria Lactobacillus rhamnosus as described in Example 1. The polynucleotides and polypeptides of the present invention including cell components selected from the group consisting of: peptidoglycans, teichoic acids, lipoteichoic acids, polysaccharides, adhesion proteins, secreted proteins, surface layer or S-layer proteins, collagen binding proteins and other cell surface proteins, and antibacterial substances such as bacteriocins and organic acids produced by these bacteria. Such bacterial cell components are known to mediate immune modulation, cell adhesion and antibacterial activities, producing many beneficial effects including: resistance to enteric pathogens: modulation of cancer, including colon cancer: anti-mutagenesis effects; reduction of small bowel bacterial overgrowth; modulation of auto-immune disorders; reduction in allergic disorders; modulation of urogenital infections, inflammatory bowel disorder, irritable bowel syndrome, Helicobacter pylori infection and hepatic encephalopathy; reduction of infection with pathogens; regulation of colonocyte proliferation and differentiation; reduction of mucosal permeability; and relief of constipation and diarrhea. Polynucleotides involved in the synthesis of these proteins and in the synthesis, modification, regulation, transport, synthesis and/or accumulation of precursor molecules for these proteins are used to modulate the immune effects, antibacterial, cell adhesion and competitive exclusion effects of the bacteria or of components that might be produced by these bacteria.
- In order to function effectively as probiotic bacteria, L. rhamnosus HN001 survives environmental stress conditions in the gastrointestinal tract, as well as commercial and industrial processes. Modification of particular polynucleotides or regulatory processes has been shown to be effective against a number of stresses including oxidative stress, pH, osmotic stress, dehydration, carbon starvation, phosphate starvation, nitrogen starvation, amino acid starvation, heat or cold shock and mutagenic stress. Polynucleotides involved in stress resistance often confer multistress resistance, i.e., when exposed to one stress, surviving cells are resistant to several non-related stresses. Bacterial genes and/or processes shown to be involved in multistress resistance include:
- Intracellular phosphate pools—inorganic phosphate starvation leads to the induction of pho regulon genes, and is linked to the bacterial stringent response. Gene knockouts involving phosphate receptor genes appear to lead to multistress resistance.
- Intracellular guanosine pools—purine biosynthesis and scavenger pathways involve the production of phosphate-guanosine compounds that act as signal molecules in the bacterial stringent response. Gene knockouts involving purine scavenger pathway genes appear to confer multistress resistance.
- Osmoregulatory molecules—small choline-based molecules, such as glycine-betaine, and sugars, such as trehalose, are protective against osmotic shock and are rapidly imported and/or synthesized in response to increasing osmolarity.
- Acid resistance—lactobacilli naturally acidify their environment through the excretion of lactic acid, mainly through the cit operon genes responsible for citrate uptake and utilization.
- Stress response genes—a number of genes appear to be induced or repressed by heat shock, cold shock, and increasing salt through the action of specific promoters.
- The isolated polynucleotides of the present invention and genetic constructs comprising such polynucleotides may be employed to produce bacteria having desired phenotypes, including increased resistance to stress and improved fermentation properties. Such genetic constructs may be used to increase production of selected polypeptides of the present invention in modified bacteria, or to produce modified levels and/or compositions of polypeptides related to polypeptides of the present invention in modified bacteria. Bacteria having desired phenotypes may also be produced by modulating the transcription and/or expression of polynucleotides of the present invention and incorporating modified regulatory elements in modified bacteria. Additionally, production of selected polypeptides of the present invention may be reduced or blocked in modified bacteria using techniques that are well known in the art, such as antisense RNAi or other gene silencing techniques. Such modified bacteria are also encompassed by the present invention.
- Many enzymes are known to influence dairy product flavor, functional and textural characteristics, as well as general fermentation characteristics such as speed of growth, acid production and survival. These enzymes include those involved in the metabolism of lipids, polysaccharides, amino acids and carbohydrates, as well as those involved in the lysis of the bacterial cells.
- The isolated polynucleotides and polypeptides of the present invention have been demonstrated to have the identities, functions and utilities described throughout this application and in the Examples. The polynucleotide and polypeptide SEQ ID NOs of the present invention, and corresponding identification and functional information is provided below in Table 1A.
TABLE 1A SEQ ID SEQ ID NO: NO: Polynucleotide Polypeptide Category Identification and Description 1 42 Flavor, Peptidases are enzymes that break the peptide bonds nutrition linking the amino group of one amino acid with the carboxy group (acid group) of an adjacent amino acid in a peptide chain. Peptidases are important in the process of cheese ripening and the development of cheese flavor. 2 43 Flavor, Homolog isolated from L. rhamnosus of citM malic carbohydrate enzyme that catalyzes L-malate oxidative metabolism decarboxylation and pyruvate reductive carboxylation. It is part of the noncyclic, branched pathway “tricarboxylic acid cycle” that is characteristic of anaerobic citrate metabolism and is part of the pathway that converts L-malate to L-lactate. 3 44 Flavor Homolog isolated from L. rhamnosus of her esterase that catalyzes the lipolysis of milk fat in dairy products such that the triglycerides are hydrolyzed to free fatty acids and glycerol or mono- and diglycerides. The protein plays an essential role in the development of flavor in cheese 4 45 Survival Homolog isolated from L. rhamnosus of JP28/pbH phosphoryl carrier protein HPR, involved in the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) that is responsible for the uptake and phosphorylation of a number of carbohydrates. The gene is up-regulated on heatshock 5 46 Amino acid Homolog isolated from L. rhamnosus of metC metabolism, cystathione beta-lyase (EC 4.4.1.8) that is involved in flavor metabolism of sulpher-containing compounds with important flavor impacts. 6 47 Adhesion Homolog isolated from L. rhamnosus of basic surface protein bspA (1) that is involved in adhesion to intestinal epithelial cells and binds mucin. 7 48 Amino acid Homolog isolated from L. rhamnosus of serine metabolism, dehydratase sdhB, beta subunit that is involved in the flavor production of pyruvate from serine. It plays a role in metabolism, flavor and survival in carbohydrate poor media (including milk). 7 49 Amino acid Homolog isolated from L. rhamnosus metabolism, of the serine dehydratse alpha subunit sdhA flavor that is involved in the production of pyruvate from serine. It plays a role in metabolism, flavor and survival in carbohydrate poor media (including milk). 8 50 Amino acid Homolog isolated from L. rhamnosus of metabolism, Aminotransferase HisC that is involved in histidine flavor biosynthesis. It plays a role in the development flavor and biogenic amines. 9 51 Flavor, Homolog isolated from L. rhamnosus of malate carbohydrate dehydrogenase citH that is involved in amino acid metabolism biosynthesis as well as L-malate utilization pathways. It is important for carbohydrate metabolism and production of flavor intermediates. 10 52 Adhesion Homolog isolated from L. rhamnosus of autoaggregation protein aggH that plays a role in colonization of intestinal surface by excluding other bacteria from binding sites. 11 53 Amino acid Homolog isolated from L. rhamnosus of aromatic metabolism, amino acid transferase araT (1) that is involved in flavor production of flavor compounds. 12 54 Amino acid Homolog isolated from L. rhamnosus of aspartate metabolism, aminotransferase aspB that produces alpha- flavor ketoglutarate using L-glutamate as an amino donor. It is involved in production of important flavor determinants. 13 55 Amino acid Homolog isolated from L. rhamnosus of metabolism, dihydrodipicolinate synthase dapA (EC 4.2.1.52) that flavor converts L-aspartate 4-semialdehyde and pyruvate to l- 2,3-dihydrodipicolinate as part of the lysine biosynthesis pathway. L-aspartate 4-semialdehyde is also the first step of the glycine, serine and threonine metabolic pathways. It is involved in production of important flavor determinants. 14 56 Amino acid Homolog isolated from L. rhamnosus of metabolism, dihydrodipicolinate reductase dapB (EC 1.3.1.26) that flavor converts L-2,3-dihydrodipicolinate to L- tetrahydropicolinate as part of the lysine biosynthesis pathway. L-aspartate 4-semialdehyde is also the first step of the glycine, serine and threonine metabolic pathways. It is involved in production of important flavor determinants. 15 57 Health Homolog isolated from L. rhamnosus of Aflatoxin B1 aldehyde reductase afar that metabolizes the carcinogen aflatoxin B1 (AFB1) and that is associated with AFB1-resistance. Afar is active against particular ketones, aromatic and aliphatic aldehydes and is an antocarcinogenic. It is also useful for the production of flavor compounds. 16 58 Adhesion Homolog isolated from L. rhamnosus of pebB that mediates binding to epithelial cells, excludes binding of other bacteria and prevents pathogenic infection. It is involved in colonization of intestinal surfaces. 17 59 Amino acid Homolog isolated from L. rhamnosus of Pyridoxal-5′- metabolism, phosphate-dependent aminotransferase patB (1) that flavor has both aminotransferase and regulatory activities, including the transamination of methionine and regulation of maltose utilization. It plays a role in production of flavor intermediates and growth on particular sugars. 18 60 Amino acid Homolog isolated from L. rhamnosus of Pyridoxal-5′- metabolism, phosphate-dependent aminotransferase patB (2) that flavor has both aminotransferase and regulatory activities, including the transamination of methionine and regulation of maltose utilization. It plays a role in production of flavor intermediates and growth on particular sugars. 19 61 Survival Homolog isolated from L. rhamnosus of Tagatose 1,6-diphosphate aldolase lacD (EC 4.1.2.40), a glycolytic enzyme that is up-regulated by stress conditions and is involved in stress resistance and carbohydrate utilization. 20 62 Survival Homolog isolated from L. rhamnosus of Phosphoenolpyruvate hydratase eno (EC 4.2.1.11), a glycolytic enzyme up-regulated by stress conditions. It is involved in stress resistance and carbohydrate utilization. 21 63 Survival Homolog isolated from L. rhamnosus of triosephosphate isomerase tpi (EC 5.3.1.1), a glycolytic enzyme up-regulated by stress conditions. It is involved in stress resistance and carbohydrate utilization. 22 64 Survival Homolog isolated from L. rhamnosus of phosphoglycerate kinase pgk (EC 2.7.2.3), a glycolytic enzyme up-regulated by stress conditions. It is involved in stress resistance and carbohydrate utilization. 23 65 Cell wall Homolog isolated from L. rhamnosus of structure and Glyceraldehyde-3-phosphate dehydrogenase gapdh function (EC 1.2.1.12), a glycolytic enzyme up-regulated by stress conditions. It is involved in stress resistance and carbohydrate utilization and is also a major cell wall component. 24 66 Regulation Homolog isolated from L. rhamnosus of the positive regulator sorR in the sorbose operon. It is important in the control of carbohydrate metabolism and useful for inducible promoter for novel vectors. 25 67 Survival Homolog isolated from L. rhamnosus of Formamidopyrimidine-DNA-glycosylase fpg that is important in protecting bacterial DNA against oxidative free radicals. It removes oxidized purine residues present in DNA, including the highly mutagenic C8-oxo-guanine (8-oxoG) generated in DNA by active oxygen during metabolism. It plays an important role in stress resistance. 26 68 Adhesion Homolog isolated from L. rhamnosus of hia, that mediates binding to epithelial cells and excludes binding of other bacteria. It is involved in colonization of intestinal surfaces. 27 69 Adhesion Homolog isolated from L. rhamnosus of Outer membrane protein rompA, a surface bound molecule required for adhesion. 28 70 Adhesion Homolog isolated from L. rhamnosus of MLC36/emb that mediates binding to epithelial cells, excludes binding of other bacteria and prevents pathogenic infection. It is involved in colonization of intestinal surfaces and is involved in plasminogen binding. It plays a role in immune impacts. 29 71 Survival Homolog isolated from L. rhamnosus of Fructose- bisphosphate aldolase fba (EC 4.1.2.13), a glycolytic enzyme that catalyzes the elimination reaction of D- Fructose 1,6-bisphosphate to glycerone phosphate andD-glyceraldehyde 3-phosphate. It is Up-regulatedby shock and is involved in metabolism, as a flavor intermediates and in stress resistance. 30 72 Stress Homolog isolated from L. rhamnosus of chaperone resistance protein dnaK that plays a role in enhanced bacterial survival in industrial processes, improved colonization of human intestinal environment, altered protein translation characteristics and control of plasmid stability. 31 73 Bacterial Homolog isolated from L. rhamnosus of 6-phospho-β- growth, galactosidase (EC 3.2.1.85) that catalyzes the nutrition, hydrolysis of O-glycosyl bonds of 6-phospho-beta-D- flavor galactosides to give alcohols and 6-phospho-D- development galactose, and is involved in lactose utilization. It is useful for flavor and aroma enhancement, nutritional enhancement, altered bacterial metabolic/growth characteristics and removal of bitter or undesirable flavors. 32 74 Flavor Homolog isolated from L. rhamnosus of Acetoin dehydrogenase butA (EC 1.1.1.5) that catalyzes the reduction of diacetyl to acetoin, and acetoin to 2,3- butanediol as part of the pyruvate to 2,3-butanediol pathway. Diacetyl is an important dairy flavor component. 33 75 Flavor Homolog isolated from L. rhamnosus of Acetyl kinase ackA (EC 2.7.2.1) that catalyzes the phosphotransfer between ADP and acetyl phosphate to yield ATP and acetate. - Isolated polynucleotides of the present invention include the polynucleotides identified herein as SEQ ID NO: 1-33; isolated polynucleotides comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO: 1-33; isolated polynucleotides comprising at least a specified number of contiguous residues (x-mers) of any of the polynucleotides identified as SEQ ID NO: 1-33; isolated polynucleotides comprising a polynucleotide sequence that is complementary to any of the above polynucleotides; isolated polynucleotides comprising a polynucleotide sequence that is a reverse sequence or a reverse complement of any of the above polynucleotides; antisense sequences corresponding to any of the above polynucleotides; and variants of any of the above polynucleotides, as that term is described in this specification.
- The word “polynucleotide(s),” as used herein, means a single or double stranded polymer of deoxyribonucleotide or ribonucleotide bases and includes DNA and corresponding RNA molecules, including mRNA molecules, both sense and antisense strands of DNA and RNA molecules, and comprehends cDNA, genomic DNA and recombinant DNA, as well as wholly or partially synthesized polynucleotides. A polynucleotide of the present invention may be an entire gene, or any portion thereof. A gene is a DNA sequence which codes for a functional protein or RNA molecule. Operable antisense polynucleotides may comprise a fragment of the corresponding polynucleotide, and the definition of “polynucleotide” therefore includes all operable antisense fragments. Antisense polynucleotides and techniques involving antisense polynucleotides are well known in the art and are described, for example, in Robinson-Benion, et al., “Antisense techniques,” Methods in Enzymol. 254(23): 363-375, 1995; and Kawasaki, et al., Artific. Organs 20 (8): 836-848, 1996.
- The definitions of the terms “complement,” “reverse complement,” and “reverse sequence,” as used herein, are best illustrated by the following examples. For the sequence 5′
AGGACC 3′, the complement, reverse complement, and reverse sequences are as follows:complement 3′ TCCTGG 5′ reverse complement 3′ GGTCCT 5′ reverse sequence 5′ CCAGGA 3′ - Preferably, sequences that are complements of a specifically recited polynucleotide sequence are complementary over the entire length of the specific polynucleotide sequence.
- Identification of genomic DNA and heterologous species DNA can be accomplished by standard DNA/DNA hybridization techniques, under appropriately stringent conditions, using all or part of a DNA sequence as a probe to screen an appropriate library. Alternatively, PCR techniques using oligonucleotide primers that are designed based on known DNA and protein sequences can be used to amplify and identify other identical or similar DNA sequences. Synthetic DNA corresponding to the identified sequences or variants thereof may be produced by conventional synthesis methods. All of the polynucleotides described herein are isolated and purified, as those terms are commonly used in the art.
- The polynucleotides identified as SEQ ID NO: 1-33 contain open reading frames (“ORFs”), or partial open reading frames, encoding polypeptides. The open reading frames are specifically identified in Example 1, below. Additionally, polynucleotides identified as SEQ ID NO: 1-33 may contain non-coding sequences such as promoters and terminators that may be useful as control elements. The open reading frames contained in polynucleotides of the present invention may be isolated and/or synthesized. Expressible genetic constructs comprising the open reading frames and suitable promoters, initiators, terminators, etc., which are well known in the art, may then be constructed. Such genetic constructs may be introduced into a host cell to express the polypeptide encoded by the open reading frame. Expression of quantities of a polypeptide of the present invention using recombinant methodologies is useful, for example, when polynucleotides of the present invention are used as nutritional supplements, as flavor and/or texture enhancers, or the like.
- Genetic constructs may be also designed and constructed, as is known in the art, to enhance or silence expression of an identified polypeptide. Antisense and gene silencing genetic constructs may be designed and constructed, for example, to reduce or silence expression of polypeptides of the present invention. Genetic constructs of the present invention may thus be assembled using techniques known in the art to enhance or reduce expression of polypeptides of the present invention encoded by polynucleotides of the present invention. Suitable host cells may include various prokaryotic and eukaryotic cells. In vitro expression of polypeptides is also possible, as well known in the art.
- As used herein, the term “oligonucleotide” refers to a relatively short segment of a polynucleotide sequence, generally comprising between 6 and 60 nucleotides, and comprehends both probes for use in hybridization assays and primers for use in the amplification of DNA by polymerase chain reaction.
- As used herein, the term “x-mer,” with reference to a specific value of “x,” refers to a polynucleotide or polypeptide comprising at least a specified number (“x”) of contiguous residues of any of the polynucleotides and polypeptides identified as SEQ ID NO: 1-33 and 42-75. The value of x may be from about 20 to about 600, depending upon the specific sequence.
- In another aspect, the present invention provides isolated polypeptides encoded, or partially encoded, by the above polynucleotides, including the polypeptides identified as SEQ ID NO: 42-75. As used herein, the term “polypeptide” encompasses amino acid chains of any length, including full-length proteins, wherein the amino acid residues are linked by covalent peptide bonds. The term “polypeptide encoded by a polynucleotide” as used herein, includes polypeptides encoded by a polynucleotide which comprises an isolated polynucleotide sequence or variant provided herein. Polypeptides of the present invention may be naturally purified products, or may be produced partially or wholly using recombinant techniques. Such polypeptides may be glycosylated with bacterial, fungal, mammalian or other eukaryotic carbohydrates or may be non-glycosylated.
- Polypeptides of the present invention may be produced recombinantly by inserting a polynucleotide that encodes the polypeptide into an expression vector and expressing the polypeptide in an appropriate host. Any of a variety of expression vectors known to those of ordinary skill in the art may be employed. Expression may be achieved in any appropriate host cell that has been transformed or transfected with an expression vector containing a polynucleotide encoding a recombinant polypeptide. Suitable host cells include prokaryotes, yeast and higher eukaryotic cells. Preferably, the host cells employed are Escherichia coli, Lactococcus lactis, Lactobacillus, insect, yeast or mammalian cell lines, such as COS and CHO. The polynucleotide(s) expressed in this manner may encode naturally occurring polypeptides, portions of naturally occurring polypeptides, or other variants thereof.
- In a related aspect, polypeptides are provided that comprise at least a functional portion of a polypeptide having an amino acid sequence encoded by a polynucleotide of the present invention. As used herein, a “functional portion” of a polypeptide is that portion which contains the active site essential for affecting the function of the polypeptide, for example, the portion of the molecule that is capable of binding one or more reactants. The active site may be made up of separate portions present on one or more polypeptide chains and will generally exhibit high binding affinity.
- Functional portions of a polypeptide may be identified by first preparing fragments of the polypeptide by either chemical or enzymatic digestion of the polypeptide, or by mutation analysis of the polynucleotide that encodes the polypeptide and subsequent expression of the resulting mutant polypeptides. The polypeptide fragments or mutant polypeptides are then tested to determine which portions retain biological activity, using, for example, the representative assays provided below.
- Portions and other variants of the inventive polypeptides may be generated by synthetic or recombinant means. Synthetic polypeptides having fewer than about 100 amino acids, and generally fewer than about 50 amino acids, may be generated using techniques that are well known to those of ordinary skill in the art. For example, such polypeptides may be synthesized using any of the commercially available solid-phase techniques, such as the Merrifield solid-phase synthesis method, where amino acids are sequentially added to a growing amino acid chain (See Merrifield, J. Am. Chem. Soc. 85:2149-2154, 1963). Equipment for automated synthesis of polypeptides is commercially available from suppliers such as Perkin Elmer/Applied Biosystems, Inc. (Foster City, Calif.), and may be operated according to the manufacturer's instructions. Variants of a native polypeptide may be prepared using standard mutagenesis techniques, such as oligonucleotide-directed site-specific mutagensis (Kunkel, Proc. Natl. Acad. Sci. USA 82: 488-492, 1985). Sections of DNA sequences may also be removed using standard techniques to permit preparation of truncated polypeptides.
- In general, the polypeptides disclosed herein are prepared in an isolated, substantially pure form. Preferably, the polypeptides are at least about 80% pure; more preferably at least about 90% pure; and most preferably at least about 99% pure.
- As used herein, the term “variant” comprehends polynucleotide or polypeptide sequences different from the specifically identified sequences, wherein one or more nucleotides or amino acid residues is deleted, substituted, or added. Variants may be naturally occurring allelic variants, or non-naturally occurring variants. Variant polynucleotide and polypeptide sequences preferably exhibit at least 75%, more preferably at least 80%, more preferably yet at least 85%, and most preferably at least 90% or 95% identity to a sequence of the present invention. The percentage identity is determined by aligning the two sequences to be compared as described below, determining the number of identical residues in the aligned portion, dividing that number by the total number of residues in the inventive (queried) sequence, and multiplying the result by 100.
- Polynucleotide and polypeptide sequences may be aligned, and the percentage of identical residues in a specified region may be determined against another polynucleotide or polypeptide, using computer algorithms that are publicly available. Two exemplary algorithms for aligning and identifying the similarity of polynucleotide sequences are the BLASTN and FASTA algorithms. Polynucleotides may also be analyzed using the BLASTX algorithm, which compares the six-frame conceptual translation products of a nucleotide query sequence (both strands) against a protein sequence database. The percentage identity of polypeptide sequences may be examined using the BLASTP algorithm. The BLASTN, BLASTX and BLASTP programs are available on the NCBI anonymous FTP server and from the National Center for Biotechnology Information (NCBI), National Library of Medicine, Building 38A, Room 8N805, Bethesda, Md. 20894, USA. The BLASTN algorithm Version 2.0.11 [Jan. 20, 2000], set to the parameters described below, is preferred for use in the determination of polynucleotide variants according to the present invention. The BLASTP algorithm, set to the parameters described below, is preferred for use in the determination of polypeptide variants according to the present invention. The use of the BLAST family of algorithms, including BLASTN, BLASTP and BLASTX, is described at NCBI's website and in the publication of Altschul, et al., Nucleic Acids Res. 25: 3389-3402, 1997.
- The computer algorithm FASTA is available on the Internet and from the University of Virginia by contacting the Vice Provost for Research, University of Virginia, P.O. Box 9025, Charlottesville, Va. 22906-9025, USA. FASTA Version 2.0u4 [February 1996], set to the default parameters described in the documentation and distributed with the algorithm, may be used in the determination of variants according to the present invention. The use of the FASTA algorithm is described in Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444-2448, 1988; and Pearson, Methods in Enzymol. 183: 63-98, 1990.
- The following running parameters are preferred for determination of alignments and similarities using BLASTN that contribute to the E values and percentage identity for polynucleotide sequences: Unix running command: blastall -p blastn -d embldb -e 10 -G0 -E0 -r 1 -v 30 -b 30 -i queryseq -o results; the parameters are: -p Program Name [String]; -d Database [String]; -e Expectation value (E) [Real]; -G Cost to open a gap (zero invokes default behavior) [Integer]; -E Cost to extend a gap (zero invokes default behavior) [Integer]; -r Reward for a nucleotide match (BLASTN only) [Integer]; -v Number of one-line descriptions (V) [Integer]; -b Number of alignments to show (B) [Integer]; -i Query File [File In]; and -o BLAST report Output File [File Out] Optional.
- The following running parameters are preferred for determination of alignments and similarities using BLASTP that contribute to the E values and percentage identity of polypeptide sequences: blastall -p blastp -d swissprotdb -e 10 -G 0 -E 0 -v 30 -b 30 -i queryseq -o results; the parameters are: -p Program Name [String]; -d Database [String]; -e Expectation value (E) [Real]; -G Cost to open a gap (zero invokes default behavior) [Integer]; -E Cost to extend a gap (zero invokes default behavior) [Integer]; -v Number of one-line descriptions (v) [Integer]; -b Number of alignments to show (b) [Integer]; -I Query File [File In]; -o BLAST report Output File [File Out] Optional. The “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 sequences. The hits are arranged in order of the degree of similarity and the length of sequence overlap. Hits to a database sequence generally represent an overlap over only a fraction of the sequence length of the queried sequence.
- The BLASTN, FASTA, and BLASTP algorithms also produce “Expect” values for alignments. The Expect value (E) indicates the number of hits one can “expect” to see over a certain number of contiguous sequences by chance when searching a database of a certain size. The Expect value is used as a significance threshold for determining whether the hit to a database, such as the preferred EMBL database, indicates true similarity. For example, an E value of 0.1 assigned to a polynucleotide hit is interpreted as meaning that in a database of the size of the EMBL database, one might expect to see 0.1 matches over the aligned portion of the sequence with a similar score simply by chance. 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 probability of finding a match by chance in the EMBL database is 1% or less using the BLASTN or FASTA algorithm.
- According to one embodiment, “variant” polynucleotides and polypeptides, with reference to each of the polynucleotides and polypeptides of the present invention, preferably comprise sequences producing an E value of 0.01 or less using the BLASTN, FASTA, or BLASTP algorithms set at parameters described above when compared to the polynucleotide or polypeptide of the present invention. According to a preferred embodiment, a variant polynucleotide is a sequence having an E value of 0.01 or less using the BLASTN or FASTA algorithms set at parameters described above when analyzed against a polynucleotide of the present invention. Similarly, according to a preferred embodiment, a variant polypeptide is a sequence having an E value of 0.01 or less using the BLASTP algorithm set at the parameters described above when analyzed against a polynucleotide of the present invention.
- 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 above, and identifying the number of identical nucleic or amino acids over the aligned portions; dividing the number of identical nucleic or amino acids by the total number of nucleic or amino acids of the polynucleotide or polypeptide sequence of the present invention; and then multiplying by 100 to determine the percentage identity. For example, a polynucleotide of the present invention having 220 nucleic acids has a hit to a polynucleotide sequence in the EMBL database having 520 nucleic acids over a stretch of 23 nucleotides in the alignment produced by the BLASTN algorithm using the parameters described above. The 23 nucleotide hit includes 21 identical nucleotides, one gap and one different nucleotide. The percentage identity of the polynucleotide of the present invention to the hit in the EMBL library is thus 21/220
times 100, or 9.5%. The polynucleotide sequence in the EMBL database is thus not a variant of a polynucleotide of the present invention. - In addition to having a specified percentage identity to an inventive polynucleotide or polypeptide sequence, variant polynucleotides and polypeptides preferably have additional structure and/or functional features in common with the inventive polynucleotide or polypeptide. Polypeptides having a specified degree of identity to a polypeptide of the present invention share a high degree of similarity in their primary structure and have substantially similar functional properties. In addition to sharing a high degree of similarity in their primary structure to polynucleotides of the present invention, polynucleotides having a specified degree of identity to, or capable of hybridizing to an inventive polynucleotide preferably have at least one of the following features: (i) they contain an open reading frame or partial open reading frame encoding a polypeptide having substantially the same functional properties as the polypeptide encoded by the inventive polynucleotide; or (ii) they contain identifiable domains in common.
- Alternatively, variant polynucleotides of the present invention hybridize to the polynucleotide sequences recited in SEQ ID NO: 1-33, or complements, reverse sequences, or reverse complements of those sequences under stringent conditions. As used herein, “stringent conditions” refers to prewashing in a solution of 6×SSC, 0.2% SDS; hybridizing at 65° C., 6×SSC, 0.2% SDS overnight; followed by two washes of 30 minutes each in 1×SSC, 0.1% SDS at 65° C. and two washes of 30 minutes each in 0.2×SSC, 0.1% SDS at 65° C.
- The present invention also encompasses polynucleotides that differ from the disclosed sequences but that, as a consequence of the discrepancy of the genetic code, encode a polypeptide having similar enzymatic activity as a polypeptide encoded by a polynucleotide of the present invention. Thus, polynucleotides comprising sequences that differ from the polynucleotide sequences recited in SEQ ID NO: 1-33, or complements, reverse sequences, or reverse complements of those sequences as a result of conservative substitutions are encompassed within the present invention. Additionally, polynucleotides comprising sequences that differ from the inventive polynucleotide sequences or complements, reverse complements, or reverse sequences as a result of deletions and/or insertions totaling less than 10% of the total sequence length are also contemplated by and encompassed within the present invention. Similarly, polypeptides comprising sequences that differ from the inventive polypeptide sequences as a result of amino acid substitutions, insertions, and/or deletions totaling less than 15% of the total sequence length are contemplated by and encompassed within the present invention, provided the variant polypeptide has similar activity to the inventive polypeptide.
- The polynucleotides of the present invention may be isolated from various libraries, or may be synthesized using techniques that are well known in the art. The polynucleotides may be synthesized, for example, using automated oligonucleotide synthesizers (e.g., Beckman Oligo 1000M DNA Synthesizer) to obtain polynucleotide segments of up to 50 or more nucleic acids. A plurality of such polynucleotide segments may then be ligated using standard DNA manipulation techniques that are well known in the art of molecular biology. One conventional and exemplary polynucleotide synthesis technique involves synthesis of a single stranded polynucleotide segment having, for example, 80 nucleic acids, and hybridizing that segment to a synthesized complementary 85 nucleic acid segment to produce a 5-nucleotide overhang. The next segment may then be synthesized in a similar fashion, with a 5-nucleotide overhang on the opposite strand. The “sticky” ends ensure proper ligation when the two portions are hybridized. In this way, a complete polynucleotide of the present invention may be synthesized entirely in vitro.
- Polynucleotides and polypeptides of the present invention comprehend polynucleotides and polypeptides comprising at least a specified number of contiguous residues α-mers) of any of the polynucleotides and polypeptides identified as SEQ ID NO: 1-33 and 42-75 or their variants. According to preferred embodiments, the value of x is preferably at least 20, more preferably at least 40, more preferably yet at least 60, and most preferably at least 80. Thus, polynucleotides and polypeptides of the present invention include polynucleotides comprising a 20-mer, a 40-mer, a 60-mer, an 80-mer, a 100-mer, a 120-mer, a 150-mer, a 180-mer, a 220-mer a 250-mer, or a 300-mer, 400-mer, 500-mer or 600-mer of a polynucleotide or polypeptide identified as SEQ ID NO: 1-75 or a variant of one of the polynucleotides or polypeptides identified as SEQ ID NO: 1-33 and 42-75.
- Oligonucleotide probes and primers complementary to and/or corresponding to SEQ ID NO: 1-33, and variants of those sequences, are also comprehended by the present invention. Such oligonucleotide probes and primers are substantially complementary to the polynucleotide of interest. An oligonucleotide probe or primer is described as “corresponding to” a polynucleotide of the present invention, including one of the sequences set out as SEQ ID NO: 1-33 or a variant, if the oligonucleotide probe or primer, or its complement, is contained within one of the sequences set out as SEQ ID NO: 1-33 or a variant of one of the specified sequences.
- Two single stranded sequences are said to be substantially complementary when the nucleotides of one strand, optimally aligned and compared, with the appropriate nucleotide insertions and/or deletions, pair with at least 80%, preferably at least 90% to 95%, and more preferably at least 98% to 100%, of the nucleotides of the other strand. Alternatively, substantial complementarity exists when a first DNA strand will selectively hybridize to a second DNA strand under stringent hybridization conditions. Stringent hybridization conditions for determining complementarity include salt conditions of less than about 1 M, more usually less than about 500 mM and preferably less than about 200 mM. Hybridization temperatures can be as low as 5° C., but are generally greater than about 22° C., more preferably greater than about 30° C. and most preferably greater than about 37° C. Longer DNA fragments may require higher hybridization temperatures for specific hybridization. Since the stringency of hybridization may be affected by other factors such as probe composition, presence of organic solvents and extent of base mismatching, the combination of parameters is more important than the absolute measure of any one alone. DNA-DNA hybridization studies may performed using either genomic DNA or DNA derived by preparing cDNA from the RNA present in a sample to be tested.
- In addition to DNA-DNA hybridization, DNA-RNA or RNA-RNA hybridization assays are also possible. In the first case, the mRNA from expressed genes would then be detected instead of genomic DNA or cDNA derived from mRNA of the sample. In the second case, RNA probes could be used. In addition, artificial analogs of DNA hybridizing specifically to target sequences could also be used.
- In specific embodiments, the oligonucleotide probes and/or primers comprise at least about 6 contiguous residues, more preferably at least about 10 contiguous residues, and most preferably at least about 20 contiguous residues complementary to a polynucleotide sequence of the present invention. Probes and primers of the present invention may be from about 8 to 100 base pairs in length or, preferably from about 10 to 50 base pairs in length or, more preferably from about 15 to 40 base pairs in length. The primers and probes may be readily selected using procedures well known in the art, taking into account DNA-DNA hybridization stringencies, annealing and melting temperatures, potential for formation of loops and other factors, which are well known in the art. Tools and software suitable for designing probes, and especially suitable for designing PCR primers, are available on the Internet. In addition, a software program suitable for designing probes, and especially for designing PCR primers, is available from Premier Biosoft International, 3786 Corina Way, Palo Alto, Calif. 94303-4504. Preferred techniques for designing PCR primers are also disclosed in Dieffenbach and Dyksler, PCR primer: a laboratory manual, CSHL Press: Cold Spring Harbor, N.Y., 1995.
- A plurality of oligonucleotide probes or primers corresponding to a polynucleotide of the present invention may be provided in a kit form. Such kits generally comprise multiple DNA or oligonucleotide probes, each probe being specific for a polynucleotide sequence. Kits of the present invention may comprise one or more probes or primers corresponding to a polynucleotide of the present invention, including a polynucleotide sequence identified in SEQ ID NO: 1-33.
- In one embodiment useful for high-throughput assays, the oligonucleotide probe kits of the present invention comprise multiple probes in an array format, wherein each probe is immobilized in a predefined, spatially addressable location on the surface of a solid substrate. Array formats which may be usefully employed in the present invention are disclosed, for example, in U.S. Pat. Nos. 5,412,087, 5,545,531, and PCT Publication No. WO 95/00530, the disclosures of which are hereby incorporated by reference.
- Oligonucleotide probes for use in the present invention may be constructed synthetically prior to immobilization on an array, using techniques well known in the art (See, for example, Gait, ed., Oligonucleotide synthesis a practical approach, IRL Press: Oxford, England, 1984). Automated equipment for the synthesis of oligonucleotides is available commercially from such companies as Perkin Elmer/Applied Biosystems Division (Foster City, Calif.) and may be operated according to the manufacturer's instructions. Alternatively, the probes may be constructed directly on the surface of the array using techniques taught, for example, in PCT Publication No. WO 95/00530.
- The solid substrate and the surface thereof preferably form a rigid support and are generally formed from the same material. Examples of materials from which the solid substrate may be constructed include polymers, plastics, resins, membranes, polysaccharides, silica or silica-based materials, carbon, metals and inorganic glasses. Synthetically prepared probes may be immobilized on the surface of the solid substrate using techniques well known in the art, such as those disclosed in U.S. Pat. No. 5,412,087.
- In one such technique, compounds having protected functional groups, such as thiols protected with photochemically removable protecting groups, are attached to the surface of the substrate. Selected regions of the surface are then irradiated with a light source, preferably a laser, to provide reactive thiol groups. This irradiation step is generally performed using a mask having apertures at predefined locations using photolithographic techniques well known in the art of semiconductors. The reactive thiol groups are then incubated with the oligonucleotide probe to be immobilized. The precise conditions for incubation, such as temperature, time and pH, depend on the specific probe and can be easily determined by one of skill in the art. The surface of the substrate is washed free of unbound probe and the irradiation step is repeated using a second mask having a different pattern of apertures. The surface is subsequently incubated with a second, different, probe. Each oligonucleotide probe is typically immobilized in a discrete area of less than about 1 mm2. Preferably each discrete area is less than about 10,000 mm2, more preferably less than about 100 mm2. In this manner, a multitude of oligonucleotide probes may be immobilized at predefined locations on the array.
- The resulting array may be employed to screen for differences in organisms or samples or products containing genetic material as follows. Genomic or cDNA libraries are prepared using techniques well known in the art. The resulting target DNA is then labeled with a suitable marker, such as a radiolabel, chromophore, fluorophore or chemiluminescent agent, using protocols well known for those skilled in the art. A solution of the labeled target DNA is contacted with the surface of the array and incubated for a suitable period of time.
- The surface of the array is then washed free of unbound target DNA and the probes to which the target DNA hybridized are determined by identifying those regions of the array to which the markers are attached. When the marker is a radiolabel, such as 32P, autoradiography is employed as the detection method. In one embodiment, the marker is a fluorophore, such as fluorescein, and the location of bound target DNA is determined by means of fluorescence spectroscopy. Automated equipment for use in fluorescence scanning of oligonucleotide probe arrays is available from Affymetrix, Inc. (Santa Clara, Calif.) and may be operated according to the manufacturer's instructions. Such equipment may be employed to determine the intensity of fluorescence at each predefined location on the array, thereby providing a measure of the amount of target DNA bound at each location. Such an assay would be able to indicate not only the absence and presence of the marker probe in the target, but also the quantitative amount as well.
- The significance of such a high-throughput screening system is apparent for applications such as microbial selection and quality control operations in which there is a need to identify large numbers of samples or products for unwanted materials, to identify microbes or samples or products containing microbial material for quarantine purposes, etc., or to ascertain the true origin of samples or products containing microbes. Screening for the presence or absence of polynucleotides of the present invention used as identifiers for tagging microbes and microbial products can be valuable for later detecting the genetic composition of food, fermentation and industrial microbes or microbes in human or animal digestive system after consumption of probiotics, etc.
- In this manner, oligonucleotide probe kits of the present invention may be employed to examine the presence/absence (or relative amounts in case of mixtures) of polynucleotides in different samples or products containing different materials rapidly and in a cost-effective manner. Examples of microbial species which may be examined using the present invention, include lactic acid bacteria, such as Lactobacillus rhamnosus, and other microbial species.
- Another aspect of the present invention involves collections of a plurality of polynucleotides of the present invention. A collection of a plurality of the polynucleotides of the present invention, particularly the polynucleotides identified as SEQ ID NO: 1-33, may be recorded and/or stored on a storage medium and subsequently accessed for purposes of analysis, comparison, etc. Suitable storage media include magnetic media such as magnetic diskettes, magnetic tapes, CD-ROM storage media, optical storage media, and the like. Suitable storage media and methods for recording and storing information, as well as accessing information such as polynucleotide sequences recorded on such media, are well known in the art. The polynucleotide information stored on the storage medium is preferably computer-readable and may be used for analysis and comparison of the polynucleotide information.
- Another aspect of the present invention thus involves storage medium on which are recorded a collection of the polynucleotides of the present invention, particularly a collection of the polynucleotides identified as SEQ ID NO: 1-33. According to one embodiment, the storage medium includes a collection of at least 20, of the polynucleotides of the present invention, preferably at least 20 of the polynucleotides identified as SEQ ID NO: 1-33, including variants of those polynucleotides.
- Another aspect of the present invention involves a combination of polynucleotides, the combination containing at least 5, preferably at least 10, more preferably at least 20 different polynucleotides of the present invention, including polynucleotides selected from SEQ ID NO: 1-33, and variants of these polynucleotides.
- In another aspect, the present invention provides genetic constructs comprising, in the 5′-3′ direction, a gene promoter sequence and an open reading frame coding for at least a functional portion of a polypeptide encoded by a polynucleotide of the present invention. In certain embodiments, the genetic constructs of the present invention also comprise a gene termination sequence. The open reading frame may be oriented in either a sense or antisense direction. Genetic constructs comprising a non-coding region of a gene coding for a polypeptide encoded by the above polynucleotides or a nucleotide sequence complementary to a non-coding region, together with a gene promoter sequence, are also provided. A terminator sequence may form part of this construct. Preferably, the gene promoter and termination sequences are functional in a host organism. More preferably, the gene promoter and termination sequences are common to those of the polynucleotide being introduced. The genetic construct may further include a marker for the identification of transformed cells.
- Techniques for operatively linking the components of the genetic constructs are well known in the art and include the use of synthetic linkers containing one or more restriction endonuclease sites as described, for example, by Sambrook et al., in Molecular cloning: a laboratory manual, Cold Spring Harbor Laboratories Press: Cold Spring Harbor, N.Y., 1989. The genetic constructs of the present invention may be linked to a vector having at least one replication system, for example, E. coli, whereby after each manipulation, the resulting construct can be cloned and sequenced and the correctness of the manipulation determined.
- Transgenic microbial cells comprising the genetic constructs of the present invention are also provided by the present invention, together with microbes comprising such transgenic cells, products and progeny of such microbes, and materials including such microbes. Techniques for stably incorporating genetic constructs into the genome of target microbes, such as Lactobacillus species, Lactococcus lactis or E. coli, are well known in the art of bacterial transformation and are exemplified by the transformation of E. coli for sequencing in Example 1, as well as the transformations described in numerous of the examples provided below.
- Transgenic, non-microbial, cells comprising the genetic constructs of the present invention are also provided, together with organisms comprising such transgenic cells, and products and progeny of such organisms. Genetic constructs of the present invention may be stably incorporated into the genomes of non-microbial target organisms, such as fungi, using techniques well known in the art.
- In preferred embodiments, the genetic constructs of the present invention are employed to transform microbes used in the production of food products, ingredients, processing aids, additives or supplements and for the production of microbial products for pharmaceutical uses, particularly for modulating immune system function and immunological effects; and in the production of chemoprotectants providing beneficial effects, probiotics and health supplements. The inventive genetic constructs may also be employed to transform bacteria that are used to produce enzymes or substances such as polysaccharides, flavor compounds, and bioactive substances, and to enhance resistance to industrial processes such as drying and to adverse stimuli in the human digestive system. The genes involved in antibiotic production, and phage uptake and resistance in Lactobacillus rhamnosus are considered to be especially useful. The target microbe to be used for transformation with one or more polynucleotides or genetic constructs of the present invention is preferably selected from the group consisting of bacterial genera Lactococcus, Lactobacillus, Streptococcus, Oenococcus, Lactosphaera, Trichococcus, Pediococcus and others potentially useful in various fermentation industries selected, most preferably, from the group consisting of Lactobacillus species in the following list: Lactobacillus acetotolerans, Lactobacillus acidophilus, Lactobacillus agilis, Lactobacillus alimentarius, Lactobacillus amylolyticus, Lactobacillus amylophilus, Lactobacillus amylovorus, Lactobacillus animalis, Lactobacillus arizonae, Lactobacillus aviarius, Lactobacillus bavaricus, Lactobacillus bifermentans, Lactobacillus brevis, Lactobacillus buchneri, Lactobacillus bulgaricus, Lactobacillus casei, Lactobacillus collinoides, Lactobacillus coryniformis, Lactobacillus crispatus, Lactobacillus curvatus, Lactobacillus delbrueckii, Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus delbrueckii subsp. lactis, Lactobacillus farciminis, Lactobacillus fermentum, Lactobacillus fructivorans, Lactobacillus gallinarum, Lactobacillus gasseri, Lactobacillus graminis, Lactobacillus hamsteri, Lactobacillus helveticus, Lactobacillus helveticus subsp. jugurti, Lactobacillus hetero, Lactobacillus hilgardii, Lactobacillus homohiochii, Lactobacillus japonicus, Lactobacillus johnsonii, Lactobacillus kefiri, Lactobacillus lactis, Lactobacillus leichmannii, Lactobacillus lindneri, Lactobacillus mali, Lactobacillus maltaromicus, Lactobacillus manihotivorans, Lactobacillus mucosae, Lactobacillus murinus, Lactobacillus oris, Lactobacillus panis, Lactobacillus paracasei, Lactobacillus paracasei subsp. pseudoplantarum, Lactobacillus paraplantarum, Lactobacillus pentosus, Lactobacillus plantarum, Lactobacillus pontis, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus niminis, Lactobacillus sake, Lactobacillus salivarius, Lactobacillus salivarius subsp. salicinius, Lactobacillus salivarius subsp. salivarius, Lactobacillus sanfranciscensis, Lactobacillus sharpeae, Lactobacillus thermophilus, Lactobacillus vaginalis, Lactobacillus vermiforme, Lactobacillus zeae.
- In yet a further aspect, the present invention provides methods for modifying the concentration, composition and/or activity of a polypeptide in a host organism, such as a microbe, comprising stably incorporating a genetic construct of the present invention into the genome of the host organism by transforming the host organism with such a genetic construct. The genetic constructs of the present invention may be used to transform a variety of organisms.
- Thus, in yet another aspect, transgenic cells comprising the genetic constructs of the present invention are provided, together with organisms, such as microbes, comprising such transgenic cells, and products and progeny of such microbes. Techniques for stably incorporating genetic constructs into the genome of target organisms are well known in the art. Once the cells are transformed, cells having the genetic construct incorporated in their genome are selected. Transgenic cells may then be cultured in an appropriate medium, using techniques well known in the art.
- Polynucleotides of the present invention may also be used to specifically suppress gene expression by methods such as RNA interference (RNAi), which may also include cosuppression and quelling. This and other techniques of gene suppression are well known in the art. A review of this technique is found in Science 288:1370-1372, 2000. Traditional methods of gene suppression, employing antisense RNA or DNA, operate by binding to the reverse sequence of a gene of interest such that binding interferes with subsequent cellular processes and thereby blocks synthesis of the corresponding protein. RNAi also operates on a post-transcriptional level and is sequence specific, but suppresses gene expression more efficiently.
- Studies have demonstrated that one or more ribonucleases specifically bind to and cleave double-stranded RNA into short fragments. The ribonuclease(s) remains associated with these fragments, which in turn specifically bind to complementary mRNA, i.e. specifically bind to the transcribed mRNA strand for the gene of interest. The mRNA for the gene is also degraded by the ribonuclease(s) into short fragments, thereby obviating translation and expression of the gene. Additionally, an RNA polymerase may act to facilitate the synthesis of numerous copies of the short fragments, which exponentially increases the efficiency of the system. A unique feature of this gene suppression pathway is that silencing is not limited to the cells where it is initiated. The gene-silencing effects may be disseminated to other parts of an organism and even transmitted through the germ line to several generations.
- Specifically, polynucleotides of the present invention are useful for generating gene constructs for silencing specific genes. Polynucleotides of the present invention may be used to generate genetic constructs that encode a single self-complementary RNA sequence specific for one or more genes of interest. Genetic constructs and/or gene-specific self-complementary RNA sequences may be delivered by any conventional method known in the art. Within genetic constructs, sense and antisense sequences flank an intron sequence arranged in proper splicing orientation making use of donor and acceptor splicing sites. Alternative methods may employ spacer sequences of various lengths rather than discrete intron sequences to create an operable and efficient construct. During post-transcriptional processing of the gene construct product, intron sequences are spliced-out, allowing sense and antisense sequences, as well as splice junction sequences, to bind forming double-stranded RNA. Select ribonucleases bind to and cleave the double-stranded RNA, thereby initiating the cascade of events leading to degradation of specific mRNA gene sequences, and silencing specific genes. Alternatively, rather than using a gene construct to express the self-complementary RNA sequences, the gene-specific double-stranded RNA segments are delivered to one or more targeted areas to be internalized into the cell cytoplasm to exert a gene silencing effect.
- Using this cellular pathway of gene suppression, gene function may be studied and high-throughput screening of sequences may be employed to discover sequences affecting gene expression. Additionally, genetically modified microbes and higher order organisms may be generated.
- In another aspect, the present invention provides methods for using one or more of the inventive polypeptides or polynucleotides to treat disorders in a mammal, such as a human.
- In this aspect, the polypeptide or polynucleotide is generally present within a composition, such as a pharmaceutical or immunogenic composition. Pharmaceutical compositions may comprise one or more polypeptides, each of which may contain one or more of the above sequences (or variants thereof), and a physiologically acceptable carrier. Immunogenic compositions may comprise one or more of the above polypeptides and an immunostimulant, such as an adjuvant or a liposome, into which the polypeptide is incorporated.
- Alternatively, a composition of the present invention may contain DNA encoding one or more polypeptides described herein, such that the polypeptide is generated in situ. In such compositions, the DNA may be present within any of a variety of delivery systems known to those of ordinary skill in the art, including nucleic acid expression systems, and bacterial and viral expression systems. Appropriate nucleic acid expression systems contain the necessary DNA sequences for expression in the patient (such as a suitable promoter and terminator signal). Bacterial delivery systems involve the administration of a bacterium (such as Bacillus Calmette-Guerin) that expresses an immunogenic portion of the polypeptide on its cell surface. In a preferred embodiment, the DNA may be introduced using a viral expression system (e.g., vaccinia or other poxvirus, retrovirus, or adenovirus), which may involve the use of a non-pathogenic, or defective, replication competent virus. Techniques for incorporating DNA into such expression systems are well known in the art. The DNA may also be “naked,” as described, for example, in Ulmer et al., Science 259:1745-1749, 1993 and reviewed by Cohen, Science 259:1691-1692, 1993. The uptake of naked DNA may be increased by coating the DNA onto biodegradable beads, which are efficiently transported into the cells.
- While any suitable carrier known to those of ordinary skill in the art may be employed in the pharmaceutical compositions of this invention, the type of carrier will vary depending on the mode of administration. For parenteral administration, such as subcutaneous injection, the carrier preferably comprises water, saline, alcohol, a lipid, a wax or a buffer. For oral administration, any of the above carriers or a solid carrier, such as mannitol, lactose, starch, magnesium stearate, sodium saccharine, talcum, cellulose, glucose, sucrose, and magnesium carbonate, may be employed. Biodegradable microspheres (e.g., polylactic galactide) may also be employed as carriers for the pharmaceutical compositions of this invention. Suitable biodegradable microspheres are disclosed, for example, in U.S. Pat. Nos. 4,897,268 and 5,075,109.
- Any of a variety of adjuvants may be employed in the immunogenic compositions of the present invention to non-specifically enhance an immune response. Most adjuvants contain a substance designed to protect the antigen from rapid catabolism, such as aluminum hydroxide or mineral oil, and a non-specific stimulator of immune responses, such as lipid A, Bordetella pertussis or M. tuberculosis. Suitable adjuvants are commercially available as, for example, Freund's Incomplete Adjuvant and Freund's Complete Adjuvant (Difco Laboratories, Detroit, Mich.), and Merck Adjuvant 65 (Merck and Company, Inc., Rahway, N.J.). Other suitable adjuvants include alum, biodegradable microspheres, monophosphoryl lipid A and Quil A.
- Routes and frequency of administration, as well as dosage, vary from individual to individual. In general, the inventive compositions may be administered by injection (e.g., intradermal, intramuscular, intravenous or subcutaneous), intranasally (e.g., by aspiration) or orally. In general, the amount of polypeptide present in a dose (or produced in situ by the DNA in a dose) ranges from about 1 pg to about 100 mg per kg of host, typically from about 10 pg to about 1 mg per kg of host, and preferably from about 100 pg to about 1 μg per kg of host. Suitable dose sizes will vary with the size of the patient, but will typically range from about 0.1 ml to about 2 ml.
- The following examples are offered by way of illustration and not by way of limitation.
- Lactobacillus rhamnosus strain HN001 DNA libraries were constructed and screened as follows.
- DNA was prepared in large scale by cultivating the bacteria in 2×100 ml cultures with 100 ml MRS broth (Difco Laboratories, Detroit Mich.) and 1 ml Lactobacillus glycerol stock as inoculum, placed into 500 ml culture flasks and incubated at 37° C. for approx. 16 hours with shaking (220 rpm).
- The cultures were centrifuged at 3500 rpm for 10 min to pellet the cells. The supernatant was removed and the cell pellet resuspended in 40 ml fresh MRS broth and transferred to clean 500 ml culture flasks. Fresh MRS broth (60 ml) was added to bring the volume back to 100 ml and flasks were incubated for a further 2 hrs at 37° C. with shaking (220 rpm). The cells were pelleted by centrifugation (3500 rpm for 10 min) and supernatant removed. Cell pellets were washed twice in 20 ml buffer A (50 mM NaCl, 30 mM Tris pH 8.0, 0.5 mM EDTA).
- Cells were resuspended in 2.5 ml buffer B (25% sucrose (w/v), 50 mM Tris pH 8.0, 1 mM EDTA, 20 mg/ml lysozyme, 20 μg/ml mutanolysin) and incubated at 37° C. for 45 min. Equal volumes of EDTA (0.25 M) was added to each tube and allowed to incubate at room temperature for 5 min. 20% SDS (1 ml) solution was added, mixed and incubated at 65° C. for 90 min. 50 μl Proteinase K (Gibco BRL, Gaithersburg, Md.) from a stock solution of 20 mg/ml was added and tubes incubated at 65° C. for 15 min.
- DNA was extracted with equal volumes of phenol:chloroform:isoamylalcohol (25:24:1). Tubes were centrifuged at 3500 rpm for 40 min. The aqueous phase was removed to clean sterile Oak Ridge centrifuge tubes (30 ml). Crude DNA was precipitated with an equal volume of cold isopropanol and incubated at −20° C. overnight.
- After resuspension in 500 μl TE buffer, DNase-free RNase was added to a final concentraion of 100 μg/ml and incubated at 37° C. for 30 min. The incubation was extended for a further 30 min after adding 100 μl Proteinase K from a stock solution of 20 mg/ml. DNA was precipitated with ethanol after a phenol:chloroform:isoamylalcohol (25:24:1) and a chloroform:isoamylalcohol (24:1) extraction and dissolved in 250 μl TE buffer.
- DNA was digested with Sau3AI at a concentration of 0.004 U/μg in a total volume of 1480 μl, with 996 μl DNA, 138.75 μl 10×
REACT 4 buffer and 252.75 μl H2O. Following incubation for 1 hour at 37° C., DNA was divided into two tubes. 31 μl 0.5 M EDTA was added to stop the digestion and 17 μl samples were taken for agarose gel analysis. Samples were put into 15 ml Falcon tubes and diluted to 3 ml for loading onto sucrose gradient tubes. - Sucrose gradient size fractionation was conducted as follows. 100 ml of 50% sucrose (w/v) was made in TEN buffer (1M NaCl, 20 mM Tris pH 8.0, 5 mM EDTA) and sterile filtered. Dilutions of 5, 10, 15, 20, 25, 30, 35 and 40% sucrose were prepared and overlaid carefully in Beckman Polyallomer tubes, and kept overnight at 4° C. TEN buffer (4 ml) was loaded onto the gradient, with 3 ml of DNA solution on top. The gradients were centrifuged at 26K for 18 hours at 4° C. in a Centricon T-2060 centrifuge using a Kontron TST 28-38 rotor. After deceleration without braking (approx. 1 hour), the gradients were removed and fractions collected using an auto Densi-Flow (Haake-Buchler Instruments). Agarose gel was used to analyse the fractions. The best two pairs of fractions were pooled and diluted to contain less than 10% sucrose. TEN buffer (4 ml) was added and DNA precipitated with 2 volumes of 100% ice cold ethanol and an overnight incubation at −20° C.
- DNA pellets were resuspended in 300 μl TE buffer and re-precipitated for approx. 6 hours at −20° C. after adding 1/10 volume 3 M NaOAC pH 5.2 and 2 volumes of ethanol. DNA was pelleted at top speed in a microcentrifuge for 15 min, washed with 70% ethanol and pelleted again, dried and resuspended in 10 μl TE buffer.
- DNA was ligated into dephosphorylated BamHI-digested pBluescript SK II+ and dephosphorylated BamHI-digested lambda ZAP Express using standard protocols. Packaging of the DNA was done using Gigapack III Gold packaging extract (Stratagene, La Jolla, Calif.) following the manufacturer's protocols. Packaged libraries were stored at 4° C.
- Mass excision from the primary packaged phage library was done using XL1-Blue MRF′ cells and ExAssist Helper Phage (Stratagene). The excised phagemids were diluted with NZY broth (Gibco BRL, Gaithersburg, Md.) and plated out onto LB-kanamycin agar plates containing 5-bromo-4-chloro-3-indolyl-β-D-galactoside (X-gal) and isopropylthio-beta-galactoside (IPTG). After incubation, single colonies were picked for PCR size determination before the most suitable libraries were selected for sequencing.
- Of the colonies picked for DNA minipreps and subsequent sequencing, the large majority contained an insert suitable for sequencing. Positive colonies were cultured in LB broth with kanamycin or ampicillin depending on the vector used, and DNA was purified by means of rapid alkaline lysis minipreps (solutions: Qiagen, Venlo, The Netherlands; clearing plates, Millipore, Bedford, Mass.). Agarose gels at 1% were used to screen sequencing templates for chromosomal contamination and concentration. Dye terminator sequencing reactions were prepared using a Biomek 2000 robot (Beckman Coulter, Inc., Fullerton, Calif.) and Hydra 96 (Robbins Scientific, Sunnyvale, Calif.) for liquid handling. DNA amplification was done in a 9700 PCR machine (Perkin Elmer/Applied Biosystems, Foster City, Calif.) according to the manufacturer's protocol.
- The sequence of the genomic DNA fragments were determined using a Perkin Elmer/Applied Biosystems Division Prism 377 sequencer. The DNA clones were sequenced from the 5′ and/or 3′ end, and are identified as SEQ ID NO: 1-33.
- This example not only shows how the sequences were obtained, but also that a bacterium (E. coli) can be stably transformed with any desired DNA fragment of the present invention for permanent marking for stable inheritance.
- The determined DNA sequences were compared to and aligned with known sequences in the public databases. Specifically, the polynucleotides identified in SEQ ID NO: 1-33 were compared to polynucleotides in the EMBL database as of the end of July 2001, using BLASTN algorithm Version 2.0.11 [Jan. 20, 2000], set to the following running parameters: Unix running command: blastall -p blastn -d embldb -e 10 -G 0 -E 0 -r 1 -v 30 -b 30 -i queryseq -o results. Multiple alignments of redundant sequences were used to build up reliable consensus sequences. Based on similarity to known sequences, the isolated polynucleotides of the present invention identified as SEQ ID NO: 1-33 were identified as encoding polypeptides.
- Numerous of the sequences provided in SEQ ID NO: 1-33 were found to be “full-length” and to contain open reading frames (ORFs). These full-length sequences, the location of ORFs (by nucleotide position) contained within these sequences, and the corresponding amino acid sequences are provided in Table 1B below.
TABLE 1B Polynucleotide Polypeptide SEQ ID NO: ORF SEQ ID NO: 1 1,128-3,026 42 2 196-924 43 3 145-1,098 44 4 82-348 45 5 103-1,239 46 6 122-934 47 7 94-759 48 7 807-1,676 49 8 126-1,232 50 9 181-1,086 51 10 23-1,510 52 11 209-1,381 53 12 1-1,179 54 13 1-650 55 14 1-768 56 15 163-1,167 57 16 64-888 58 17 47-1,219 59 18 45-1,295 60 19 175-1,173 61 20 48-1,352 62 21 1,705-2,280 63 22 60-1,250 64 23 71-1,093 65 24 120-1,074 66 25 86-934 67 26 2,209-7,434 68 27 74-4,465 69 28 821-6,460 70 29 141-1,022 71 30 83-607 72 31 27-875 73 32 96-881 74 33 1-1,191 75 - The polynucleotide and polypeptide sequences of SEQ ID NO: 1-33 and 42-75 were compared to sequences in the EMBL and SwissProt databases using the BLAST computer algorithms version 2.0.11 [Jan. 20, 2000]. Comparisons of polynucleotide sequences provided in SEQ ID NO: 1-33 to sequences in the EMBL database were made as of August 2001. Comparisons of amino acid sequences provided in SEQ ID NO: 42-75 to sequences in the SwissProt database were made as of August 2001. Analysis of six-frame translations of the polynucleotides of SEQ ID NO: 1-33 were also compared to and aligned with the six-frame translations of polynucleotides in the SwissProt database using the BLASTX program.
- BLASTN Polynucleotide Analysis
- The polynucleotide sequences of SEQ ID NO: 1-3, 5-23 and 25-33 were determined to have less than 50% identity, determined as described above, to sequences in the EMBL database using the computer algorithm BLASTN, as described above. The polynucleotide sequence of SEQ ID NO: 24 was determined to have less than 90% identity, determined as described above, to sequences in the EMBL database using BLASTN, as described above. The polynucleotide sequence of SEQ ID NO: 4 was determined to have less than 98% identity, determined as described above, to sequences in the EMBL database using BLASTN, as described above.
- BLASTP Amino Acid Analysis
- The amino acid sequences of SEQ ID NO: 43, 45-47, 51-53, 58, 60, 61, 63, 67, 68, 70, 71, 73 and 74 were determined to have less than 50% identity, determined as described above, to sequences in the SwissProt database using the BLASTP computer algorithm as described above. The amino acid sequences of SEQ ID NO: 48-50, 55-56, 62, 64, 66, 69, 72 and 75 were determined to have less than 75% identity, determined as described above, to sequences in the SwissProt database using the BLASTP computer algorithm as described above. The amino acid sequences of SEQ ID NO: 57 and 65 were determined to have less than 90% identity, determined as described above, to sequences in the SwissProt database using the computer algorithm BLASTP, as described above. The amino acid sequence of SEQ ID NO: 54 and 59 was determined to have less than 98% identity, determined as described above, to sequences in the SwissProt database using the computer algorithm BLASTP, as described above.
- BLASTX Analysis
- The six-frame translations of the polynucleotide sequences of SEQ ID NO: 1-33 were compared to and aligned with six-frame translations of polynucleotides in the EMBL database using the BLASTX program version 2.0.11 [Jan. 20, 2000] set to the following running parameters: Unix running command: blastall -p blastn -d embldb -e 10 -G 0 -E 0 -v 30 -b 30 -i queryseq -o results. The translations of the polynucleotides of SEQ ID NO: 1, 3, 5-9, 11-19, 21 and 25-32 were determined to have less than 50% identity, determined as described above, to translations of polynucleotides in the EMBL database using the computer algorithm BLASTX. The translations of the polynucleotides of SEQ ID NO: 2, 4, 10, 20, 22, 23 and 33 were determined to have less than 75% identity, determined as described above, to translations of polynucleotides in the EMBL database using the computer algorithm BLASTX. The translations of the polynucleotide sequence of SEQ ID NO: 24 was determined to have less than 90% identity, determined as described above, to translations of polynucleotides in the EMBL database using the computer algorithm BLASTX.
- The full-length gene sequence of a peptidase believed to be related to pepO, and referred to herein as “pepO” from L. rhamnosus strain HN001 (given in SEQ ID NO: 1 and shown in
FIG. 80 ) was isolated essentially as described in Example 1. Primers were designed to this sequence and employed to amplify pepO from L. rhamnosus HN001 using standard PCR methodology. PepO was cloned in the vector pTRKH2 (obtained from Dr Todd Klaenhammer, North Carolina State University, North Carolina, USA) and transformed into E. coli. Competent cells of L. rhamnosus HN001 were transformed with the pTRKH2+pepO construct to overexpress the gene in strain HN001. The amino acid sequence of the expressed protein is provided in SEQ ID NO: 42 and shown inFIG. 81 . - Cell extracts of the HN001 strain constructs with enhanced levels of the peptidase enzyme showed enhanced enzyme activity on the casein peptide, αS1-casein(1-17). Specifically, αS1-casein(1-17) was incubated with non-transformed strain HN001 (referred to as DR20 WT) and strain HN001 transformed with the pepO construct described above (referred to as DR20 PepO:1 and DR20 PepO:4). HPLC separation of the resulting peptide products was performed using a Vydac reverse phase C18 column, 4.6 mm×250 mm. The solvent system was solvent A, 0.1% TFA in water, solvent B, 0.08% TFA in acetonitrile and the gradient employed was 15-40% solvent B over 20 minutes. A major peak was observed at 11 minutes, together with other non-identified minor peaks corresponding to hydrolysis products of the original substrate.
- With non-transformed HN001 (
DR 20 WT), the major peak of unhydrolysed αs1-casein(1-17) had a height of approximately 250 mAU. With each of the two transformed strains of HN001 (DR 20 PepO:1 andDR 20 PepO:4) the major peak of unhydrolysed αsi-casein(1-17) had a height of approximately 150 mAU, demonstrating that HN001 transformed with the pepO construct has enhanced peptidase activity compared to non-transformed HN001. - The peptidase of SEQ ID NO: 42 was not active on bradykinin, a standard substrate for measuring pepO activity (Pritchard et al., Microbiol. 140:923-30,1994). The enzyme of SEQ ID NO: 42 thus has a specificity that is significantly different from the homologous enzyme from Lactococcus.
- The polypeptide of SEQ ID NO: 42 and the polynucleotide of SEQ ID NO: 1 have utility in processing food products to develop new characteristics in food products, and as supplements and additives to food products, including cheese and hydrolyzed milk protein products. This enzyme may also be used to develop non-food products. The attributes conferred by this enzyme, and the applications for use of this enzyme, include: flavor and aroma enhancement; removal of bitter peptides and undesirable flavors; nutritional enhancement; enhanced texture and functionality; production of bioactive peptides; and removal of allergenic peptides or proteins.
- These attributes may be produced in food, such as dairy products, (including milk protein hydrolysates and cheese) by directed activity of the enzyme, introduced in a bacterial strain (including strain HN001 or starter cultures) comprising a polynucleotide of SEQ ID NO: 1, or as an enzyme preparation comprising a polypeptide of SEQ ID NO: 42.
- The full-length polynucleotide sequence of an esterase gene, given in SEQ ID NO: 3, was used to amplify the AA 7 esterase gene from L. rhamnosus HN001 using standard PCR methodology.
FIG. 1 shows the nucleotide sequence of L. rhamnosus strain HN001 esterase gene AA 7, with the ATG initiation and translation stop codons shown boxed. - The AA7 esterase gene sequence was cloned into the pUniBlunt/V5-HisTopo vector (Invitrogen, Auckland, NZ) and transformed into the E. coli strain PIR1 OneShot competent cells (Invitrogen). To construct an expression plasmid the pUniBlunt/V5-HisTopo vector construct was recombined with the pBad/Thio-E vector (Invitrogen) and transformed into the E. coli strain TOP10 competent cells (Invitrogen) according to the manufacturer's instructions. The gene product was therefore cloned as a fusion protein tagged with a His-patch polypeptide and thioredoxin protein. The esterase fusion protein was expressed and purified using a Ni-NTA column (Qiagen, Auckland, NZ) according to the manufacturer's instructions and protein expression checked by SDS-PAGE. The amino acid sequence of the esterase AA7 polypeptide is given in SEQ ID NO: 44 and shown in
FIG. 2 . - Esterase activity was assessed using the para-nitrophenyl butyrate assay as described in Lee and Lee, Biotech. Appl. Biochem. 11:552-563, 1989, with some modifications. Briefly, esterase activity was measured spectrophotometrically using p-nitrophenyl butyrate (Sigma Chemical Co., St Louis, Mo.) as substrate. Substrate was prepared by sonicating 1 ml of 50 mM methanolic p-nitrophenyl butyrate in 18
ml 50 mM sodium phosphate buffer (pH 7.5). Aliquots of 1.9 ml were placed in cuvettes, allowed to stabilize at 30° C., and between 5 and 20 μl of purified AA7 esterase added. Changes in optical density (OD) 410 nm were determined. Based on the results, enzyme activity was calculated, with one unit (U) of enzyme defined as the amount required to hydrolyze 1 μmol substrate per minute. - Esterase activity of the AA7 fusion protein was compared to the activity of a known esterase enzyme from Streptococcus thermophilus (ST1, as described in Liu et al., Int. Dairy J. 11:27-35, 2001), a non-esterase HN001 enzyme also expressed as a His-patch/Thioredoxin fusion protein and buffer-only.
- The results are shown in
FIG. 3 and the enzyme activities are given in Table 1C.FIG. 3 demonstrates the production of ethyl butyrate from para-nitrophenyl butyrate substrate as measured by change in OD at 410 nm. As shown inFIG. 3 , while buffer only (♦) and the HN001 non-esterase fusion protein (●) showed minimal esterase activity, the ST1 esterase from Streptococcus thermophilus (σ) and the AA7 esterase fusion protein (ν) showed strong activity. Thus, the AA7 esterase fusion protein showed strong esterase activity compared to the positive control, and negligible amounts of esterase was produced by the two negative controls (buffer-only and the non-esterase fusion protein).TABLE 1C Esterase activity of the AA7 fusion protein Enzyme activity Protein Δ OD410/min (μmol/min/ml) AA7 fusion protein 0.41 3.7 ST1 esterase control 0.49 4.0 Non-esterase control 0.05 0.4 Buffer-only control 0.02 0.2 - The esterase activity exhibited by the AA7 fusion protein was not due to background hydrolysis of the substrate as the buffer-only control showed little or no activity. The specific enzyme activity of the His-patch/Thio/AA7 fusion protein was 1.42 μmol/min/mg protein compared with 0.03 μmol/min/mg for the non-esterase fusion protein, showing an almost 50-fold difference in esterase activity. Therefore, AA7 esterase activity was not due to the His-patch/Thioredoxin fusion protein tag.
- The dose-response of the AA7 fusion protein was determined by comparing the esterase activity in a series of three two-fold dilutions of the purified enzyme. Results are shown in
FIG. 4 and the rate of change in optical density at 410 nm and enzyme activities given in Table 2. As shown inFIG. 4 , while buffer-only (λ) showed no esterase activity, increasing amounts of His-patch/Thio/AA7 fusion protein; 5 μl (υ), 10 μl (σ) and 20 μl (ν) purified protein showed increasing rates of substrate hydrolysis. The increase in substrate hydrolysis was proportional to amount of AA7 fusion protein added.TABLE 2 Esterase activity for increasing amounts of AA7 fusion protein Enzyme activity Protein Δ OD410/min (μmol/min/ml) 5 μl His-patch/Thio/AA7 0.18 5.9 10 μl His-patch/Thio/AA7 0.40 6.7 20 μl His-patch/Thio/AA7 0.68 5.6 1.Buffer-only 0.00 0.0 - Results indicated the rate of change in OD at 410 nm was proportional to the amount of enzyme added, whilst enzyme activity remained relatively constant. Therefore, esterase activity was dependent on the amount of esterase AA7 fusion protein present.
- The effect of the serine esterase inhibitor PMSF was determined using the p-nitrophenyl butyrate assay. Esterase activity of the AA7 fusion protein was assessed in the presence and absence of 10 mM PMSF. Results are shown in
FIG. 5 , and the rate of change in OD at 410 nm and enzyme activities given in Table 3. These results indicate that the PMSF inhibitor caused a 17.9% reduction in the esterase activity of the AA7 fusion protein. Therefore, AA7 esterase activity was inhibited by the serine esterase-specific inhibitor PMSF.TABLE 3 Effect of PMSF inhibitor on AA7 fusion protein esterase activity Enzyme activity Protein Δ OD410/min (μmol/min/ml) AA7 0.68 5.6 AA7 + 10 mM PMSF 0.50 4.1 Buffer-only 0.00 0.0 - The enzymatic breakdown of milk fat plays an essential role in the development of flavor in cheese. Esterases and lipases catalyze the lipolysis of milk fat in dairy products such that the triglycerides are hydrolyzed to free fatty acids and glycerol or mono- and diglycerides. Although exogenous esterases and lipases of mammalian and fungal origins are often used to encourage extensive lipolysis in cheeses, esterases and lipases from cheese microorganisms may also contribute to lipolysis (reviewed in Fox and Wallace, Adv. Appl. Microbiol. 45:17-85, 1997 and McSweeney and Wallace, Lait 80:293-324, 2000).
- The polypeptide of SEQ ID NO: 44 and the polynucleotide of SEQ ID NO: 3 have utility for processing food products and as supplements and additives to food products. This esterase may also be used to develop non-food products. The attributes conferred by this enzyme include: enhanced flavor and aroma; removal of off-flavors; altered levels of butyric acid; and altered metabolic characteristics.
- These attributes may be produced in food, such as dairy products, by directed activity of the enzyme, introduced in a bacterial strain (including strain HN001 or starter cultures) comprising a polynucleotide of SEQ ID NO: 3 or a variant, or as an enzyme preparation comprising a polypeptide of SEQ ID NO: 44 or a variant.
- The full-length polynucleotide sequence of an autoaggregation protein from L. rhamnosus strain HN001, given in SEQ ID NO: 10, was used to amplify the AG5 autoaggregation gene from L. rhamnosus HN001 DNA using standard PCR methodology. The full-length polynucleotide sequence of L. rhamnosus strain HN001 autoaggregation gene AG5, showing ATG initiation and translation stop codons (boxed) is shown in
FIG. 6 . - AG5 was then cloned into the EcoRI and SalI sites of the pGEX-6P-3 expression vector (Pharmacia Biotech, Auckland, NZ) and transformed into the E. coli strain K12 XL-1Blue competent cells according to standard laboratory protocols. The amino acid sequence of the autoaggregation protein AG5 is given in SEQ ID NO: 52 and shown in
FIG. 7 . - The autoaggregation AG5 protein was expressed as a fusion protein with glutathione S-transferase (GST), isolated and purified using Glutathione Sepharose 4B resin (Pharmacia Biotech) according to the manufacturer's instructions and protein expression checked by SDS-PAGE.
- An assay for aggregation was adapted from Roos et al., Mol. Microbiol. 32:427-436, 1999. A 10 ml overnight culture of L. rhamnosus strain HN001 was grown in Man-Rogosa-Sharpe (MRS) broth (Oxoid) with glucose at a final concentration of 1%. The bacteria were washed five times in sterile deionized water resulting in loss of endogenous aggregation. Bacteria were suspended in 1 ml PBS, and 5 μl of the purified the HN001 autoaggregation protein AG5 fusion protein or an irrelevant (ie. non-adhesion) GST-fusion protein were added to 20 μl aliquots of the bacterial suspension, and placed on microscope slides. The slides were rocked gently for 13 min, and aggregation monitored by light microscopy.
- As shown in
FIG. 8A , in the presence of the AG5 autoaggregation GST-fusion protein, L. rhamnosus strain HN001 cells readily aggregated.FIG. 8A illustrates an image of a phase-contrast photomicrograph (exposure ⅛ sec, final magnification×240) showing obvious clumping of washed L. rhamnosus strain HN001 cells in the presence of AG5 autoaggregation protein tagged with GST. If an irrelevant (ie. non-adhesion) GST-fusion protein was used, no aggregation occurred.FIG. 8B illustrates an image of a phase-contrast photomicrograph (exposure ⅛ sec, final magnification×240) showing no clumping of washed L. rhamnosus strain HN001 cells in the presence of an irrelevant (non-adhesion) HN001 protein tagged with GST, as a negative control. The GST-tagged HN001 autoaggregation protein AG5 did not form observable clumps in the absence of bacterial cells (data not shown). Thus, the HN001 autoaggregation protein AG5 mediated the autoaggregation of L. rhamnosus strain HN001 cells. - The L. rhamnosus strain HN001 is known to have probiotic properties (see Tannock et al., Appl. Environ. Microbiol. 66:2,578-2,588, 2000; Gill et al., Br. J. Nutr. 83:167-176, 2000; Prasad et al., Int. Dairy J. 8:993-1002, 1998). In order to function effectively as probiotic bacteria, L. rhamnosus HN001 must colonize (at least transiently) the gut environment, as well as exert positive health benefits, possibly through the exclusion of pathogenic bacteria from intestinal surfaces. The ability to form aggregates may be important for both survival in the gut environment and functionality of L. rhamnosus HN001. The ability to autoaggregate may assist in the formation of biofilms of L. rhamnosus HN001 and/or related species, improving the chances of colonization in the highly competitive gut environment, and then exclusion of competing bacteria, including pathogens.
- The polypeptide of SEQ ID NO: 52, and the polynucleotide of SEQ ID NO: 10 have utility for processing food products and as supplements and additives to food products. This protein may also be used to develop non-food products. The attributes conferred by this enzyme include: as a prebiotic to enhance the growth of L. rhamnosus HN001 or other Lactobacillus species in the gut; as an agent to promote clumping of L. rhamnosus HN001 in media to improve survival in industrial processes; and as an agent to help prevent pathogenic colonization of mucosal surfaces.
- These attributes may be produced in food, such as dairy products, by directed activity of the autoaggregation protein, introduced in a bacterial strain (including strain HN001 or starter cultures) comprising a polynucleotide of SEQ ID NO: 10 or a variant, or as an enzyme preparation comprising a polypeptide of SEQ ID NO: 52 or a variant.
- The full-length polynucleotide sequence of malic enzyme AA5, given in SEQ ID NO: 2, was amplified from L. rhamnosus HN001 DNA using standard PCR methodology. The polynucleotide sequence of L. rhamnosus strain HN001 malic enzyme gene AA5 showing ATG initiation and translation stop codons (boxed) is shown in
FIG. 9 . The upstream and downstream primers were tagged with EcoRI and BamHI restriction endonuclease recognition sequences to facilitate cloning. - The AA5 gene was then cloned into the EcoRI and BamHI sites of the pGEX-6P-3 expression vector (Pharmacia Biotech) and transformed into the E. coli strain DH-5α competent cells according to standard laboratory protocols. Cells were lysed by sonication and the AA5 protein, expressed as a GST fusion protein, was checked by SDS-PAGE analysis. The polypeptide sequence is given in SEQ ID NO: 43 and shown in
FIG. 10 . - Malic enzyme activity was assessed determining the rate of pyruvate reduction in transformed strains of an E. coli mutant. The E. coli strain EJ1321 contains multiple mutations that affect both NAD- and NADP-dependent malic enzyme activity, as well as malic enzyme regulation (Hansen and Juni, Biochem. Biophys. Res. Comm. 65:559-566, 1975). The strain was obtained from the E. coli Genetic Stock Centre (Yale University, USA), and transformed with the pGEX-6P-3 vector construct encoding the HN001 malic enzyme AA5. Transformants were selected by resistance to 100 μg/ml ampicillin on M9 plates supplemented with 0.5% glucose (ie. permissive growth conditions). Ampicillin resistant EJ132 colonies were picked and grown overnight at 37° C. in 10 ml LB broth with 100 μg/ml ampicillin and 2 ml then used to inoculate 100 ml LB broth with 100 μg/ml ampicillin. Cultures were incubated at 37° C. with shaking until OD at 600 nm reached approximately 0.4 whereupon expression of the AA5 protein was induced by the addition of 100 μl of 1 M IPTG. After a further 4 hours culture at 37° C. with shaking, 10 ml aliquots were taken, spun at 4000 rpm for 5 min, supernatants removed and cells resuspended in 5 ml PBS. Cultures were then sonicated to produce crude lysates.
- Malic enzyme activity in the crude lysates was measured according to Kobayashi et al., J. Biol. Chem. 264:3200-3205, 1989, with modifications. Briefly, total protein content of the lysates were quantitated using the BCA Protein Assay Reagent kit (Pierce, Rockford, Ill., USA) according to the manufacturer's instructions, and 3.5 mg total protein added to 990 μl reaction solution containing 100 μM MOPS buffer (pH 6.1), 100 μM Na2CO3, 50 μM NADH and 5 μM MgCl2 (Sigma). Lastly, 10 μl of 1 M sodium pyruvate was added as substrate and utilization of NADH measured as change in OD at 340 nm.
- Malic enzyme activity was compared between PBS buffer only (20 μl), crude lysate from wild type EJ1321 cells (ie. non-transformed), EJ1321 cells transformed with pGEX-6P-3 encoding an irrelevant protein (AD5), and EJ1321 cells transformed with pGEX-6P-3 encoding HN001 malic enzyme AA5 (
FIG. 11 ). Specific activities are given in Table 4, with a unit of enzyme was defined as μmole NADH used per min per mg protein. - The results indicate that although NADH was stable (ie. no change in OD in the presence of NADH and substrate), some background NADH reduction occurred when crude lysates from wild-type EJ1321 cells or EJ1321 cells expressing an irrelevant protein. Nonetheless, clear malic enzyme activity was observed when crude lysate from EJ1321 cells expressing AA5 protein was used, with over 6-fold more enzyme activity compared to background. Therefore, AA5 encodes a malic enzyme.
-
FIG. 11 shows malate enzyme activity measured as rate of pyruvate reduction by crude lysate preparations of EJ1321 cell transformants. ν PBS buffer-only; σ 3.5 μg wild-type EJ1321 cell lysate; υ 3.5 μg cell lysate of EJ1321 transformed with pGEX-6P-3 construct encoding an irrelevant HN001 protein (AD5); λ 3.5 μg cell lysate of EJ1321 transformed with pGEX-6P-3 construct encoding HN001 malic enzyme AA5.TABLE 4 Malic enzyme activity in crude lysates of transformed and non-transformed EJ1321 cells Enzyme activity Lysate Δ OD340/min (μmol/min/ml) Buffer-only 0.00 0.00 Wild-type EJ1321 0.01 2.0 × 102 EJ1321 with pGEX-6P-3 0.02 4.2 × 102 encoding an irrelevant protein EJ1321 with pGEX-6P-3 0.12 26.8 × 102 encoding AA5 - The malic enzyme assay was repeated with increasing amounts of crude lysate from EJ1321 cells expressing AA5 protein to determine whether malic enzyme activity was proportional to amount of AA5 protein present (
FIG. 12 and Table 5). - Results from
FIG. 12 and Table 5 indicate that increased amounts of crude lysate of EJ1321 E. coli strain transformed with HN001 malic enzyme AA5 led to increased malic enzyme activity. However, as the amount of substrate became limiting at higher amounts of lysate, the increases in activity were not strictly proportional. Nonetheless, these results support the evidence that AA5 encodes the HN001 malic enzyme. -
FIG. 12 shows data illustrating the effect of increasing amounts of EJ1321 crude lysate on malic enzyme activity. ν 5 μl wild-type EJ1321 cell lysate; σ 5 μl cell lysate of EJ1321 transformed with pGex-6P-3 encoding AA5; υ 50 μl cell lysate of EJ1321 transformed with pGex-6P-3 encoding AA5; υ 200 μl cell lysate of EJ1321 transformed with pGex-6P-3 encoding AA5.TABLE 5 Malic enzyme activity with increasing amounts of cell lysate Enzyme activity Lysate Δ OD340/min (μmol/min/ml) 5 μl wild-type EJ1321 0.004 3.2 × 102 5 μl EJ1321 with pGEX-6P-3 0.032 25.8 × 102 encoding AA5 2.50 μl EJ1321 with pGEX-6P- 0.216 17.3 × 102 3 encoding AA5 3.200 μl EJ1321 with pGEX- 0.232 4.6 × 102 6P-3 encoding AA5 - The NAD-dependent malic enzyme (EC 1.1.1.38) catalyzes L-malate oxidative decarboxylation and pyruvate reductive carboxylation (Murai, T. et al, Biochem. Biophys. Res. Comm. 43:875-881, 1971) and is central to citrate metabolism.
- The polypeptide of SEQ ID NO: 43 and the polynucleotide of SEQ ID NO: 2 have utility for processing food products and as supplements and additives to food products, as well as in industrial processing. This malic enzyme may also be used to develop non-food products and in non-food processing systems. The attributes conferred by this enzyme include: manipulation of energy production and growth in particular media; altered survival characteristics in industrial processes; formation of common intermediates of various flavor compounds; and lactic acid production, important for antibacterial effects and acid tolerance.
- These attributes may be produced in food or in other environments by directed activity of the enzyme, introduced in a bacterial strain (including strain HN001, or starter cultures) comprising a polynucleotide of SEQ ID NO: 2 or a variant, or as an enzyme preparation comprising a polypeptide of SEQ ID NO: 43 or a variant.
- The full-length polynucleotide sequence of malic enzyme, given in SEQ ID NO: 9, was amplified from the AG3 malate dehydrogenase gene from L. rhamnosus HN001 DNA using standard PCR methodology.
FIG. 13 shows the polynucleotide sequence of L. rhamnosus strain HN001 malate dehydrogenase gene AG3 showing the TTG initiation and translation stop codons (boxed). - AG3 was then cloned into the pUniBlunt/V5-HisTopo vector (Invitrogen) and transformed into the E. coli strain PIR1 OneShot competent cells (Invitrogen) according to the manufacturer's instructions. To construct an expression plasmid, the pUniBlunt/V5-HisTopo vector construct was recombined with the pBad/Thio-E Echo vector (Invitrogen) and transformed into the E. coli strain TOP10 competent cells (Invitrogen) according to the manufacturer's instructions. The AG3 gene product was therefore cloned as a fusion protein tagged with a His-patch polypeptide and thioredoxin protein. The fusion protein was expressed and purified using a Ni-NTA column (Qiagen, Auckland, NZ) according to the manufacturer's instructions and protein expression checked by SDS-PAGE. The polypeptide sequence is given in SEQ ID NO: 51 and shown in
FIG. 14 . - Malate dehydrogenase activity was assessed by gene complementation of the mutant E. coli strain UTH4606 that lacks a functional malate dehydrogenase gene (Heard et al., J. Bacteriol. 122:329-331, 1975; Shaw et al., Mutation Res., 18:247-250, 1973), provided by the E. coli Genetic Stock Centre (Yale University, USA). UTH4606 strain cells cannot utilize malate as a carbon source, in contrast to wild-type E. coli. pBAD-Thio-E construct containing the HN001 malate dehydrogenase AG3 gene or empty pBAD-Thio-E vector was transformed into the UTH4606 E. coli strain and plated onto M9 media plates containing 100 μg/ml kanamycin and 0.5% glucose. Transformant colonies were picked, and plated out onto a series of selective M9 agar plates containing 100 μg/ml Kanamycin and/or 0.5% glucose or 0.5% malate. Growth of the UTH4606 transformed with pBAD-Thio-E encoding the AG3 protein was compared with wild-type UTH4606 cells and UTH4606 cells transformed with empty pBAD-Thio-E vector. Plates were incubated aerobically at 37° C. overnight. Growth was assessed for malate dehydrogenase complementation.
- Results are shown in Table 6 and indicate that wild-type UTH780 cells grew on M9 media supplemented with glucose, but not on M9 media supplemented with malate, or on media containing Kanamycin. This confirmed the phenotype of the UTH780 strain of being unable to utilize malate as a carbon source due to the loss of malate dehydrogenase function. Transformation with empty pBAD/Thio-E vector allowed growth on media containing Kanamycin, but did not complement the malate dehydrogenase mutation. Transformation with pBAD/Thio-E encoding the HN001 malate dehydrogenase AG3 allowed growth on Kanamycin, indicating the presence of the plasmid, and on malate, indicating that the AG3 protein complemented the E. coli malate dehydrogenase deficiency. Therefore, the HN001 protein AG3 has malate dehydrogenase activity.
TABLE 6 Results of LB agar plate assay for malate dehydrogenase gene complementation M9 agar plates containing: E. coli UTH780 0.5% 0.5% Glucose + 0.5% Malate + 0.5% transformed with: Glucose Kanamycin Kanamycin Malate — + − − − pBAD/Thio-E + + − − pBAD/Thio-E + + + + encoding AG3
+: growth;
−: no growth
- Malate dehydrogenase (EC 1.1.1.37) catalyzes the reversible oxidation of malate to oxaloacetate with the concomitant reduction of NAD. As lactobacilli appear not to have a functioning Krebs cycle, the enzyme may be involved in amino acid biosynthesis or L-malate utilization pathways.
- The polypeptide of SEQ ID NO: 51, and the polynucleotide of SEQ ID NO: 9 have utility for processing food and other products and as supplements and additives to food products and in industrial processing. The attributes conferred by this enzyme include: manipulation of energy production and growth in particular media; altered survival characteristics in industrial processes; and formation of common intermediates of various flavor compounds.
- These attributes may be produced in food products, or in industrial processing, by directed activity of the enzyme, introduced in a bacterial strain (including strain HN001, or starter cultures) comprising a polynucleotide of SEQ ID NO: 9 or a variant, or as an enzyme preparation comprising a polypeptide of SEQ ID NO: 51 or a variant.
- The full-length polynucleotide sequence of dihydrodipicolinate synthase from L. rhamnosus HN001, given in SEQ ID NO: 13 and shown in
FIG. 15 with ATG initiation and translation stop codons (boxed), was used to amplify the AI2 dihydrodipicolinate synthase gene from L. rhamnosus HN001 DNA using standard PCR methodology. The upstream and downstream primers were tagged with EcoRI and SalI restriction endonuclease recognition sequences to facilitate cloning. - AI2 was then cloned into the EcoRI and SalI sites of the pGEX-6P-3 expression vector (Pharmacia Biotech) and transformed into the E. coli strain K12 XL-1Blue competent cells according to standard laboratory protocols. The dihydrodipicolinate synthase AI2 protein was expressed as a fusion protein with glutathione S-transferase (GST), bound to Glutathione Sepharose 4B resin (Pharmacia Biotech), and PreScission protease used to cleave off dihydrodipicolinate synthase AI2 protein, according to the manufacturer's instructions. An aliquot of the purified AI2 protein was checked by SDS-PAGE analysis. The polypeptide sequence is given in SEQ ID NO: 55 and is shown in
FIG. 16 . - Dihydrodipicolinate synthase activity was assessed by gene complementation of the mutant E. coli strain AT997 deficient in dihydrodipicolinate synthase gene function (Bukhari and Taylor, J. Bacteriol. 105:844-854, 1971), provided by the E. coli Genetic Stock Centre (Yale University, USA). AT997 cells require diaminopimelic acid (DAP) for growth, in contrast to wild-type E. coli that is DAP-independent. pGEX-6P-3 construct containing the HN001 dihydrodipicolinate synthase AI2 gene or empty pGEX-6P-3 vector was transformed into the AT997 E. coli strain. Transformed AT997 cells were plated onto LB agar plates containing ampicillin (100 μg/ml) only or ampicillin and 45 μg/ml DAP, at dilutions designed to allow the visualization of distinct colonies (ie. <200 colonies/plate). Plates were incubated aerobically at 37° C. overnight and growth assessed as the presence of distinct colonies.
- Results in Table 7 indicate that while AT997 cells transformed with either empty pGEX-6P-3 or pGEX-6P-3 containing the HN001 dihydrodipicolinate synthase AI2 grew in the presence of DAP, only cells transformed with vector containing AI2 grew without DAP. Therefore, the HN001 dihydrodipicolinate synthase protein AI2 complemented the dihydrodipicolinate synthase gene mutation in E. coli strain AT997.
TABLE 7 Results of LB agar plate assay for dihydrodipicolinate synthase gene complementation LB agar plates containing: E. coli AT997 transformed with: Ampicillin and DAP Ampicillin only pGEX-6P-3 + − pGEX-6P-3 with AI2 + +
+: growth;
−: no growth
- Dihydrodipicolinate synthase (EC 4.2.1.52) converts L-aspartate 4-semialdehyde and pyruvate to 1-2,3-dihydrodipicolinate as part of the lysine biosynthesis pathway. L-aspartate 4-semialdehyde is also the first step of the glycine, serine and threonine metabolic pathways.
- The polypeptide of SEQ ID NO: 55 and the polynucleotide of SEQ ID NO: 13 have utility for processing food products, as additives for industrial processing, and in the commercial production of lysine or intermediates. The attributes conferred by this enzyme include: altered amino acid content, with important flavor and metabolic impacts; commercial production of lysine or intermediates; manipulation of energy production and growth in particular media; and altered survival characteristics in industrial processes.
- These attributes may be produced in food products and used in food and other types of industrial processing, by directed activity of the enzyme, introduced in a bacterial strain (including strain HN001, or starter cultures) comprising a polynucleotide of SEQ ID NO: 13 or a variant, or as an enzyme preparation comprising a polypeptide of SEQ ID NO: 55 or a variant.
- The full-length polynucleotide sequence of dihydrodipicolinate reductase from L. rhamnosus strain HN001, given in SEQ ID NO: 14 and shown in
FIG. 72 with ATG initiation and translation stop codons (boxed), was used to amplify the AI3 dihydrodipicolinate reductase gene from L. rhamnosus HN001 DNA using standard PCR methodology. The upstream and downstream primers were tagged with EcoRI and SalI restriction endonuclease recognition sequences to facilitate cloning. - AI3 was then cloned into the EcoRI and SalI sites of the pGEX-6P-3 expression vector (Pharmacia Biotech) and transformed into the E. coli strain K12 XL-1Blue competent cells according to standard laboratory protocols. The polypeptide sequence of dihydrodipicolinate reductase AI3 is given in SEQ ID NO: 56 and is shown in
FIG. 73 . The dihydrodipicolinate reductase AI3 protein was expressed as a fusion protein with glutathione S-transferase (GST), bound to Glutathione Sepharose 4B resin (Pharmacia Biotech), and PreScission protease used to cleave off dihydrodipicolinate reductase AI3 protein, according to the manufacturer's instructions. An aliquot of the purified AI3 protein was checked by SDS-PAGE analysis. - Dihydrodipicolinate reductase activity was assessed by gene complementation of the mutant E. coli strain AT999 deficient in dihydrodipicolinate reductase gene function (Bukhari and Taylor, J. Bacteriol. 105:844-854, 1971), provided by the E. coli Genetic Stock Centre (Yale University, USA). AT999 cells require diaminopimelic acid (DAP) for growth, in contrast to wild-type E. coli that is DAP-independent. pGEX-6P-3 construct containing the HN001 dihydrodipicolinate reductase AI3 gene or empty pGEX-6P-3 vector was transformed into the AT999 E. coli strain. Transformed AT999 cells were plated onto LB agar plates containing ampicillin (100 μg/ml) only or ampicillin and 45 μg/ml DAP, at dilutions designed to allow the visualization of distinct colonies (ie. <200 colonies/plate). Plates were incubated aerobically at 37° C. overnight and growth assessed as the presence of distinct colonies (Table 8).
- The results in Table 8 indicate that while AT999 cells transformed with either empty pGEX-6P-3 or pGEX-6P-3 containing the HN001 dihydrodipicolinate reductase AI3 grew in the presence of DAP, only cells transformed with vector containing AI3 grew without DAP. Therefore, the HN001 dihydrodipicolinate reductase protein AI3 complemented the dihydrodipicolinate reductase gene mutation in E. coli strain AT999.
TABLE 8 Results of LB agar plate assay for dihydrodipicolinate reductase gene complementation. LB agar plates containing: E. coli AT999 transformed with: Ampicillin and DAP Ampicillin only pGEX-6P-3 + − pGEX-6P-3 with AI3 + +
+: growth;
−: no growth
- Dihydrodipicolinate reductase (EC 1.3.1.26) converts L-2,3-dihydro-dipicolinate to L-tetrahydropicolinate as part of the lysine biosynthesis pathway.
- The polypeptide of SEQ ID NO: 56, and the polynucleotide of SEQ ID NO: 14 have utility for processing food products, as additives for industrial processing, and in the commercial production of lysine or intermediates. The attributes conferred by this enzyme include: altered amino acid content, with important flavor and metabolic impacts; commercial production of lysine or intermediates; manipulation of energy production and growth in particular media; and altered survival characteristics in industrial processes.
- These attributes may be produced in food products and used in food and other types of industrial processing, by directed activity of the enzyme, introduced in a bacterial strain (including strain HN001, or starter cultures) comprising a polynucleotide of SEQ ID NO: 14 or a variant, or as an enzyme preparation comprising a polypeptide of SEQ ID NO: 56 or a variant.
- The full-length gene sequence of aspartate aminotransferase from L. rhamnosus strain HN001, given in SEQ ID NO: 12 and shown in
FIG. 17 with GTG initiation and translation stop codons (boxed), was used to amplify the AH9 aspartate aminotransferase gene from L. rhamnosus HN001 DNA using standard PCR methodology. The upstream and downstream primers were tagged with EcoRI and SalI restriction endonuclease recognition sequences to facilitate cloning. - AH9 was then cloned into the EcoRI and SalI sites of the pGEX-6P-3 expression vector (Pharmacia Biotech) and transformed into the E. coli strain K12 XL-1Blue competent cells according to standard laboratory protocols. The aspartate aminotransferase AH9 protein was expressed as a fusion protein with glutathione S-transferase (GST), bound to Glutathione Sepharose 4B resin (Pharmacia Biotech), and PreScission protease used to cleave off the aspartate aminotransferase AH9 protein, according to the manufacturer's instructions. An aliquot of the purified AH9 protein was checked by SDS-PAGE analysis. The polypeptide sequence is given in SEQ ID NO: 54 and is shown in
FIG. 18 . - AH9 activity was assayed according to the previously published malate dehydrogenase-coupled method (Karmen, J. Clin. Invest. 34:131-133, 1955) with modifications. Briefly, 1 ml reaction mixtures containing 100 μmol Tris hydrochloride buffer (pH 8.0), 100 μmol L-aspartate, 10 μmol of α-ketoglutarate, 0.2 μmol NADH, 0.015 μmol pyrodoxal 5′-phosphate (PLP), and 3 μg (3.6 U) malate dehydrogenase (all chemicals from Sigma Chemical Co.) were incubated at 37° C. with increasing amounts (0 to 142.5 ng) of the purified HN001 aspartate aminotransferase AH9 protein. The rationale of the assay is that aspartate aminotransferase converts α-ketoglutarate and L-aspartate to oxaloacetate and L-glutamate. The oxaloacetate is then substrate for the malate dehydrogenase, which oxidizes one molecule of NADH to NAD+ for every molecule of oxaloacetate converted to L-malate. As the first step is rate limiting, the amount of NADH oxidized in the second step is directly proportional to the aspartate aminotransferase-dependent production of oxaloacetate from α-ketoglutarate in the first step. The reaction was monitored by the decrease in absorbance at 340 nm, and results used to calculate the μmol NADH oxidized per minute. One unit of enzyme was defined as the amount of enzyme that catalyzed the production of 1 μmol of oxaloacetate per minute at 37° C.
- Results in Table 9 indicate that, while in the absence of purified HN001 aspartate aminotransferase AH9 protein there was some background oxidation of NADH, the addition of AH9 protein led to increased rates of aspartate aminotransferase-dependent NADH oxidation. Increased amounts of AH9 increased NADH oxidation in a dose-dependent manner. A similar background rate observed in reaction mixtures without the addition of AH9 protein was also observed in reaction mixtures without both AH9 protein and α-ketoglutarate substrate (data not shown), indicating that the background NADH oxidation was not aspartate aminotransferase-dependent. The addition of 142.5 ng of AH9 protein led to an over 19-fold increase in NADH oxidation. The activity of the purified HN001 aspartate aminotransferase AH9 protein was calculated to be 31 U/mg protein. Therefore, HN001 protein AH9 is an aspartate aminotransferase.
TABLE 9 Results of the malate dehydrogenase-coupled aspartate aminotransferase assay. Concentration of purified HN001 aspartate aminotransferase AH9 NADH oxidized (ng/ml) (μmol/min/ml) 0 0.20 × 10−3 23.8 0.80 × 10−3 47.5 1.13 × 10−3 71.2 1.61 × 10−3 95.0 1.95 × 10−3 118.8 2.57 × 10−3 142.5 3.82 × 10−3 - α-Ketoglutarate is an important chemical mediator in lactic acid bacteria, and the addition of this compound to cheese curd has positive impacts on cheese flavor (Yvon et al., Int. Dairy J. 8:889-898, 1998). The formation of α-ketoglutarate using L-glutamate as an amino donor, catalysed by aspartate aminotransferase, is an important pathway in maintaining intracellular α-ketoglutarate levels.
- The polypeptide of SEQ ID NO: 54 and the polynucleotide of SEQ ID NO: 12 have utility for processing food products and as supplements and additives to food products. This esterase may also be used to develop non-food products. The attributes conferred by this enzyme include: altered amino acid content, with important flavor and metabolic impacts; manipulation of energy production and growth in particular media; and altered survival characteristics in industrial processes, including food processing These attributes may be produced in food, such as dairy products, and in other industrial processes, by directed activity of the enzyme, introduced in a bacterial strain (including strain HN001, or starter cultures) comprising a polynucleotide of SEQ ID NO: 12 or a variant, or as an enzyme preparation comprising a polypeptide of SEQ ID NO: 54 or a variant.
- The full-length polynucleotide sequence of serine dehydratase subunits α and β, given in SEQ ID NO: 7, was used to amplify the AF8 serine dehydratase cc subunit and AF7 serine dehydratase β subunit from L. rhamnosus HN001 DNA as a single operon using standard PCR methodology. The polynucleotide sequence of L. rhamnosus strain HN001 serine dehydratase subunits α (AF8) and β (AF7) is shown in
FIG. 19 , with ATG translation initiation codons and termination codons shown boxed for AF8 and shaded for AF7. - The AF8 serine dehydratase cc subunit and AF7 serine dehydratase β subunit were amplified from L. rhamnosus HN001 DNA as a single operon using standard PCR methodology. The AF8 and AF7 genes were cloned in the vector pTRKH2 (obtained from Dr Todd Klaenhammer, North Carolina State University, North Carolina, USA) and transformed into E. coli DH5α cells. Positive transformants were selected, grown overnight and the plasmid isolated by standard laboratory techniques. Competent L. rhamnosus HN001 cells were then transformed with the pTRKH2 construct containing the HN001 serine dehydratase subunits α and β to overexpress the genes in strain HN001. The amino acid sequences of the expressed proteins AF8 serine dehydratase α and AF7 serine dehydratase β are given in SEQ ID NO: 49 and 48, respectively, and shown in
FIGS. 22A and 22B , respectively. - Serine dehydratase enzyme activity was assessed by comparing serine utilization in liquid cultures of HN001 strain cells transformed with either the pTRKH2 construct containing the HN001 serine dehydratase or empty pTRKH2 vector only.
- The results shown in
FIGS. 20 and 21 indicate that the presence of the expression plasmid encoding HN001 serine dehydratase subunits α (AF8) and β (AF7) significantly increased the utilization of serine by HN001 strain cells, compared to cells transformed with empty expression vector only. Therefore the HN001 genes AF7 and AF8 encode the serine dehydratase enzyme.FIG. 20 shows the percentage serine utilization by HN001 strain in liquid culture with 5 mM initial serine concentration: ν HN001 transformed with vector only; υ pTRKH2 construct containing HN001 serine dehydratase.FIG. 21 shows the percentage serine utilization by HN001 strain in liquid culture with 12 mM initial serine concentration: ν HN001 transformed with vector only, υ pTRKH2 construct containing HN001 serine dehydratase. - Serine dehydratase (EC 4.2.1.13), comprising α and β subunits, catalyzes the irreversible deamination of serine to pyruvate and ammonia (Ogawa et al., J. Biol. Chem. 264:15818-15822, 1989; Grabowski et al., Trends in Biochem. Sci. 18:297-300, 1993).
- The polypeptides of SEQ ID NO: 48 and 49, and the polynucleotide of SEQ ID NO: 7 have utility for processing food products and in other types of industrial processing, and in the production of ammonia. Applications for the HN001 serine dehydratase subunits AF7 and AF8 of the present invention include: energy supply from amino acids present in growth media or environment; production of ammonia, regarded as a flavor compound; altered pyruvate levels—pyruvate is a highly reactive compound, and is important in a number of flavor pathways; and altered survival characteristics in industrial processes.
- These applications may be implemented by directed activity of the enzyme, introduced in a bacterial strain (including strain HN001, or starter cultures) comprising a polynucleotide of SEQ ID NO: 7 or a variant, or as an enzyme preparation comprising a polypeptide of SEQ ID NO: 48 and/or 49, or variants.
- The full-length polynucleotide sequence of histidinol-phosphate amino-transferase from L. rhamnosus strain HN001, given in SEQ ID NO: 8 and shown in
FIG. 23 with ATG initiation and translation stop codons (boxed), was used to amplify the AG2 histidinol-phosphate aminotransferase gene from L. rhamnosus HN001 DNA using standard PCR methodology. The upstream and downstream primers were tagged with EcoRI and BamHI restriction endonuclease recognition sequences to facilitate cloning. - AG2 was then cloned into the EcoRI and BamHI sites of the pGEX-6P-3 expression vector (Pharmacia Biotech) and transformed into E. coli strain DH-5α at competent cells according to standard laboratory protocols. Cells were lysed by sonication and the presence of AG2 protein, expressed as a GST fusion protein, checked by SDS-PAGE analysis. The polypeptide sequence of AG2 is given in SEQ ID NO: 50 and shown in
FIG. 24 . - Histidinol-phosphate aminotransferase activity was assessed by gene complementation of the mutant E. coli strain UTH780 that lacks a functional his C gene that encodes histidinol-phosphate aminotransferase (Goldschmidt et al., Genetics, 66:219-229, 1970), provided by the E. coli Genetic Stock Centre (Yale University, USA). UTH780 cells require L-histidine for growth, in contrast to wild-type E. coli that is L-histidine-independent. pGEX-6P-3 construct encoding HN001 histidinol-phosphate aminotransferase AG2 was transformed into the UTH780 E. coli strain and plated onto LB agar plates containing 100 μg/ml ampicillin. Ampicillin-resistant transformant colonies were picked and plated out onto selective media (ie. M9 media plates with and without 100 μg/ml L-histidine, with and without 100 μg/ml ampicillin). Growth of UTH780 transformed with AG2 was compared with the growth of wild-type UTH780 cells and UTH780 cells transformed with a pGex-6P-3 construct encoding a non-histidinol-phosphate aminotransferase (AE8). Plates were incubated aerobically at 37° C. overnight and growth assessed as the presence of distinct colonies.
- Results in Table 10 indicate that while wild-type UTH780 cells grew in the presence of histidine, no growth was observed when ampicillin was added to the media. Therefore, ampicillin resistance in transformed UTH780 was due to the presence of pGEX-6P-3 vector. UTH780 cells transformed with either empty pGEX-6P-3 or pGEX-6P-3 encoding an irrelevant protein (AE9) grew in the presence of histidine and ampicillin, but remained auxotrophic for hisitidine, indicating that the H is C phenotype was not complemented. UTH780 cells transformed with pGEX-6P-3 encoding HN001 histidinol-phosphate aminotransferase AG2 grew on M9 media without histidine. Therefore, the AG2 protein complemented the his C mutation of UTH780 strain E. coli cells.
TABLE 10 Results of M9 agar plate assay for histidinol-phosphate aminotransferase gene complementation. M9 agar plates containing: E. coli UTH780 Ampicillin and L- transformed with: L-histidine only histidine Ampicillin only — + − − pGEX-6P-3 + + − pGEX-6P-3 + + − encoding AE9 pGEX-6P-3 + + + encoding AG2
+: growth;
−: no growth
- Histidinol-phosphate aminotransferase (EC 2.6.1.9) catalyzes the transamination of histidinol phosphate and 2-oxoglutarate to 3-(Imidazol-4-yl)-2-oxopropyl phosphate and glutamate, as the eighth step in histidine biosynthesis (Martin et al., J. Bio. Chem. 242:1168-1174, 1967). Some lactic acid bacteria are known to decarboxylate amino acids, such that histidine can be converted to histamine, which has undesirable physiological effects (Lonvaud-Funel, FEMS Microb. Lett. 199:9-13, 2001).
- The polypeptide of SEQ ID NO: 50 and the polynucleotide of SEQ ID NO: 8 have utility for processing food products and as supplements and additives to food products. This histidinol-phosphate aminotransferase may also be used to develop non-food products. The attributes conferred by this enzyme include: altered levels of particular amino acids, leading to flavor and metabolic changes; affect aromatic amino acid metabolism, a source of important flavor compounds; and modulate production of biogenic amines.
- These attributes may be produced in food, and other products, by directed activity of the enzyme, introduced in a bacterial strain (including strain HN001, or starter cultures) comprising a polynucleotide of SEQ ID NO: 8 or a variant, or as an enzyme preparation comprising a polypeptide of SEQ ID NO: 50 or a variant.
- The full-length polynucleotide sequence of malY aminotransferase from L. rhamnosus strain HN001, given in SEQ ID NO: 17 and shown in
FIG. 25 with ATG initiation and translation stop codons (boxed), was used to amplify the AJ6 aminotransferase gene from L. rhamnosus HN001 DNA using standard PCR methodology. The upstream and downstream primers were tagged with EcoRI and BamHI restriction endonuclease recognition sequences to facilitate cloning. - AJ6 was then cloned into the EcoRI and BamHI sites of the pGEX-6P-3 expression vector (Pharmacia Biotech) and transformed into E. coli strain DH-5α competent cells according to standard laboratory protocols. Cells were lysed by sonication and the presence of AJ6 protein, expressed as a GST fusion protein, checked by SDS-PAGE analysis. The polypeptide sequence of AJ6 is given in SEQ ID NO: 59 and shown in
FIG. 26 . - A feature of malY-aminotransferases is the ability to complement mutations of the E. coli cystathione β-lyase protein metC (Zdych et al., J. Bacteriol. 177:5035-5039, 1995). Therefore, AJ6 activity was assessed by suppression of the metC− phenotype in the E. coli strain CAG18527 (Singer et al., Microbiol. Rev. 53:1-24, 1989) provided by the E. coli Genetic Stock Centre (Yale University, USA). CAG18527 cells require L-methionine for growth, in contrast to wild-type E. coli that is L-methionine-independent. A pGEX-6P-3 construct encoding the HN001 aminotransferase AJ6 was transformed into the CAG18527 E. coli strain and plated onto LB agar plates containing 100 μg/ml ampicillin. Ampicillin-resistant transformant colonies were picked and plated out onto selective media (M9 plates with and without 1 mM L-methionine, with and without 5 μg/ml ampicillin). Growth of the CAG18527 transformed with AJ6 was compared with the growth of wild-type CAG18527 cells and CAG18527 cells transformed with a pGEX-6P-3 construct encoding a non-aminotransferase irrelevant protein. Plates were incubated aerobically at 37° C. for 48 hrs and growth assessed.
- Results in Table 11 indicate that while wild-type CAG18527 cells grew in the presence of methionine, no growth was observed in the presence of ampicillin. This confirmed the ampicillin-sensitive, methionine-auxotrophic phenotype of the CAG18527 strain. CAG18527 cells transformed with either empty pGEX-6P-3 or pGEX-6P-3 encoding an irrelevant HN001 protein (AC9) grew in the presence of methionine and ampicillin, but not in the absence of methionine, indicating that the metC− phenotype was not suppressed. CAG18527 cells transformed with pGEX-6P-3 encoding HN001 aminotransferase AJ6 were ampicillin resistant and grew on M9 media without methionine. Therefore, the AJ6 protein suppressed the metC− mutation of CAG18527 strain E. coli cells.
TABLE 11 Results of M9 agar plate assay for suppression of the metC phenotype. M9 agar plates containing: E. coli CAG18527 L-methionine Ampicillin and L- transformed with: only methionine Ampicillin only — + − − pGEX-6P-3 + + − pGEX-6P-3 + + − encoding AC9 pGEX-6P-3 + + + encoding AJ6
+: growth;
−: no growth
- The malY/PatB pyridoxal-5′-phosphate-dependent aminotransferase family (EC 2.6.1.-) appear to have both aminotransferase and regulatory activities (Mehta and Christen, Eur. J. Biochem. 203 :373-376, 1993), including the transamination of methionine and regulation of maltose utilization (Reidl and Boos, J. Bacteriol. 173:4862-4876, 1991), as well as other activities (Chu et al., Infect. Imm. 63: 4448-4455, 1995).
- The polypeptide of SEQ ID NO: 59 and the polynucleotide of SEQ ID NO: 17 have utility for processing food products and as supplements and additives to food products. This aminotransferase may also be used to develop non-food products. The attributes conferred by this enzyme include: altered levels of particular amino acids, leading to flavor and metabolic changes; altered expression of catabolite or other regulons; modulation of hemolytic activity; and probiotic effects.
- These attributes may be produced in food products by directed activity of the enzyme, introduced in a bacterial strain (including strain HN001, or starter cultures) comprising a polynucleotide of SEQ ID NO: 17 or a variant, or as an enzyme preparation comprising a polypeptide of SEQ ID NO: 59 or a variant.
- The full-length polynucleotide sequence of a second malY-aminotransferase from L. rhamnosus strain HN001, given in SEQ ID NO: 18 and shown in
FIG. 27 with ATG initiation and translation stop codons (boxed), was used to amplify the AJ7 aminotransferase gene from L. rhamnosus HN001 DNA using standard PCR methodology. The upstream and downstream primers were tagged with EcoRI and BamHI restriction endonuclease recognition sequences to facilitate cloning. - AJ7 was then cloned into the EcoRI and BamHI sites of the pGEX-6P-3 expression vector (Pharmacia Biotech) and transformed into E. coli strain DH-5α competent cells according to standard laboratory protocols. Cells were lysed by sonication and the presence of AJ7 protein, expressed as a GST fusion protein, checked by SDS-PAGE analysis. The polypeptide sequence of AJ7 is given in SEQ ID NO: 60 and shown in
FIG. 28 . - A feature of malY-aminotransferases is the ability to complement mutations of the E. coli cystathione β-lyase protein metC (Zdych et al., J. Bacteriol. 177:5035-5039, 1995). Therefore, AJ7 activity was assessed by suppression of the metC− phenotype in the E. coli strain CAG18527 (Singer et al., Microbiol. Rev. 53:1-24, 1989) provided by the E. coli Genetic Stock Centre (Yale University, USA). CAG18527 cells require L-methionine for growth, in contrast to wild-type E. coli that is L-methionine-independent. pGEX-6P-3 construct encoding the HN001 aminotransferase AJ7 was transformed into the CAG18527 E. coli strain and plated onto LB agar plates containing 100 μg/ml ampicillin. Ampicillin-resistant transformant colonies were picked and plated out onto selective media (M9 plates with and without 1 mM L-methionine, with and without 5 μg/ml ampicillin). Growth of the CAG18527 transformed with AJ7 was compared with the growth of wild-type CAG18527 cells and CAG18527 cells transformed with a pGEX-6P-3 construct encoding a irrelevant protein. Plates were incubated aerobically at 37° C. for 48 hrs and growth assessed.
- Results in Table 12 indicate that while wild-type CAG18527 cells grew in the presence of methionine, no growth was observed in the presence of ampicillin. This confirmed the ampicillin-sensitive, methionine-auxotrophic phenotype of the CAG18527 strain. CAG18527 cells transformed with either empty pGEX-6P-3 or pGEX-6P-3 encoding an irrelevant HN001 protein (AC9) grew in the presence of methionine and ampicillin, but not in the absence of methionine, indicating that the metC− phenotype was not suppressed. CAG18527 cells transformed with pGEX-6P-3 encoding HN001 aminotransferase AJ7 were ampicillin resistant and grew on M9 media without methionine. Therefore, the AJ7 protein suppressed the metC− mutation of CAG18527 strain E. coli cells.
TABLE 12 Results of M9 agar plate assay for suppression of the metC phenotype. M9 agar plates containing: E. coli CAG18527 L-methionine Ampicillin and L- transformed with: only methionine Ampicillin only — + − − pGEX-6P-3 + + − pGEX-6P-3 + + − encoding AC9 pGEX-6P-3 + + + encoding AJ7
+: growth;
−: no growth
- The malY/PatB pyridoxal-5′-phosphate-dependent aminotransferase family (EC 2.6.1.-) appear to have both aminotransferase and regulatory activities (Mehta and Christen, Eur. J. Biochem. 203:373-376, 1993), including the transamination of methionine and regulation of maltose utilization (Reidl and Boos, J. Bacteriol. 173:4862-4876, 1991), as well as other activities (Chu et al., Infect. Imm. 63: 4448-4455, 1995).
- The polypeptide of SEQ ID NO: 60 and the polynucleotide of SEQ ID NO: 18 have utility for processing food products and as supplements and additives to food products. This aminotransferase may also be used to develop non-food products. The attributes conferred by this enzyme include: altered levels of particular amino acids, leading to flavor and metabolic changes; altered expression of catabolite or other regulons; modulation of hemolytic activity; and probiotic effects.
- These attributes may be produced in food, such as dairy products, by directed activity of the enzyme, introduced in a bacterial strain (including strain HN001, or starter cultures) comprising a polynucleotide of SEQ ID NO: 18 or a variant, or as an enzyme preparation comprising a polypeptide of SEQ ID NO: 60 or a variant.
- The full-length polynucleotide sequence of cystathione β-lyase from L. rhamnosus strain HN001, given in SEQ ID NO: 5 and shown in
FIG. 29 with ATG initiation and translation stop codons (boxed), was used to amplify the AC8 cystathione α-lyase gene from L. rhamnosus HN001 DNA using standard PCR methodology. - AC8 was cloned into the pUniBlunt/V5-HisTopo vector (Invitrogen) and transformed into the E. coli strain PIR1 OneShot competent cells (Invitrogen). To construct an expression plasmid, the pUniBlunt/V5-HisTopo vector construct was recombined with the pBad/Thio-E vector (Invitrogen) and transformed into the E. coli strain TOP10 competent cells (Invitrogen) according to the manufacturer's instructions. The AC gene product was therefore cloned as a fusion protein tagged with a His-patch polypeptide and thioredoxin protein. The AC8 fusion protein was expressed and purified using a Ni-NTA column (Qiagen, Auckland, NZ) according to the manufacturer's instructions and protein expression checked by SDS-PAGE. The polypeptide sequence of AC8 is given in SEQ ID NO: 46 and shown in
FIG. 30 . - Cystathione β-lyase activity was assessed according to the method of Uren, Methods in Enzymol. 143:483-496, 1987, with modifications. Briefly, aliquots of the purified AC8-GST fusion protein were added to 1 ml cuvettes containing 780 μl of 0.1 M Tris-HCl pH 9.0, 200 μl of 10 mM L-cystathionine, and 20 μl of 0.1 M potassium phosphate, with pyridoxal-5′-phosphate added to a final concentration of 20 μM, on ice. Change in OD was measured at 412 nm over time, and one unit of enzyme defined as the formation of 1 μmol of mercaptide per minute at 37° C. Cystathione β-lyase activity of the AC8 fusion protein was compared with activity of an irrelevant protein (pBAD/Thio-E/Uni-CAT expression control vector, Invitrogen), and reactions containing water or Ni-NTA column elution buffer.
- The results are shown in
FIG. 31 , with rates of change of OD and enzyme activity given in Table 13. These results indicate that similar background rates of mercaptide formation were observed in reactions containing water only, elution buffer only or 10 μl purified CAT fusion protein. Significantly greater mercaptide formation was observed in reactions containing 10 μl purified HN001 cystathione α-lyase AC8 fusion protein. Therefore, AC8 protein has cystathione α-lyase activity.FIG. 31 shows cystathione β-lyase activity measured as rate of mercaptide formation.υ 10 μl purified HN001 cystathione β-lyase AC8 fusion protein;ν 10 μl purified CAT fusion protein;σ 10 μl H2O only;λ 10 μl elution buffer only.TABLE 13 Cystathione β-lyase activity of AC8 compared with irrelevant protein, H2O and elution buffer controls. Enzyme activity Protein Δ OD412/min (μmol/min/ml) AC8 fusion protein 0.00328 16.31 CAT fusion protein 0.00242 12.03 H2O only 0.00232 11.53 Elution buffer-only 0.00233 11.58 - The dose-response of the HN001 cystathione β-lyase activity AC8 was determined by comparing mercaptide formation in a series of dilutions of the purified enzyme. Results are shown in
FIG. 32 , and the rate of change in optical density and enzyme activities given in Table 14. - These results indicate that the increased rate of mercaptide peptide was proportional to the amount of AC8 fusion protein, supporting that AC8 encodes HN001 cystathionine β-lyase.
FIG. 32 shows the experimentally determined dose-response of the AC8 fusion protein. Cystathione β-lyase activity of increasing amounts of His-patch/Thio/AC8 fusion protein; 10 μl (υ), 25 μl (ν) and 50 μl (σ) purified protein showed increasing rates of mercaptide formation. The increase in mercaptide formation was proportional to amount of AC8 fusion protein added.TABLE 14 Cystathione β-lyase activity in increasing amounts of AC8 protein Amount of purified AC8 Enzyme activity fusion protein Δ OD410/min (μmol/min/ml) 10 μl 0.00319 15.8 25 μl 0.00378 18.8 50 μl 0.00496 24.7 - Cystathionine β-lyase (EC 4.4.1.8) deaminates cystathionine to L-homocysteine, ammonia and pyruvate (Dwivedi et al., Biochem. 21:3064-3069, 1982), and may also have active on L-cystine and related substrates (Uren, Methods in Enzymol. 143:483-486, 1987; Alting et al., Appl. Environ. Microbiol. 61:4037-4042, 1995). Thus, cystathionine β-lyase is involved in a number of pathways including methionine metabolism and catabolism of sulphur-containing compounds. L-homocysteine has been shown to have important health impacts in humans (Nittynen et al., Ann. Med. 31:318-326, 1999; Giles et al., Am. Heart J. 139:446-453, 2000).
- The polypeptide of SEQ ID NO: 46 and the polynucleotide of SEQ ID NO: 5 have utility for processing food products and as supplements and additives to food products. This lyase may also be used to develop non-food products. The attributes conferred by this enzyme include: altered flavor and metabolic characteristics through changes in levels of particular amino acids; altered levels of important sulphur-containing flavor compounds; and health impacts through the modulation of L-homocysteine levels.
- These attributes may be produced in food products by directed activity of the enzyme, introduced in a bacterial strain (including strain HN001, or starter cultures) comprising a polynucleotide of SEQ ID NO: 5 or a variant, or as an enzyme preparation comprising a polypeptide of SEQ ID NO: 46 or a variant.
- HN001 phosphoenolpyruvate hydratase AK4 was isolated by a series of experiments designed to identify HN001 strain proteins that were up-regulated in response to physiological stresses encountered during industrial processes. Cells were subjected to heat or osmotic shock, proteins radiolabeled with [35S]-methionine and [35S]-cysteine (Amersham, USA), and cell-free extracts from shocked and non-shocked HN001 cultures compared by 2-D analysis and N-terminal sequencing as below.
- Shock proteins were radiolabeled according to standard laboratory methods. Heat shock was performed by incubation at 50° C. on both log phase and stationary phase HN001 strain cultures, and salt (osmotic) shock on late log phase by HN001 strain cultures by transfer into MRS broth containing 0.6 M sodium chloride. Immediately after heat or osmotic shock, approximately 5 μCi ml−1 each of L-[35S]-methionine and L-[35S]-cysteine were added to the culture medium and incubated for 30 min, followed by the addition of excess of cold 1 mM L-cysteine hydrochloride and 1 mM L-methionine, and cultures then placed on ice. Radiolabeled cells were collected by centrifugation, washed twice in washing buffer (0.1 M Tris-HCl, 1 mM EDTA, pH 7.5) and resuspended in resuspension buffer (10 mM Tris-HCl, 5 mM MgCl2, 2 mM PMSF, pH 7.5). About 0.5 ml cell suspension was mixed with 0.5 g of 0.17-0.18 mm glass beads and homogenized using Shake-it-Baby (Biospec products). After homogenization for 25 min, the suspension was centrifuged and the supernatant was collected. 2-D Gel electrophoresis was performed on the cell free extract containing 50-75 μg of protein. Excess chilled methanol was added and kept at −80° C. for 1 hr followed by centrifugation at 13,000 rpm to collect the pellet. The pellet was vacuum-dried and resuspended in rehydration buffer (8M urea, 2
% Triton X 100, 0.5% (v/v) IPG buffer (Amersham Pharmacia Biotech, USA) and few grains of bromophenol blue). Endonuclease (Sigma) was added (150 U) to the rehydrated sample and incubated at room temperature for 20 min. The solution was then added to IPG strips and rehydrated overnight at 20° C. The rehydrated IPG strips were placed on a flat bed electrophoresis unit (Amersham Pharmacia Biotech, USA) and focused at 300 Volts for 30 min followed by 3,000 volts for 4 hrs. The focused strips were equilibrated (15 min) in equilibration buffer (50 mM Tris-HCl, pH 8.8, 6 M Urea, 30% (v/v) glycerol, 2% (w/v) SDS and few grains bromophenol blue) containing either dithioerythritol (1.0% w/v) or iodoacetamide (2.5% w/v). After equilibration, the strips were placed on the second dimensional (vertical SDS-PAGE homogeneous) gels a using PROTEAN II xi cell (Bio-Rad). The second dimension was carried out at 20 mA per plate for 15 min and 40 mA per plate for 4 hrs. - Gels were then equilibrated in protein transfer buffer (24.8 mM Tris, pH 8.3, 192 mM Glycine and 10% (v/v) methanol) and blotted on a PVDF membrane using a Trans-blot apparatus (Bio-Rad) at 24 volts overnight at 4° C. PVDF membranes were exposed to Hyperfilm-βmax (Amersham Pharmacia Biotech, USA) for up to two weeks using standard procedures. Resultant autoradiograms were scanned using the Fluor-S Multimager system (Bio-Rad) and patterns compared using PDQuest software. For N-terminal sequencing, membranes were stained with Coommassie Brilliant Blue R-250. The desired spots were excised and N-terminal sequencing carried out using a protein sequencer (Applied BioSystems, Model 476A) according to standard methods.
- A protein up-regulated by heat and osmotic shock was N-terminal sequenced and the amino acid sequence is given in SEQ ID NO: 83. This sequence was used to search an HN001 sequence database using the TBLASTN program (NCBI) and the corresponding polynucleotide and polypeptide sequences are given in SEQ ID NO: 20 and 62, and shown in
FIGS. 33 and 34 , respectively. Similarity searching using BLAST software revealed closest amino acid sequence similarity to phosphoenolpyruvate hydratase sequences but with significant differences. - Phosphoenolpyruvate hydratase (EC 4.2.1.11) is a glycolytic pathway enzyme that hydrolyzes 2-phospho-D-glycerate to give phosphoenolpyruvate (Malmstroem, B. G, The Enzymes, 2nd. Ed., Boyer, P. D., Lardy, H., Myrbäck, K., eds., 5:471-494, 1961).
- The polypeptide of SEQ ID NO: 62 and the polynucleotide of SEQ ID NO: 20 have utility for processing food products and as supplements and additives to food products. This hydratase may also be used to develop non-food products. The attributes conferred by this enzyme include: enhanced bacterial survival in industrial processes; improved colonization of human intestinal environment; and altered metabolic characteristics through changes in carbohydrate utilization.
- These attributes may be produced in food products by directed activity of the enzyme, introduced in a bacterial strain (including strain HN001, or starter cultures) comprising a polynucleotide of SEQ ID NO: 20 or a variant, or as an enzyme preparation comprising a polypeptide of SEQ ID NO: 62 or a variant.
- HN001 tagatose bisphosphate aldolase AK1 was isolated by a series of experiments designed to identify HN001 strain proteins that were up-regulated in response to physiological stresses encountered during industrial processes. Cells were subjected to heat or osmotic shock, proteins radiolabeled with [35S]-methionine and [35S]-cysteine (Amersham, USA), and cell-free extracts from shocked and non-shocked HN001 cultures compared by 2-D analysis and N-terminal sequencing as described for Example 15 (HN001 phosphoenolpyruvate hydratase AK4).
- A protein up-regulated by heat and osmotic shock was N-terminal sequenced and the polypeptide sequence is given in SEQ ID NO: 81. This was used to search an HN001 sequence database using the TBLASTN program (NCBI) and the corresponding polynucleotide and polypeptide sequences are given in SEQ ID NO: 19 and 61, and shown in
FIGS. 35 and 36 , respectively. Similarity searching using BLAST software revealed closest amino acid sequence similarity to tagatose bisphosphate aldolase sequences but with significant differences. - Tagatose bisphosphate aldolase (EC 4.1.2.40) is involved in the tagatose 6-phosphate pathway of lactose catabolism, and converts D-
tagatose 1,6-bisphosphate to glycerone phosphate and D-glyceraldehyde 3-phosphate (Anderson and Markwell, Methods in Enzymol. 90:232-234, 1982). - The polypeptide of SEQ ID NO: 61 and the polynucleotide of SEQ ID NO: 19 have utility for processing food and other products, and as supplements and additives to food products. This aldolase may also be used to develop non-food products. The attributes conferred by this enzyme include: enhanced bacterial survival in industrial processes; improved colonization of human intestinal environment; and altered metabolic characteristics through changes in carbohydrate utilization.
- These attributes may be produced in food products or in supplements, by directed activity of the enzyme, introduced in a bacterial strain (including strain HN001, or starter cultures) comprising a polynucleotide of SEQ ID NO: 19 or a variant, or as an enzyme preparation comprising a polypeptide of SEQ ID NO: 61 or a variant.
- HN001 phosphoglycerate kinase AK6 was isolated by a series of experiments designed to identify HN001 strain proteins that were up-regulated in response to physiological stresses encountered during industrial processes. Cells were subjected to heat or osmotic shock, proteins radiolabeled with [35S]-methionine and [35S]-cysteine (Amersham, USA), and cell-free extracts from shocked and non-shocked HN001 cultures compared by 2-D analysis and N-terminal sequencing as described for Example 15 (HN001 phosphoenolpyruvate hydratase AK4).
- A protein up-regulated by heat and osmotic shock was N-terminal sequenced and the polypeptide sequence is given in SEQ ID NO: 82. This was used to search an HN001 sequence database using the TBLASTN program (NCBI) and the corresponding polynucleotide and polypeptide sequences are given in SEQ ID NO: 22 and 64, and shown in
FIGS. 37 and 38 , respectively. Similarity searching using BLAST software revealed closest amino acid sequence similarity to phosphoglycerate kinase sequences but with significant differences. - Phosphoglycerate kinase (EC 2.7.2.3) is involved in the glycolysis pathway, and catalyzes the phospho-transfer reaction of ATP and 3-phospho-D-glycerate to ADP and 3-phospho-D-glyceroyl phosphate (bacterial enzyme reviewed in Suzuki and Imahori, Methods in Enzymol. 90:126-130, 1982).
- The polypeptide of SEQ ID NO: 64 and the polynucleotide of SEQ ID NO: 22 have utility for processing food products, and as supplements and additives to food products. This kinase may also be used to develop non-food products. The attributes conferred by this enzyme include: enhanced bacterial survival in industrial processes; improved colonization of human intestinal environment; and altered metabolic characteristics through changes in carbohydrate utilization.
- These attributes may be produced in food products and supplements by directed activity of the enzyme, introduced in a bacterial strain (including strain HN001, or starter cultures) comprising a polynucleotide of SEQ ID NO: 22 or a variant, or as an enzyme preparation comprising a polypeptide of SEQ ID NO: 64 or a variant.
- HN001 triosephosphate isomerase AK5 was isolated by a series of experiments designed to identify HN001 strain proteins that were up-regulated in response to physiological stresses encountered during industrial processes. Cells were subjected to heat or osmotic shock, proteins radiolabeled with [35S]-methionine and [35S]-cysteine (Amersham, USA), and cell-free extracts from shocked and non-shocked HN001 cultures compared by 2-D analysis and N-terminal sequencing as described for Example 15 (HN001 phosphoenolpyruvate hydratase AK4).
- A protein up-regulated by heat and osmotic shock was N-terminal sequenced. The polypeptide sequence is given in SEQ ID NO: 76. This sequence was used to search an HN001 sequence database using the TBLASTN program (NCBI) and the corresponding polynucleotide and polypeptide sequences are given in SEQ ID NO: 21 and 63, and shown in
FIGS. 39 and 40 , respectively. Similarity searching using BLAST software revealed closest amino acid sequence similarity to triosephosphate isomerase sequences but with significant differences.FIG. 39 shows the nucleotide sequence of L. rhamnosus strain HN001 triosephosphate isomerase AK5 showing ATG initiation and translation stop codons (boxed). - Triosephosphate isomerase (EC 5.3.1.1) is involved in the glycolysis pathway, and catalyzes the isomerisation reaction of D-glyceraldehyde 3-phosphate to glycerone phosphate (Fahey et al., Biochem. J. 124:77P, 1971).
- The polypeptide of SEQ ID NO: 63 and the polynucleotide of SEQ ID NO: 21 have utility for processing food and other products and as supplements and additives to food products. This isomerase may also be used to develop non-food products. The attributes conferred by this enzyme include: enhanced bacterial survival in industrial processes, including food processing; improved colonization of human intestinal environment; and altered metabolic characteristics through changes in carbohydrate utilization.
- These attributes may be produced in food products or supplements by directed activity of the enzyme, introduced in a bacterial strain (including strain HN001, or starter cultures) comprising a polynucleotide of SEQ ID NO: 21 or a variant, or as an enzyme preparation comprising a polypeptide of SEQ ID NO: 63 or a variant.
- HN001 fructose-bisphosphate aldolase AM8 was isolated by a series of experiments designed to identify HN001 strain proteins that were up-regulated in response to physiological stresses encountered during industrial processes. Cells were subjected to heat or osmotic shock, proteins radiolabeled with [35S]-methionine and [35S]-cysteine (Amersham, USA), and cell-free extracts from shocked and non-shocked HN001 cultures compared by 2-D analysis and N-terminal sequencing as described in Example 15.
- A protein upregulated by heat and osmotic shock was N-terminal sequenced. The amino acid is given in SEQ ID NO: 77. This was used to search an HN001 sequence database using the TBLASTN program (NCBI). The corresponding polynucleotide and polypeptide sequences are given in SEQ ID NO: 29 and 71, and shown in
FIGS. 74 and 75 , respectively. - Fructose-bisphosphate aldolase (EC 4.1.2.13) is involved in the glycolysis pathway, and catalyzes the elimination reaction of D-
fructose 1,6-bisphosphate to glycerone phosphate and D-glyceraldehyde 3-phosphate (bacterial enzyme reviewed in: Ujita and Kimura, Methods in Enzymol. 90: 235-241, 1982). - The polypeptide of SEQ ID NO: 71 and the polynucleotide of SEQ ID NO: 29 have utility for processing food and other products and as supplements and additives to food and other products. This aldolase may also be used to develop non-food products. The attributes conferred by this enzyme include: enhanced bacterial survival of industrial processes; improved colonization of human intestinal environment; and altered metabolic characteristics through changes in carbohydrate utilization.
- These attributes may be produced in food products and supplements by directed activity of the enzyme, introduced in a bacterial strain (including strain HN001, or starter cultures) comprising a polynucleotide of SEQ ID NO: 29 or a variant, or as an enzyme preparation comprising a polypeptide of SEQ ID NO: 71 or a variant.
- HN001 phosphoryl carrier protein HPR AA9 was isolated by a series of experiments designed to identify HN001 strain proteins that were up-regulated in response to physiological stresses encountered during industrial processes. Cells were subjected to heat or osmotic shock, proteins radiolabeled with [35S]-methionine and [35S]-cysteine (Amersham, USA), and cell-free extracts from shocked and non-shocked HN001 cultures compared by 2-D analysis and N-terminal sequencing as described for Example 15.
- A protein upregulated by heat and osmotic shock was N-terminal sequenced, with the determined amino acid sequence being given in SEQ ID NO: 78. This sequence was used to search an HN001 sequence database using the TBLASTN program (NCBI) and the corresponding polynucleotide and polypeptide sequences are given in SEQ ID NO: 4 and 45, and shown in
FIGS. 41 and 42 , respectively. Similarity searching using BLAST software revealed closest amino acid sequence similarity to phosphoryl carrier protein HPR sequences but with significant differences. - Phosphoryl carrier protein HPR is involved in the phosphoenol-pyruvate:carbohydrate phosphotransferase system (PTS) responsible for the uptake and phosphorylation of a number of carbohydrates (De Reuse et al., Gene 35:199-207, 1985; Gonzy-Treboul et al., Mol. Microbiol. 3:103-112, 1989). PTS is also involved in the regulation of various bacterial functions by various mechanisms, including catabolite repression, inducer exclusion, and inducer expulsion (reviewed in Postma et al., Microbiol. Rev. 57:543-594, 1993; Reizer et al., Crit. Rev. Microbiol. 15:297-338, 1988; Saier et al., Microbiol 142:217-230, 1996).
- The polypeptide of SEQ ID NO: 45 and the polynucleotide of SEQ ID NO: 4 have utility for processing food products and as supplements and additives to food products. This aldolase may also be used to develop non-food products. The attributes conferred by this enzyme include: enhanced bacterial survival in industrial processes; improved colonization of human intestinal environment; altered metabolic characteristics through changes in carbohydrate utilization; and control of catabolite regulation.
- These attributes may be produced in food, such as dairy products, by directed activity of the enzyme, introduced in a bacterial strain (including strain HN001, or starter cultures) comprising a polynucleotide of SEQ ID NO: 4 or a variant, or as an enzyme preparation comprising a polypeptide of SEQ ID NO: 45 or a variant.
- HN001 dnaK chaperone protein AM9 was isolated by a series of experiments designed to identify HN001 strain proteins that were up-regulated in response to physiological stresses encountered during industrial processes. Cells were subjected to heat or osmotic shock, proteins radiolabeled with [35S]-methionine and [35S]-cysteine (Amersham, USA), and cell-free extracts from shocked and non-shocked HN001 cultures compared by 2-D analysis and N-terminal sequencing as described for Example 15.
- A protein up-regulated by heat and osmotic shock was N-terminal sequenced. The determined amino acid sequence is given in SEQ ID NO: 79. This sequence was used to search an HN001 sequence database using the TBLASTN program (NCBI). The corresponding polynucleotide and polypeptide sequences are given in SEQ ID NO: 30 and 72 and shown in
FIGS. 76 and 77 , respectively. Similarity searching using BLAST software revealed closest amino acid sequence similarity to chaperone protein dnaK sequences but with significant differences. - Chaperone protein dnaK is a 70 kDa heat shock protein (HSP). DnaK chaperones act by binding and protecting exposed regions on unfolded or partially folded protein chains, and are involved in reactivating proteins that become aggregated after heat shock (reviewed in Lund, Adv. Microbial Physiol. 44:93-140, 2001). Overexpression may contribute to plasmid instability (Lobacz and Wolska, Acta Microbiol. Pol. 46:393-397, 1977).
- The polypeptide of SEQ ID NO: 72 and the polynucleotide of SEQ ID NO: 30 have utility for processing food products and as supplements and additives to food products. This chaperone protein may also be used to develop non-food products. The attributes conferred by this protein include: enhanced bacterial survival in industrial processes; improved colonization of human intestinal environment; altered protein translation characteristics; and methods to control plasmid stability.
- These attributes may be produced in food, such as dairy products, by directed activity of the enzyme, introduced in a bacterial strain (including strain HN001, or starter cultures) comprising a polynucleotide of SEQ ID NO: 30 or a variant, or as an enzyme preparation comprising a polypeptide of SEQ ID NO: 72 or a variant.
- HN0001 glyceraldehyde-3-phosphate dehydrogenase AK7 was isolated by a series of experiments designed to identify HN001 strain proteins that were up-regulated in response to physiological stresses encountered during industrial processes. Cells were subjected to heat or osmotic shock, proteins radiolabeled with [35S]-methionine and [35S]-cysteine (Amersham, USA), and cell-free extracts from shocked and non-shocked HN001 cultures compared by 2-D analysis and N-terminal sequencing as described for Example 15.
- A protein up-regulated by heat and osmotic shock was N-terminal sequenced and the determined amino acid sequence is given in SEQ ID NO: 80. This sequence was used to search an HN001 sequence database using the TBLASTN program (NCBI). The corresponding polynucleotide and polypeptide sequences are given in SEQ ID NO: 23 and 65, and shown in
FIGS. 43 and 44 , respectively. Similarity searching using BLAST software revealed the closest amino acid sequence similarity to glyceraldehyde-3-phosphate dehydrogenase sequences but with significant differences. - A second experiment was also performed to identify surface layer proteins extracted from Lactobacillus rhamnosus HN0001 strain. Surface layer proteins were extracted using the method of Turner et al., J. Bacteriol. 179:3310-3316, 1997. Briefly, 100 ml stationary phase HN001 culture was pelleted by centrifugation, washed with an equal volume of 0.15M NaCl, resuspended in 1 ml of 5M LiCl2 and kept on ice for 15 min. The crude lysate was centrifuged at 13,000 rpm using a microcentrifuge and analyzed by SDS-PAGE on a 12.5% gel. To facilitate better extraction of surface layer proteins, freeze-dried DR20 was extracted with 0.2% SDS and 5M LiCl2 as described by Brennan et al., Infect. Imm. 52:840-845, 1986 and Toba et al., J Imm. Methods 182:193-207, 1995. After 1-D electrophoresis according to standard laboratory methods, gels were blotted on a PVDF membrane using a Semi-dry blotting apparatus (Bio-Rad). A major surface protein with molecular weight between 30 and 46 kDa was excised and N-terminal sequencing performed using a protein sequencer (Applied BioSystems, Model 476A). The determined N-terminal sequence was identical to that obtained from the heat and osmotic shock experiments described above. Therefore, HN001 gene AK7 encodes glyceraldehyde-3-phosphate dehydrogenase, which is up-regulated by shock and is a major cell surface protein.
- Glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12) is part of the glycolytic pathway and catalyzes the redox reaction of D-glyceraldehyde 3-phosphate, phosphate and NAD+ to 3-phospho-D-glyceroyl phosphate and NADH (for bacterial enzyme see Amelunxen, Methods in Enzymol. 41:268-273, 1975; D'Alessio and Josse, J. Biol. Chem. 246:4326-4333, 1971). The enzyme has also been found to be a major cell-surface component of several bacterial species including Saccharomyces cerevisiae (Delgado et al., Microbiol. 147:411-417, 2001), Candida albicans (Gil-Navarro et al., J. Bacteriol. 179: 4992-4999, 1997) and group A Streptococci (Pancholi and Fischetti, Proc. Natl. Acad. Sci. USA 90:8154-8158, 1993).
- The polypeptide of SEQ ID NO: 65 and the polynucleotide of SEQ ID NO: 23 have utility for processing food products and as supplements and additives to food products. This dehydrogenase may also be used to develop non-food products. The attributes conferred by this enzyme include: flavor and aroma enhancement; enhanced bacterial survival in industrial processes; prolonged survival in storage; improved colonization of human intestinal environment; enhanced textural properties; enhanced adhesion to intestinal cell surfaces; and altered metabolic characteristics.
- These attributes may be produced in food, such as dairy products, by directed activity of the enzyme, introduced in a bacterial strain (including strain HN001, or starter cultures) comprising a polynucleotide of SEQ ID NO: 23 or a variant, or as an enzyme preparation comprising a polypeptide of SEQ ID NO: 65 or a variant.
- The full-length polynucleotide sequence of a transcription regulator sorR, given in SEQ ID NO: 24 and shown in
FIG. 45 , was used to amplify the AL3 transcription regulator sorR gene from L. rhamnosus HN001 DNA using standard PCR methodology. The upstream and downstream primers were tagged with BamHI and PstI restriction endonuclease recognition sequences to facilitate cloning. The polypeptide sequence of AL3 is given in SEQ ID NO: 66 and shown inFIG. 46 . - Full-length HN001 sorR transcription regulator AL3 was cloned into BamHI and PstI cut pFX3 vector (an E. coli/Lactococcus lactis shuttle vector as used in Xu et al., FEMS Microbiol. Lett. 61:55-59, 1991), and transformed into competent E. coli DH5α cells according to standard laboratory methods. Positive transformants were selected, grown overnight, and the plasmid construct isolated using a QIAprep Spin Miniprep Kit (Qiagen). The pFX3 construct encoding the HN001 sorR transcription regulator AL3 was digested using the restriction enzymes EcoRI and NruI, which released a 500 bp internal AL3 fragment that was cloned into the pBEryl vector cut with EcoRI and SmaI. The 3.6 kb pBEryl vector was constructed using the replicon and multiple cloning site (MCS) from the phagemid pBlueScript (pBS-SK+) (Stratagene, La Jolla Calif., USA). The ampicillin resistance gene in pBS-SK+ was removed by digestion with RcaI (Roche, Auckland, New Zealand) and the 1,953 bp fragment containing the ColE1 origin and multiple cloning site purified and treated with Klenow enzyme (Roche) to give a blunt-ended fragment. A gene encoding resistance to erythromycin (Em) was isolated on a 1.6 kb fragment obtained after cutting pVA891 (Macrina et al., Gene 25:145-50, 1983) with ClaI and HindIII and treatment with Klenow to give blunt ends. The 1.6 kb Em fragment was ligated to the 1,953 bp pBS-SK+ fragment, transformed into E. coli TG1 (Gibson T J, Studies on the Epstein-Barr virus genome. Ph.D. Thesis, University of Cambridge, Cambridge, England, 1984), and plated on LB agar plates containing 200 μg/ml Em. Maintenance of α-complementation for blue/white color selection of recombinant pBEry1 clones was confirmed by growing E. coli colonies on agar plates containing IPTG/X-gal.
- The resulting pBEryl construct encoding the HN001 sorR transcription regulator AL3 gene was transformed into competent HN001 cells and grown anaerobically for 48 hrs at 37° C. on MRS lactobacilli agar (Difco, Detroit Mich.) containing 2.5 μg/ml Em. Erythromycin-resistant HN001 were checked for integration of the plasmid construct into the sorR gene by PCR using vector-specific (T3 or T7) and AL3 internal fragment-specific primers. Colonies giving PCR patterns consistent with the insertional inactivation of the endogenous HN001 sorR transcription regulator AL3 gene were assessed for sorbose auxotrophy.
- Auxotrophy of selected HN001 mutants for metabolism of sorbose was tested by growing pure cultures (1% inoculum) overnight at 37° C. on MRS agar in the presence of 1% sorbose or 1% glucose, compared to wild-type HN001 and undefined mutant HN001 (Em-resistant cultures with intact sorR transcription regulator AL3 gene, the result of a random integration event) cultures.
- As shown in Table 15, the results indicate that the AL3− HN001 mutant strain failed to utilize sorbose as a carbon source in contrast to wild type HN001 and undefined mutant HN001 strain. This result was confirmed by growing pure cultures (1% inoculum) overnight at 37° C. in liquid MRS broth with 1% sorbose or 1% glucose and measuring absorbance at 600 nm. Again, results showed a clear difference in growth between the AL3− mutant strain, and the wild-type and undefined mutant HN001 strains containing intact the AL3 gene. Thus, the AL3 gene is required for sorbose metabolism in HN001, and encodes the sorR transcriptional regulator.
TABLE 15 Results of assessment of sorbose auxotrophy. MRS Wild type Undefined HN001 AL3− mutant plates with: HN001 mutant HN001 1% glucose + + + 1% sorbose + + −
+: growth;
−: no growth
- The sorR transcriptional regulator is required for the transcription of the sorbose operon, thereby regulating the utilization of L-sorbose as a carbon source, and its expression is induced by sorbose (Yebra et al., J. Bacteriol. 182:155-163, 2000; Sprenger and Lengeler, Mol. Gen. Genetics 209:352-359, 1987).
- The polypeptide of SEQ ID NO: 66 and the polynucleotide of SEQ ID NO: 24 have utility as transcriptional regulators in L. rhamnosus and other bacterial species. Specifically, applications for the HN001 sorR transcriptional regulator include: reagents for the control or modification of metabolic processes; and construction of sorbose-inducible HN001 expression vectors using the sorR gene promoter.
- The full-length polynucleotide sequence of formamidopyrimidine-DNA-glycosylase (fpg) from L. rhamnosus strain HN001, given in SEQ ID NO: 25 and shown in
FIG. 47 with ATG initiation and translation stop codons (boxed), was used to amplify the AL4 fpg gene from L. rhamnosus HN001 DNA using standard PCR methodology. The upstream and downstream primers were tagged with EcoRI and SalI restriction endonuclease recognition sequences to facilitate cloning. - AL4 was then cloned into the EcoRI and SalI sites of the pKK223-3 expression vector (Pharmacia Biotech) and transformed into the E. coli strain DH5α competent cells according to standard laboratory protocols. The polypeptide sequence of AL4 is given in SEQ ID NO: 67 and shown in
FIG. 48 . Expression of the fpg AL4 protein was confirmed by SDS-PAGE analysis. - AL4 fpg activity was assayed according to previously published methods (Duwat et al., Microbiol. 141:411-417, 1995; Zhang et al., Nucleic Acids Res. 26:4669-4675, 1998) that examine the ability of fpg to suppress the spontaneous mutator phenotype of fpg or mutY mutants of E. coli. The E. coli strain CSH117 (Miller, in: A short course in Bacterial Genetics, Cold Spring Harbor Press, Cold Spring Harbor, N.Y., 1992) that contains a mutated mutY gene was obtained from the E. coli Genetic Stock Centre (Yale University, USA) and transformed with the pKK223-3 construct encoding the HN001 fpg AL4 gene according to standard laboratory methods. Positive transformants were selected according to ampicillin resistance, and used to innoculate 7 ml LB broth cultures containing 100 μg/ml ampicillin and incubated aerobically at 37° C. with shaking. Cultures containing pKK223-3 constructs encoding AL4 or empty pKK223-3 vector were grown to similar OD at 600 nm, serially diluted, and plated in triplicate on LB plates with and without 100 μg/ml rifampicin (Sigma). Plates were incubated overnight at 37° C. and colonies counted. Results are shown in Table 16 as mean plate counts from three independent experiments.
- These results indicate that there was a significant difference in the frequency of mutations leading to rifampicin resistance in E. coli CSH117 transformed with pKK223-3 encoding HN001 fpg AL4 and empty pKK22-3 vector (p<0.001 by paired Student's t-test (1-tailed)). Because the presence of AL4 suppressed the spontaneous mutation rate, it was concluded that AL4 encoded the HN001 fpg protein.
TABLE 16 Spontaneous mutagenesis in E. coli CHS117 expressing the HN001 fpg AL4 gene. E. coli CSH117 Counts on Counts on LB transformed with LB plates (no plates with pKK223-3 rifampicin) rifampicin) Mutation Expt: encoding: (108/ml) (101/ml) frequency* 1 Empty 13.8 20.0 14.5 AL4 11.0 3.7 3.4 2 Empty 13.0 25.8 20 AL4 10.3 9.5 9.3 3 Empty 12.1 24.4 20.1 AL4 11.6 8.6 7.7
*expressed as the number of rifampicin-resistant mutants per 108 cells
- The fpg protein (EC 3.2.2.23) is a DNA glycosylase/AP lyase that removes oxidized purine residues present in DNA, including the highly mutagenic C8-oxo-guanine (8-oxoG) generated in DNA by active oxygen during metabolism (Laval et al., Mutation Res. 233:73-79, 1990; Boiteux et al., EMBO J. 6: 3177-3183, 1987). The fpg protein exhibits three catalytic activities in vitro (Olga et al., J. Biol. Chem. 275:9924-9929, 2000): a DNA glycosylase that excises modified nucleotide bases (Laval et al., Mutation Res. 402:93-102, 1998), an AP lyase that incises DNA at abasic sites by an elimination mechanism, and a deoxyribophosphodiesterase that removes 5′-terminal deoxyribose phosphate residues.
- The polypeptide of SEQ ID NO: 67 and the polynucleotide of SEQ ID NO: 25 have utility for processing food products, and as supplements and additives to food products. This glycosylase may also be used to develop non-food products. The applications for and attributes conferred by this enzyme include: reagents or techniques to improve the survival of HN001 in aerobic conditions; enhanced bacterial survival in industrial processes; and enhanced bacterial survival in the intestinal environment. These attributes may be produced in food, such as dairy products, by directed activity of the enzyme, introduced in a bacterial strain (including strain HN001, or starter cultures) comprising a polynucleotide of SEQ ID NO: 25 or a variant, or as an enzyme preparation comprising a polypeptide of SEQ ID NO: 67 or a variant.
- The full-length polynucleotide sequence of acetoin dehydrogenase from L. rhamnosis strain HN001, given in SEQ ID NO: 32 and shown in
FIG. 49 with ATG initiation and translation stop codons (boxed), was used to amplify the AP1 acetoin dehydrogenase gene from L. rhamnosus HN001 DNA using standard PCR methodology. The upstream and downstream primers were tagged with EcoRI and SalI restriction endonuclease recognition sequences to facilitate cloning. - AP1 was then cloned into the EcoRI and SalI sites of the pGEX-6P-3 expression vector (Pharmacia Biotech) and transformed into E. coli strain K12 XL-1Blue competent cells according to standard laboratory protocols. The polypeptide sequence of acetoin dehydrogenase AP1 is given in SEQ ID NO: 74 and shown in
FIG. 50 . The acetoin dehydrogenase AP1 protein was expressed as a fusion protein with glutathione S-transferase (GST), and purified using Glutathione Sepharose 4B resin (Pharmacia Biotech) according to the manufacturer's instructions. An aliquot of purified AP1-GST fusion protein was confirmed by SDS-PAGE analysis. - Acetoin dehydrogenase activity was assayed according to published methods (Rattray et al., Int. Dairy J. 10:781-789, 2000) with some modifications. Briefly, acetoin dehydrogenase activity was measured spectrophotometrically by monitoring the change in absorbance of the cofactor NADH at 340 nm. Aliquots of the purified AP4-GST fusion protein solution were added to reaction mixtures containing 50 mM 2[N-morpholino]ethanesulphonic acid (MES, Sigma) buffer pH 5.5 at 30° C. and the reactions started by the addition of 0.5 mM NADH and 37 mM diacetyl (Sigma) in a total volume of 1 ml. The change in optical density at 340 nm was measured, and rates of NADH utilization measured as an indicator of acetoin dehydrogenase activity. Enzyme activity was calculated as the amount of protein required to convert 1 μmol diacetyl and NADH to acetoin and NAD+ per minute at pH 5.5 at 30° C. Enzyme activity of AP1-GST fusion protein was compared to that of an irrelevant GST-fusion protein, GST protein and elution buffer only.
- Results presented in
FIG. 51 and Table 17 indicate significant background utilization of NADH in the reactions. Similar rates were observed for elution buffer, GST protein and irrelevant fusion protein, indicating that the GST fusion protein did not exhibit acetoin reductase activity. Nonetheless, presence of the AP1-GST fusion protein gave significantly greater acetoin dehydrogenase activity than background, indicating that HN001 AP1 protein encodes acetoin dehydrogenase.FIG. 51 shows the results of an acetoin reductase assay as measured by oxidation of NADH co-factor by OD at 340 nm in the presence of acetoin substrate: λ, elution buffer only; ν, purified irrelevant GST-fusion protein; σ, purified GST protein; υ, purified AP1-GST fusion protein.TABLE 17 Acetoin reductase activity of AP1 GST-fusion protein compared to elution buffer, GST protein, and irrelevant GST-fusion protein controls. Δ OD/min Enzyme activity (μmol/min/ml) AP1-GST fusion protein 3.54 × 10−3 14.07 Irrelevant GST-fusion 2.77 × 10−3 11.13 protein GST protein 2.68 × 10−3 10.77 Elution buffer only 2.38 × 10−3 9.56 - Acetoin dehydrogenase (EC 1.1.1.5) catalyzes the reduction of diacetyl to acetoin, and acetoin to 2,3-butanediol as part of the pyruvate to 2,3-butanediol pathway (reviewed in Sarmiento and Burgos, Methods in Enzymol. 89:516-523, 1982). Diacetyl is an important flavor component in a variety of dairy products including butter, buttermilk, sour cream, fermented cream and cheese. Like its metabolites or related compounds acetoin, acetaldehyde and 2,3-butanediol, diacetyl plays a role in flavor when present in trace amounts (reviewed in Escamilla-Hurtado et al., Rev. Latinoamerican Microbiol. 38:129-37, 1996). A mixture of all these compounds is produced during lactic acid fermentation, and particular proportions of these compounds lead to characteristic flavors in dairy products.
- The polypeptide of SEQ ID NO: 74 and the polynucleotide of SEQ ID NO: 32 have utility for processing food products, and as supplements and additives to food products. This dehydrogenase may also be used to develop non-food products. The attributes conferred by and applications for use of this enzyme include: modulation of the production of important flavor compounds; modification of pyruvate metabolic pathways; industrial production of flavor compounds; and control of diacetyl levels in dairy products.
- These attributes may be produced in food, such as dairy products, and the applications implemented by directed activity of the enzyme, introduced in a bacterial strain (including strain HN001, or starter cultures) comprising a polynucleotide of SEQ ID NO: 32 or a variant, or as an enzyme preparation comprising a polypeptide of SEQ ID NO: 74 or a variant.
- The full-length polynucleotide sequence of aflatoxin B1 aldehyde reductase from L. rhamnosus strain HN001, given in SEQ ID NO: 15 and shown with ATG initiation and translation stop codons (boxed) in
FIG. 52 , was used to amplify the AI7 aflatoxin B1 aldehyde reductase gene from L. rhamnosus HN001 DNA using standard PCR methodology. The upstream and downstream primers were tagged with EcoRI and SalI restriction endonuclease recognition sequences to facilitate cloning. - AI7 was then cloned into the EcoRI and SalI sites of the pGEX-6P-3 expression vector (Pharmacia Biotech) and transformed into E. coli strain DH5α competent cells according to standard laboratory protocols. The polypeptide sequence of aflatoxin B, aldehyde reductase AI7 is given in SEQ ID NO: 57 and shown in
FIG. 53 . The aflatoxin B1 aldehyde reductase AI7 protein was expressed as a fusion protein with glutathione S-transferase (GST) and purified using Glutathione Sepharose 4B resin (Pharmacia Biotech) according to the manufacturer's instructions. An aliquot of the purified AI7 protein was checked by SDS-PAGE analysis. - AI7 activity was assayed by the previously published method of Ellis and Hayes (Biochem. J. 312:535-541, 1995) with some modifications. Briefly, the aldehyde- and ketone-reducing activity of aflatoxin B1 aldehyde reductase was assayed using 4-nitrobenzyl alcohol as substrate and NADPH as a cofactor. Enzyme activity was assessed spectrophotometrically by monitoring the utilization of NADPH at an OD of 340 nm. Reaction volumes of 1 ml containing 100 mM sodium phosphate pH 6.6 and 0.2 mM NADPH were prepared, aliquots of purified AI7 protein added, and the changes in OD measured. Enzyme activity was compared between reactions containing AI7-GST fusion protein, irrelevant GST-fusion protein, elution buffer used during protein purification and water only. Enzyme activity was calculated as μmol NADP used/min/ml.
- As shown in
FIG. 54 and Table 18, the results indicate that HN001 AI7 protein fused with GST exhibited significant aldehyde reductase activity, while the irrelevant GST-fusion protein, as well as the water and elution buffer controls, showed no activity whatsoever. Therefore, the aflatoxin B1 aldehyde reductase activity of the AP4-GST fusion protein was due to the AI7 moiety rather than the GST. Also, the results showed increased rate of substrate utilization proportional to the amount of AI7 protein added, indicating that aflatoxin B1 aldehyde reductase activity of AI7 was dose dependent. Therefore, AI7 encodes HN001 aflatoxin B1 aldehyde reductase. -
FIG. 54 shows the experimental results of aflatoxin B1 aldehyde reductase assay according to oxidation of the NADPH co-factor in the presence of acetoin substrate. X, water only; +, Sepharose column elution buffer only; λ, irrelevant GST-fusion protein; ν, 10 μl purified AP4-GST fusion protein;σ 20 μl purified AP4-GST fusion protein.TABLE 18 Aflatoxin B1 aldehyde reductase activity of AI7 GST-fusion protein compared to elution buffer, water and irrelevant GST-fusion protein controls. Δ OD/min at 340 nm Enzyme activity (μmol/min/ml) Elution 0.00 0 buffer only Water only 0.00 0 10 μl 0.00 0 Irrelevant protein 10 μl AI7 0.038 606 20 μl AI7 0.087 690 - Aflatoxin B1 aldehyde reductase metabolizes the carcinogen aflatoxin B1 (AFB1) by converting the protein-binding dialdehyde form of AFB1-dihydrodiol to the non-binding di-alcohol metabolite, and is associated with AFB1-resistance in animal studies (Ellis et al., Proc. Natl. Acad. Sci. USA 90:10350-10354, 1993; Hayes et al., Cancer Res. 53:3887-3894, 1993). The enzyme is also active against other substrates including a particular class of ketone (ketone groups on adjacent carbon atoms, e.g. 9,10-phenanthrenequinone), as well as aromatic and aliphatic aldehydes (Ellis and Hayes, Biochem. J. 312:535-541, 1995).
- The polypeptide of SEQ ID NO: 57 and the polynucleotide of SEQ ID NO: 15 have utility for processing food products, and as supplements and additives to food products. This reductase also has applications in non-food products and processes. The attributes conferred by and applications for use of this enzyme include: anti-carcinogenic or chemoprotectant reagents; probiotic bacterial strains with anti-cancer effects; research tools for cancer research; enhanced flavor or aroma characteristics; removal of undesirable flavors; and description and application of novel metabolic pathways.
- These attributes may be produced and applications implemented by directed activity of the enzyme, introduced in a bacterial strain (including strain HN001, or starter cultures) comprising a polynucleotide of SEQ ID NO: 15 or a variant, or as an enzyme preparation comprising a polypeptide of SEQ ID NO: 57 or a variant.
- The full-length polynucleotide sequence of 6-phospho-β-galactosidase, given in SEQ ID NO: 31 and shown with the translation stop codon (boxed) in
FIG. 78 , was used to amplify the A05 6-phospho-α-galactosidase gene from L. rhamnosus HN001 DNA using standard PCR methodology. The upstream and downstream primers were tagged with EcoRI and SalI restriction endonuclease recognition sequences to facilitate cloning. - A05 was then cloned into the EcoRI and SalI sites of the pGEX-6P-3 expression vector (Pharmacia Biotech) and transformed into E. coli strain DH5α competent cells according to standard laboratory protocols. The polypeptide sequence of AO5 is given in SEQ ID NO: 73 and shown in
FIG. 79 . The 6-phospho-β-galactosidase AO5 protein was expressed as a fusion protein with glutathione S-transferase (GST) and purified using Glutathione Sepharose 4B resin (Pharmacia Biotech) according to the manufacturer's instructions. An aliquot of the purified AO5 protein was checked by SDS-PAGE analysis. - A05 activity was assayed using standard laboratory methods as follows. Briefly, crude cell lysates were prepared by resuspending a 10 ml overnight culture of E. coli DH5α cells in 1 ml lysis buffer (50 mM potassium phosphate pH 7.8, 400 mM NaCl, 100 mM KCl, 10% glycerol, 0.5% Triton X-100, 10 mM imidazole). Cells were sonicated and spun to sediment cell debris according to standard laboratory methods. Aliquots of 50 μl of cell lysate were added to 900 μl reaction buffer (100 mM KH2PO4 pH 7.0, 2 mM MgCl2) and 50 μl substrate O-nitrophenyl β-D-glycopyranoside (ONPG) (Sigma). Utilization of OPNG was measured spectrophotometrically by monitoring change in absorbance at 420 nm and enzyme activity was calculated as μmol OPNG used/min/ml. 6-Phospho-β-galactosidase enzyme activity was compared in crude lysates from E. coli DH5α transformed with pGEX-6P-3 encoding A05, pGEX-6P-3 encoding an irrelevant protein, and lysis buffer only.
- Experimental results presented in
FIG. 55 and Table 19 indicate that while reactions containing crude lysates from cells transformed with an irrelevant GST-fusion protein or lysis buffer only exhibited little or no enzyme activity, crude lysate from E. coli expressing AO5-GST fusion protein showed significant enzyme activity.FIG. 55 shows the experimental determination of 6-phospho-β-galactosidase enzyme activity as measured by substrate utilization using crude lysates of strains transformed with pGex-6P-3 encoding A05 (υ), pGex-6P-3 encoding an irrelevant protein (ν), or using lysis buffer only (X).TABLE 19 6-Phospho-β-galactosidase enzyme activity in crude cell lysates Enzyme activity Crude cell lysate expressing: Δ OD/min at 420 nm (μmol/min/ml) AO5-GST fusion protein 0.074 0.60 Irrelevant GST-fusion protein 0.001 0.01 Lysis buffer only 0.000 0.00 - Enzyme activity was also measured in increasing amounts of crude cell lysates to assess dose-dependency. Results shown in
FIG. 56 and Table 20 indicate that increasing amounts of cell lysates from cells expressing the A05-GST fusion protein led to proportional increases in 6-phospho-β-galactosidase enzyme activity. Therefore, A05 encodes HN001 6-phospho-β-galactosidase.FIG. 56 shows 6-phospho-β-galactosidase enzyme activity as measured experimentally by substrate utilisation using increasing amounts of crude lysate from strains transformed with pGex-6P-3 encoding A05-GST fusion protein. υ, 50 μl lysate; ν, 100 μl lysate; σ, 200 μl lysate; λ, 200 μl lysis buffer only.TABLE 20 6-Phospho-β-galactosidase enzyme activity in increasing amounts of crude cell lysates. Crude cell lysate expressing Enzyme activity AO5-GST fusion protein Δ OD/min at 420 nm (μmol/min/ml) 50 μl 0.074 0.60 100 μl 0.113 0.92 200 μl 0.169 1.38 200 μl 0.000 0.00 Lysis buffer only - 6-Phospho-β-galactosidase (EC 3.2.1.85) catalyzes the hydrolysis of O-glycosyl bonds of 6-phospho-beta-D-galactosides to give alcohols and 6-phospho-D-galactose, and is involved in lactose utilization (Hengstenberg and Morse, Methods in Enzymol. 42:491-494, 1975).
- The polypeptide of SEQ ID NO: 73 and the polynucleotide of SEQ ID NO: 31 have utility for processing food products and as supplements and additives to food products. This galactosidase may also be used to develop non-food products. The attributes conferred by this enzyme include: flavor and aroma enhancement; nutritional enhancement; altered bacterial metabolic/growth characteristics; and removal of bitter or undesirable flavors.
- These attributes may be produced in food, such as dairy products, by directed activity of the enzyme, introduced in a bacterial strain (including strain HN001, or starter cultures) comprising a polynucleotide of SEQ ID NO: 31 or a variant, or as an enzyme preparation comprising a polypeptide of SEQ ID NO: 73 or a variant.
- The full-length polynucleotide sequence of aromatic aminotransferase of L. rhamnosus strain HN001, given in SEQ ID NO: 11 and shown in
FIG. 57 with ATG initiation and translation stop codons (boxed), was used to amplify the AH7 aromatic aminotransferase gene from L. rhamnosus HN001 DNA using standard PCR methodology. The upstream and downstream primers were tagged with EcoRI and SalI restriction endonuclease recognition sequences to facilitate cloning. - AH7 was then cloned into the EcoRI and SalI sites of the pGEX-6P-3 expression vector (Pharmacia Biotech) and transformed into E. coli strain DH5α competent cells according to standard laboratory protocols. The polypeptide sequence of aromatic aminotransferase AH7 is given in SEQ ID NO: 53 and shown in
FIG. 58 . The aromatic aminotransferase AH7 protein was expressed as a fusion protein with glutathione S-transferase (GST) and purified using Glutathione Sepharose 4B resin (Pharmacia Biotech) according to the manufacturer's instructions. An aliquot of the purified AH7 protein was checked by SDS-PAGE analysis. - Aromatic aminotransferase activity was assayed according to previously published methods (Yvon et al., Appl. Environ. Microbiol. 63:414-419, 1997) with modifications. The assay is composed of two parts: the first is an aminotransferase reaction using the aromatic amino acid phenylalanine as substrate and results in the production of glutamate from α-ketoglutarate. The second part of the assay is the colorimetric determination of the glutamate. For the phenylalanine transamination, 250 μl reaction mixtures containing 70 mM Tris-HCl pH 8.0, 3 mM L-phenylalanine, 10 mM α-ketoglutarate and 0.05 μM pyridoxal 5′ phosphate were incubated with purified proteins or elution buffer at 37° C. for 15 min. Aliquots of 20 μl were then taken and glutamate levels determined by adding to a reaction mixture containing 65 mM Tris pH 9.0, 1.3 mM EDTA, 40 mM hydrazine, 19.5 mM NAD+ and 65 mM ADP, with and without 2.4 U glutamate dehydrogenase in a total volume of 250 μl in the wells of a microtitre plate. Reactions were incubated at 37° C. for 40 min and absorbance at 340 nm measured using a plate reader (Molecular Devices, Sunnyvale Calif.). Enzyme activity of the purified AH7-His-Thio fusion protein was compared a purified irrelevant His-Thio-fusion protein, elution buffer used to elute the purified proteins from the Ni-NTA columns and water only and results are shown in Table 21. Glutamate concentrations were calculated using a standard curve, and assays on all samples and standards were performed in triplicate. Enzyme activities were calculated as μmol glutamate produced/min/ml and specific activities calculated using protein concentrations obtained using the BCA protein assay kit (Pierce) according to the manufacturer's instructions. While the irrelevant fusion protein, elution buffer and water resulted in little glutamate production, AH7 fusion protein exhibited significant aminotransferase activity using phenylalanine as substrate, demonstrating that HN001 AH7 encodes an aromatic amino acid transaminase.
TABLE 21 Aromatic amino acid transaminase activity in HN001 AH7 purified protein as measured by glutamate production. mM glutamate Enzyme Specific per activity activity reaction (μmol/min/ml) (μmol/min/μg) Water only 0.044 146 — 10 μl elution buffer 0.031 103 — 10 μl irrelevant His-Thio- 0.030 100 0.52 fusion protein solution 10 μl His-Thio-AH7 fusion 1.120 3733 17.7 protein solution - Aromatic amino acid transaminase (EC 2.6.1.57) catalyzes the transfer of amino groups between an aromatic amino acid and α-ketoglutarate to its aromatic oxo-acid and L-glutamate (Mavrides and Orr, J. Biol. Chem. 250:4128-4133, 1975). The products of enzymatic amino acid degradation play a major role in cheese flavor development. Degradation products from aromatic amino acids have both positive and negative impacts on cheese flavor (Dunn and Lindsay, J. Dairy Sci. 68:2859-2874, 1985; Engels et al., Int. Dairy J. 7:225-263, 1997). Therefore, the applications of HN001 aromatic amino acid aminotransferase AH7 include: flavor and aroma enhancement; removal of off-flavors; altered levels of biogenic amines; and altered metabolic characteristics.
- These attributes may be produced in food, such as dairy products, by directed activity of the enzyme, introduced in a bacterial strain (including strain HN001 or starter cultures) comprising a polynucleotide of SEQ ID NO: 11, or as an enzyme preparation comprising a polypeptide of SEQ ID NO: 53.
- The full-length polynucleotide sequence of acetate kinase, given in SEQ ID NO: 33 and shown in
FIG. 59 with ATG initiation and translation stop codons (boxed), was used to amplify the AP5 acetate kinase gene from L. rhamnosus strain. The upstream and downstream primers were tagged with EcoRI and SalI restriction endonuclease recognition sequences to facilitate cloning. - AP5 was then cloned into the EcoRI and SalI sites of the pGEX-6P-3 expression vector (Pharmacia Biotech) and transformed into E. coli strain K12 XL-1Blue competent cells according to standard laboratory protocols. The polypeptide sequence of the acetate kinase AP5 polypeptide is given in SEQ ID NO: 75 and shown in
FIG. 60 . This was expressed as a fusion protein with glutathione S-transferase (GST) and purified using Glutathione Sepharose 4B resin (Pharmacia Biotech) according to the manufacturer's instructions. An aliquot of the purified AP5 protein was checked by SDS-PAGE analysis. - AP5 activity was assayed based on a published method for analysis of the related carbamate kinase (Crow and Thomas, J. Bacteriol. 150:1024-1032, 1982). Briefly, the assay uses a couple reaction such that acetyl phosphate and ADP is converted to CO2, NH3 and ATP in the presence of acetate kinase. The produced ATP is then combined with glucose by the enzyme hexokinase to give glucose-6-phosphate, which in turn is reduced by glucose-6-phosphate dehydrogenase using the NADP+ cofactor. Because the hexokinase glucose-6-phosphate dehydrogenase enzymes are provided in excess, acetate kinase activity can be assessed spectrophotometrically by monitoring NADPH production at an OD of 340 nm. Reaction mixtures of 730
μl 200 mM Tris-HCL pH 7.9, 73μl 200 mM acetyl phosphate, 36.5μl 200 mM ADP, 36.5μl 200 mM MgCl2, 73μl 500 mM glucose, 7μl 100 mM NADP+ and 7 μl hexokinase glucose-6-phosphate dehydrogenase were prepared and allowed to equilibrate at 37° C. Purified AP5-GST fusion protein and sterile milliQ water was added to a final volume of 1 ml, and changes in OD at 340 nm measured. Enzyme activity was compared between purified AP5-GST fusion protein, irrelevant fusion protein, and elution buffer used to elute the purified proteins off the Sepharose column. The results are shown in Table 22. Enzyme activities were calculated as μmol NAPDH produced/min/ml, and specific activities calculated using protein concentrations obtained using the BCA protein assay kit (Pierce) according to the manufacturer's instructions. The results indicate that while elution buffer and irrelevant GST-fusion protein showed little or no enzyme activity, the AP5-GST fusion protein exhibited significant activity. Therefore, AP5 encodes HN001 acetate kinase.TABLE 22 Acetate kinase activity of HN001 protein AP5. Enzyme Specific Δ OD/min activity activity at 340 nm (μmol/min/μl) (μmol/min/μg) 20 μl elution buffer 0.0 0.0 — 1 μl irrelevant GST-fusion 0.13 0.05 0.025 protein solution 2 μl AP5-GST fusion 1.72 0.35 0.172 protein solution - Acetyl kinase (EC 2.7.2.1) catalyzes the phosphotransfer between ADP and acetyl phosphate to give ATP and acetate (Nishimura and Griffith, Methods in Enzymol. 71:311-316, 1981). Acetate, a flavor compound in its own right, can give ammonia and carbon dioxide, both of which have important flavor and texture impacts in cheese (Fox et al., Crit. Rev. Food Sci. Nutr. 29:237-53, 1990).
- The polypeptide of SEQ ID NO: 75 and the polynucleotide of SEQ ID NO: 33 have utility for processing food products and as supplements and additives to food products. This kinase may also be used to develop non-food products. The attributes conferred by this enzyme include: flavor and aroma enhancement; removal of off-flavors; altered texture characteristics; and altered metabolic characteristics.
- These attributes may be produced in food, such as dairy products, by directed activity of the enzyme, introduced in a bacterial strain (including strain HN001, or starter cultures) comprising a polynucleotide of SEQ ID NO: 33 or a variant, or as an enzyme preparation comprising a polypeptide of SEQ ID NO: 75 or a variant.
- The full-length polynucleotide sequence of basic surface protein from L. rhamnosus strain HN001, given in SEQ ID NO: 6 and shown in
FIG. 61 with ATG initiation and translation stop codons (boxed), was used to amplify the AC9 basic surface protein gene, but excluding the predicted N-terminal Type II signal sequence. The primer sequences used (SEQ ID NO: 34 and 35) and were tagged with EcoRI and BamHI restriction endonuclease recognition sequences, respectively, to facilitate cloning. AC9 sequence was then amplified from HN001 strain genomic DNA, purified, cloned into EcoRI/BamHI-cut pGEX-6P-3 expression vector, and transformed into E. coli DH5α cells according to standard laboratory methods. The polypeptide sequence of basic surface protein AC9 is given in SEQ ID NO: 47 and shown inFIG. 62 . The basic surface protein AC9 was expressed as a fusion protein with glutathione S-transferase (GST), bound to Glutathione Sepharose 4B resin (Pharmacia Biotech), and PreScission protease used to cleave off the basic surface protein AC9 protein, according to the manufacturer's instructions. An aliquot of the purified AC9 protein was checked by SDS-PAGE analysis. - Purified AC9 protein (14 μg) was labeled by radio-iodination with 0.1 mCi iodine-125 (Amersham Pharmacia) using IODO-BEADS iodination reagent (Pierce) following the manufacturer's instructions. The radio-iodinated protein was separated from unincorporated iodine-125 and excess sodium iodide-125 using a PD-10 desalting column (Amersham-Pharmacia) according to the manufacturer's instructions, except that the elution was performed in phosphate buffered saline in twelve 500 μl aliquots. Radioactivity in eluted fractions was quantitated on a Bioscan Quick Count QC-4000/XER Benchtop Radioisotope Counter (Bioscan, Inc.) and fractions containing the first peak of radioactivity (corresponding to labeled AC9 protein) were pooled and bovine serum albumin added to a final concentration of 10 mg/ml.
- To analyze the binding of polypeptide AC9 to proteins associated with intestinal surface proteins known to act as ligands for bacterial adhesins, different intestinal protein ligands were dot blotted onto a nitrocellulose membrane using a Convertible Filtration Manifold System (Life Technologies) following the manufacturer's instructions. Duplicate dots of approximately 1 μg of type I collagen from calf skin, type IV collagen from human placenta, fibronectin from human plasma, laminin from the basement membrane of Engelbreth-Holm-Swarm mouse sarcoma and type III mucin partially purified from porcine stomach and bovine serum albumin included as a negative control (all proteins were obtained from Sigma) were blotted. The blot was incubated at room temperature on an orbital shaker in 10 ml phosphate buffered saline, pH 7.4, containing 0.1
% Tween 20 and 5 mg/ml bovine serum albumin for 1 hour. Radio-iodinated AC9 protein was then added to a final concentration of approximately 500 ng/ml, and incubated at room temperature for a further hour. The blot was washed three times in approximately 40 ml phosphate buffered saline, pH 7.4, containing 0.1% Tween 20 at room temperature for 10 minutes, then autoradiographed against X-ray film at −80° C. overnight. The autoradiograph was developed and the resulting image digitized with a Fluor S MultiImager (BioRad). Binding by AC9 protein to the intestinal protein ligands was quantitated using Bio-Rad Quantity One software by measuring the density of the signal on the autoradiograph resulting from radiolabeled AC9 protein binding to the different ligands and subtracting the background density of blank film. To quantitate relative amounts of protein ligands blotted, blots were stained with Ponceau S using standard procedures (Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, 2001), and quantitated as for the autoradiograhs. The density corresponding to AC9 protein binding to individual ligands was divided by the average density of Ponceau S staining of the ligand to give the relative AC9 bound to each ligand. Binding of iodinated AC9 (1.50×107 dpm) was compared to binding of iodinated mucus adhesion promoting (mapA) protein of Lactobacillus reuteri (GenBank accession number AJ293860) as a positive control, and iodinated irrelevant HN001 protein (7.00×106 dpm) as a negative control. - As shown in Table 23, the results indicate that while the irrelevant HN001 protein did not bind to any of the intestinal adhesin ligands, both the AC9 protein and the positive control protein mapA showed significant binding to mucin. Therefore, AC9 encodes the HN001 basic surface protein. The results represent the mean of relative density of two dots.
TABLE 23 Density of autoradiographic signals from AC9 basic surface protein binding to dot blots of intestinal proteins, compared to a positive control (mapA) and negative control (irrelevant HN001 protein). Intestinal Relative Relative Relative Irrelevant Protein AC9 Binding mapA Binding protein Binding BSA 0.25 0.76 0.06 Collagen I 0.44 1.02 0.26 Collagen IV 0.34 0.78 0.09 Fibronectin 0.35 0.67 0.09 Laminin 0.53 0.83 0.12 Mucin 2.20 2.57 0.68 - The basic surface protein of Lactobacillus fermentum is a surface-bound molecule that belongs to a family of ATP-binding cassette (ABC) receptor solute binding proteins (Turner et al., J. Bacteriol. 179:3310-3316, 1997; Tam et al., Microbiol. Rev. 57:320-346, 1993). Basic surface protein has also been shown to be involved in cysteine uptake (Turner et al., J. Bacteriol. 181:2192-2198, 1999) and has been used as an attachment site for immunodominant proteins in the development of new vaccine strategies (Turner et al., Infect. Imm. 67:5486-5489, 1999).
- The polypeptide of SEQ ID NO: 47 and the polynucleotide of SEQ ID NO: 6 have utility for processing food products, and as supplements and additives. This basic surface protein may also be used to develop non-food products. The attributes conferred by this protein include: enhanced adhesion to intestinal surface and cell lines; enhanced bacterial survival in intestinal environment; altered metabolic characteristics; altered flavor or aroma characteristics; enhanced probiotic effects; reagents to block or modify adherence of bacteria to mucosal surfaces; and development of vaccine carriers.
- These attributes may be produced in food, such as dairy products, or in supplements by directed activity of the enzyme, introduced in a bacterial strain (including strain HN001, or starter cultures) comprising a polynucleotide of SEQ ID NO: 6 or a variant, or as an enzyme preparation comprising a polypeptide of SEQ ID NO: 47 or a variant.
- The full-length polynucleotide sequence of outer membrane protein A from L. rhamnosus strain HN001, given in SEQ ID NO: 27 and shown in
FIG. 63 with ATG initiation and translation stop codons (boxed) was used to amplify the N-terminal region of AL8 outer membrane protein A gene. The primer sequences are given in SEQ ID NO: 36 and 37, respectively, and were tagged with BamHI and XhoI restriction endonuclease recognition sequences, respectively, to facilitate cloning. AL8 sequence was then amplified from HN001 strain genomic DNA, purified, cloned into BamHI/XhoI-cut pGEX-6P-3 expression vector, and transformed into E. coli DH5α cells according to standard laboratory methods. The polypeptide sequence of outer membrane protein A AL8 is given in SEQ ID NO: 69 and shown inFIG. 64 . AL8 was expressed as a fusion protein with glutathione S-transferase (GST), bound to Glutathione Sepharose 4B resin (Pharmacia Biotech), and PreScission protease was used to cleave off the AL8 protein, according to the manufacturer's instructions. An aliquot of the purified AL8 protein was checked by SDS-PAGE analysis. - Purified AL8 protein (20 μg) was then labeled by radio-iodination with 0.1 mCi iodine-125 (Amersham Pharmacia) using IODO-BEADS iodination reagent (Pierce) following the manufacturer's instructions. Radio-iodinated protein was separated from unincorporated iodine-125 and excess sodium iodide-125 using a PD-10 desalting column (Amersham-Pharmacia) according to the manufacturer's instructions, except that the elution was performed in phosphate buffered saline in twelve 500 μl aliquots. Radioactivity in eluted fractions was quantitated on a Bioscan Quick Count QC-4000/XER Benchtop Radioisotope Counter (Bioscan, Inc.) and fractions containing the first peak of radioactivity (corresponding to labeled AL8 protein) were pooled and bovine serum albumin added to a final concentration of 10 mg/ml.
- To analyze AL8 protein binding to proteins associated with intestinal surface proteins known to act as ligands for bacterial adhesins, different intestinal protein ligands were dot blotted onto a nitrocellulose membrane using a Convertible Filtration Manifold System (Life Technologies) following the manufacturer's instructions. Duplicate dots of approximately 1 μg of type I collagen from calf skin, type IV collagen from human placenta, fibronectin from human plasma, laminin from the basement membrane of Engelbreth-Holm-Swarm mouse sarcoma and type III mucin partially purified from porcine stomach and bovine serum albumin included as a negative control (all proteins were obtained from Sigma) were blotted. The blot was incubated at room temperature on an orbital shaker in 10 ml phosphate buffered saline, pH 7.4, containing 0.1
% Tween 20 and 5 mg/ml bovine serum albumin for 1 hour. Radio-iodinated AL8 protein was then added to a final concentration of approximately 500 ng/ml, and incubated at room temperature for a further hour. The blot was then washed three times in approximately 40 ml phosphate buffered saline, pH 7.4, containing 0.1% Tween 20 at room temperature for 10 minutes, then autoradiographed against X-ray film at −80° C. overnight. The autoradiograph was developed and the resulting image digitized with a Fluor S Multilmager (BioRad). Binding by AL8 protein to the intestinal protein ligands was quantitated using Bio-Rad Quantity One software by measuring the density of the signal on the autoradiograph resulting from radiolabelled AL8 protein binding to the different ligands and subtracting the background density of blank film. To quantitate relative amounts of protein ligands blotted, blots were stained with Ponceau S using standard procedures (Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, 2001), and quantitated as for the autoradiograhs. The density corresponding to AL8 protein binding to individual ligands was divided by the average density of Ponceau S staining of the ligand to give the relative AL8 bound to each ligand. Binding of iodinated AL8 (3.2×107 DPM) was compared to binding of iodinated mucus adhesion promoting (mapA) protein of Lactobacillus reuteri (GenBank accession number AJ293860) (6.6×106 dpm) as a positive control, and iodinated irrelevant HN001 protein (7.0×106 DPM) as a negative control. - The results, shown in Table 24, indicate that while the irrelevant HN001 protein did not bind to any of the intestinal adhesin ligands, both the AL8 protein and the positive control protein mapA showed significant binding to mucin. Therefore, AL8 encodes the HN001 outer membrane protein A. Results represent mean of relative density of two dots.
TABLE 24 Density of autoradiographic signals from AL8 outer membrane protein A binding to dot blots of intestinal proteins, compared to a positive control (mapA) and negative control (irrelevant HN001 protein). Intestinal Relative Relative Relative Irrelevant Protein AL8 Binding mapA Binding protein Binding BSA 0.08 0.76 0.06 Collagen I 0.89 1.02 0.26 Collagen IV 0.16 0.78 0.09 Fibronectin 0.24 0.67 0.09 Laminin 0.47 0.83 0.12 Mucin 2.01 2.57 0.68 - The outer membrane protein A of Rickettsia spp. is a 190 kDa surface bound molecule required for the adhesion of Rickettsia to host cells (Li and Walker, Microbial Path. 179:3310-3316, 1998). Rickettsial outer membrane protein A is also an immunodominant protein and has been used for the serotyping of rickettsial strains (Philip et al., J. Imm. 121:1961-1968, 1978).
- The polypeptide of SEQ ID NO: 69 and the polynucleotide of SEQ ID NO: 27 have utility for processing food products and as supplements and additives. This outer membrane protein may also be used to develop non-food products. The attributes conferred by and applications for this protein include: enhanced adhesion to intestinal surface and cell lines; enhanced bacterial survival in intestinal environment; altered texture characteristics; enhanced probiotic effects; reagents to block or modify adherence of bacteria to mucosal surfaces; and development of vaccine carriers.
- These attributes may be produced in food, such as dairy products, and the applications may be implemented by directed activity of the protein, introduced in a bacterial strain (including strain HN001, or starter cultures) comprising a polynucleotide of SEQ ID NO: 27 or a variant, or as an enzyme preparation comprising a polypeptide of SEQ ID NO: 69 or a variant.
- The full-length polynucleotide sequence of extracellular matrix binding protein, AM4, from L. rhamnosus strain HN001, given in SEQ ID NO: 28 and shown in
FIG. 65 , was used to amplify the N-terminal region of AM4 extracellular matrix binding protein gene. The primer sequences used (SEQ ID NO: 38 and 39) were tagged with EcoRI and NotI restriction endonuclease recognition sequences, respectively, to facilitate cloning. AM4 sequence was then amplified from HN001 strain genomic DNA, purified, cloned into EcoRI/NotI-cut pGEX-6P-3 expression vector, and transformed into E. coli DH5α cells according to standard laboratory methods. The polypeptide sequence of extracellular matrix binding protein AM4 is given in SEQ ID NO: 70 and shown inFIG. 66 . The extracellular matrix binding protein AM4 was expressed as a fusion protein with glutathione S-transferase (GST) and purified using Glutathione Sepharose 4B resin (Pharmacia Biotech), according to the manufacturer's instructions. An aliquot of the purified AM4-GST fusion protein was checked by SDS-PAGE analysis. - Purified AM4 protein (10 μg) was labeled by radio-iodination with 0.1 mCi iodine-125 (Amersham Pharmacia) using IODO-BEADS iodination reagent (Pierce) following the manufacturer's instructions. Radio-iodinated protein was separated from unincorporated iodine-125 and excess sodium iodide-125 using a PD-10 desalting column (Amersham-Pharmacia) according to the manufacturer's instructions, except that the elution was performed in phosphate buffered saline in twelve 500 μl aliquots. Radioactivity in eluted fractions was quantitated on a Bioscan Quick Count QC-4000/XER Benchtop Radioisotope Counter (Bioscan, Inc.) and fractions containing the first peak of radioactivity (corresponding to labeled AM4 protein) were pooled and bovine serum albumin added to a final concentration of 10 mg/ml.
- To analyze binding of the AM4 protein with intestinal surface proteins known to act as ligands for bacterial adhesins, different intestinal protein ligands were dot blotted onto a nitrocellulose membrane using a Convertible Filtration Manifold System (Life Technologies) following the manufacturer's instructions. Duplicate dots were blotted of approximately 1 μg of type I collagen from calf skin, type IV collagen from human placenta, fibronectin from human plasma, and laminin from the basement membrane of Engelbreth-Holm-Swarm mouse sarcoma, with type III mucin partially purified from porcine stomach and bovine serum albumin included as a negative control (all proteins were obtained from Sigma). The blot was incubated at room temperature on an orbital shaker in 10 ml phosphate buffered saline, pH 7.4, containing 0.1
% Tween 20 and 5 mg/ml bovine serum albumin for 1 hour. Radio-iodinated AM4 protein was then added to a final concentration of approximately 500 ng/ml, and incubated at room temperature for a further hour. The blot was then washed three times in approximately 40 ml phosphate buffered saline, pH 7.4, containing 0.1% Tween 20 at room temperature for 10 minutes, and autoradiographed against X-ray film at −80° C. overnight. The autoradiograph was developed and the resulting image digitized with a Fluor S Multilmager (BioRad). Binding by AM4 protein to the intestinal protein ligands was quantitated using Bio-Rad Quantity One software by measuring the density of the signal on the autoradiograph resulting from radiolabeled AM4 protein binding to the different ligands and subtracting the background density of blank film. To quantitate relative amounts of protein ligands blotted, blots were stained with Ponceau S using standard procedures (Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, 2001), and quantitated as for the autoradiograhs. The density corresponding to AM4 protein binding to individual ligands was divided by the average density of Ponceau S staining of the ligand to give the relative AM4 bound to each ligand. Binding of iodinated AM4-GST fusion protein (3.3×107 DPM) was compared to binding of iodinated mucus adhesion promoting (mapA) protein of Lactobacillus reuteri (GenBank accession number AJ293860; 6.6×106 DPM) as a positive control, and iodinated irrelevant HN001 protein (7.0×106 dpm) as a negative control. - Results in Table 25 indicate that while the irrelevant HN001 protein did not bind to any of the intestinal adhesin ligands, the AM4 fusion protein gave a very similar binding pattern to the positive control protein mapA, with significant binding to mucin and collagen types I and IV. Therefore, AM4 encodes the HN001 extracellular matrix binding protein. Results represent mean of relative density of two dots.
TABLE 25 Density of autoradiographic signals from AM4-GST fusion protein to dot blots of intestinal proteins, compared to a positive control (mapA) and negative control (irrelevant HN001 protein). Intestinal Relative AM4-GST Relative Relative Irrelevant Protein Binding mapA Binding Protein Binding BSA 0.37 0.76 0.06 Collagen I 1.43 1.02 0.26 Collagen IV 0.94 0.78 0.09 Fibronectin 0.53 0.67 0.09 Laminin 0.65 0.83 0.12 Mucin 1.48 2.57 0.68 - The extracellular matrix binding protein is a surface bound molecule required for the adhesion of Streptococcus spp. to the extracellular matrix, exposed during tissue injury (Manganelli and van de Rijn, Infect. Imm. 67:50-56, 1999).
- The polypeptide of SEQ ID NO: 70 and the polynucleotide of SEQ ID NO: 28 have utility for processing food products and as supplements and additives. This binding protein also has other applications. The attributes conferred by and applications for this enzyme include: enhanced adhesion to intestinal surface and cell lines; enhanced bacterial survival in intestinal environment; altered texture characteristics; enhanced probiotic effects; reagents to block or modify adherence of bacteria to surfaces; and development of vaccine carriers.
- These attributes may be produced in food, such as dairy products, and other applications may be implemented by directed activity of the binding protein, introduced in a bacterial strain (including strain HN001, or starter cultures) comprising a polynucleotide of SEQ ID NO: 28 or a variant, or as an enzyme preparation comprising a polypeptide of SEQ ID NO: 70 or a variant.
- The full-length polynucleotide sequence of high-molecular-weight adhesion protein, AL7, from L. rhamnosus strain HN001, given in SEQ ID NO: 26 and shown in
FIG. 67 with ATG initiation and translation stop codons (boxed), was used to amplify the N-terminal region of AL7 high-molecular-weight adhesion protein gene. The primer sequences used (SEQ ID NO: 40 and 41) were tagged with BamHI and EcoRI restriction endonuclease recognition sequences, respectively, to facilitate cloning. AL7 sequence was amplified from HN001 strain genomic DNA, purified, cloned into BamHI/EcoRI-cut pGEX-6P-3 expression vector, and transformed into E. coli DH5α cells according to standard laboratory methods. The polypeptide sequence of high-molecular-weight adhesion protein AL7 is given in SEQ ID NO: 68 and shown inFIG. 68 . The high-molecular-weight adhesion protein AL7 was expressed as a fusion protein with glutathione S-transferase (GST) and expression was checked by SDS-PAGE analysis. - Lysates of DH5α clones containing pGEX-6P-3 expressing AL7-GST fusion protein, lysates of DH5α clones containing pGEX-6P-3 expressing irrelevant HN001 GST-fusion protein, and crude cell wall cytoplasmic HN001 protein preparations (prepared by standard laboratory methods) were separated by SDS-PAGE. Proteins were blotted onto nitrocellulose membranes using a Trans-Blot SD Semi-Dry Electrophoretic Transfer Cell (Bio-Rad) according to the manufacturer's instructions. The nitrocellulose blot was then blocked overnight at 4° C. in phosphate buffered saline, pH 7.4, 0.1% Tween 20 (PBS-T), containing 5% non-fat dried milk. Rabbit anti-sera raised against HN001 cell wall proteins (supplied by Dr. Paul O'Toole, Institute of Molecular Biosciences, Massey University, Palmerston North, New Zealand) were diluted 1:5000 in PBS-T, 5% non-fat dried milk and incubated with the blot for 1 hr at room temperature. The blot was washed three times for 15 min each in PBS-T and incubated at room temperature in 50 ml PBS-T, 5% non-fat dried milk containing a 1:3000 dilution of a horseradish peroxidase-labeled antibody against rabbit Ig (Amersham Pharmacia) for 20 min. The blot was washed six times in PBS-T at room temperature for 15 min each, and binding visualized using the ECL Western blotting detection system (Amersham Pharmacia) according to the manufacturer's instructions.
- Results of the Western blot revealed that the anti-sera detected a number of proteins from HN001 raised against the HN001 cell wall preparations. While several of these proteins were found in both the cell wall and cytoplasmic preparations of HN001, these proteins consisted of bands of approximately 66 kDa and less. In addition, a number of high molecular weight protein bands were detected in the HN001 cell wall protein preparations that were not present in the HN001 cytoplasmic protein preparations. These bands ranged from approx. 130 kDa to approx. 220 kDa or greater. Therefore the cell wall antisera specifically detected several large cell wall proteins from HN001. Of the E. coli extracts, the only signal came from the lysate of the DH5α clone containing pGEX-6P-3 expressing the N-terminal region of AL7. This strong band was approximately 97 kDa, the same size as the AL7-GST fusion protein. Lysates from E. coli clones expressing unrelated proteins showed no cross-reactivity with the HN001 cell wall anti-sera. This data indicates that AL7 encodes a high-molecular-weight adhesion protein at the cell surface.
- The high-molecular-weight adhesion protein is a homologue of the surface-bound molecule of Haemophilus influenzae shown to be involved in adhesion to human cell lines (Barenkamp and St Geme, Mol. Microbiol. 19:1215-1223, 1996; St Geme et al., Proc. Natl. Acad. Sci. USA 90:2875-2879, 1993).
- The polypeptide of SEQ ID NO: 68 and the polynucleotide of SEQ ID NO: 26 have utility for processing food products, as supplements and additives, and as reagents for several applications. The attributes conferred by and applications for use of this adhesion protein include: enhanced adhesion to intestinal surfaces and cell lines; enhanced bacterial survival in intestinal environment; altered texture characteristics; enhanced probiotic effects; reagents to block or modify adherence of bacteria to surfaces; and development of vaccine carriers. These attributes may be produced in food, such as dairy products, and implemented in other applications by directed activity of the adhesion protein, introduced in a bacterial strain (including strain HN001, or starter cultures) comprising a polynucleotide of SEQ ID NO: 26 or a variant, or as an enzyme preparation comprising a polypeptide of SEQ ID NO: 68 or a variant.
- The full-length polynucleotide sequence of a periplasmic binding protein 1 (PEB1), AJ4, from L. rhamnosus strain HN001, given in SEQ ID NO: 16 and shown in
FIG. 69 with ATG initiation and translation stop codons (boxed), was used to amplify the AJ4 PEB1 gene from HN001 strain genomic DNA by PCR according to standard laboratory methods. Primers were tagged with BamHI and EcoRI to facilitate cloning. AJ4 PCR products were purified, cloned into BamHI/EcoRI-cut pGEX-6P-3 expression vector, and transformed into E. coli DH5α cells according to standard laboratory methods. The polypeptide sequence of PEB1 AJ4 is given in SEQ ID NO: 58 and shown inFIG. 70 . AJ4 was expressed as a fusion protein with glutathione S-transferase transferase (GST), bound to Glutathione Sepharose 4B resin (Pharmacia Biotech), and PreScission protease was used to cleave off the PEB1 AJ4 protein, according to the manufacturer's instructions. An aliquot of the purified AJ4 protein was checked by SDS-PAGE analysis. - Purified AJ4 protein (10 μg) was then labeled by radio-iodination with 0.1 mCi iodine-125 (Amersham Pharmacia) using IODO-BEADS iodination reagent (Pierce) following the manufacturer's instructions. Radio-iodinated protein was separated from unincorporated iodine-125 and excess sodium iodide-125 using a PD-10 desalting column (Amersham-Pharmacia) according to the manufacturer's instructions, except that the elution was performed in phosphate buffered saline in twelve 500 μl aliquots. Radioactivity in eluted fractions was quantitated on a Bioscan Quick Count QC-4000/XER Benchtop Radioisotope Counter (Bioscan, Inc.) and fractions containing the first peak of radioactivity (corresponding to labeled AJ4 protein) were pooled and bovine serum albumin added to a final concentration of 10 mg/ml.
- To analyze the binding of the AJ4 protein to intestinal surface proteins known to act as ligands for bacterial adhesins, different intestinal protein ligands were dot blotted onto a nitrocellulose membrane using a Convertible Filtration Manifold System (Life Technologies) following the manufacturer's instructions. Duplicate dots were blotted of approximately 1 μg of type I collagen from calf skin, type IV collagen from human placenta, fibronectin from human plasma, laminin from the basement membrane of Engelbreth-Holm-Swarm mouse sarcoma, and type III mucin partially purified from porcine stomach and bovine serum albumin included as a negative control (all proteins were obtained from Sigma). The blot was incubated at room temperature on an orbital shaker in 10 ml phosphate buffered saline, pH 7.4, containing 0.1
% Tween 20 and 5 mg/ml bovine serum albumin for 1 hour. Radio-iodinated AJ4 protein was then added to a final concentration of approximately 500 ng/ml, and incubated at room temperature for a further hour. The blot was washed three times in approximately 40 ml phosphate buffered saline, pH 7.4, containing 0.1% Tween 20 at room temperature for 10 minutes, and autoradiographed against X-ray film at −80° C. overnight. The autoradiograph was developed and the resulting image digitized with a Fluor S MultiImager (BioRad). Binding by AJ4 protein to the intestinal protein ligands was quantitated using Bio-Rad Quantity One software by measuring the density of the signal on the autoradiograph resulting from radio-labeled AJ4 protein binding to the different ligands and subtracting the background density of blank film. To quantitate relative amounts of protein ligands blotted, blots were stained with Ponceau S using standard procedures (Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, 2001), and quantitated as for the autoradiograhs. The density corresponding to AJ4 protein binding to individual ligands was divided by the average density of Ponceau S staining of the ligand to give the relative AJ4 bound to each ligand. Binding of iodinated AJ4 protein (2.6×106 DPM) was compared to binding of iodinated mucus adhesion promoting (mapA) protein of Lactobacillus reuteri (GenBank accession number AJ293860; 1.3×106 dpm) as a positive control, and iodinated irrelevant HN001 protein (1.4×106 DPM) as a negative control. - Results shown in
FIG. 71 demonstrate that while the irrelevant HN001 protein showed no significant binding to the intestinal proteins, AJ4 and the positive control protein mapA showed significant binding to mucin. AJ4 also showed some binding to laminin, fibronectin, and collagen type IV. Therefore, AJ4 encodes the HN001 PEB1.FIG. 71 shows the relative density of autoradiographic signals from AJ4 protein (grey bars) to dot blots of intestinal proteins, compared to a positive control (mapA, white bars) and negative control (irrelevant HN001 protein, black bars). Results for each dot (duplicates) are shown. - The PEB1 is a surface-bound molecule required for the adhesion of Campylobacter spp. to intestinal epithelial cells and is required for effective colonization of the gut environment (Pei et al., Infect. Imm. 66:938-943, 1998; Pei and Blaser, J. Biol. Chem. 268:18717-18725, 1993).
- The polypeptide of SEQ ID NO: 58 and the polynucleotide of SEQ ID NO: 16 have utility for processing food products and as supplements and additives. This binding protein may also be used to develop non-food products. The attributes conferred by and applications for this protein include: enhanced adhesion to intestinal surface and cell lines; enhanced bacterial survival in intestinal environment; altered texture characteristics; enhanced probiotic effects; reagents to block or modify adherence of bacteria to surfaces; and development of vaccine carriers.
- These attributes may be produced in food, such as dairy products, and implemented in other applications, by directed activity of the protein, introduced in a bacterial strain (including strain HN001, or starter cultures) comprising a polynucleotide of SEQ ID NO: 16 or a variant, or as an enzyme preparation comprising a polypeptide of SEQ ID NO: 58 or a variant.
- SEQ ID NO: 1-83 are set out in the attached Sequence Listing. The codes for nucleotide sequences used in the attached Sequence Listing, including the symbol “n,” conform to WIPO Standard ST.25 (1998), Appendix 2, Table 1.
- All references cited herein, including all patent and literature references, are incorporated herein by reference in their entireties.
- While in the foregoing specification this invention has been described in relation to certain preferred embodiments, and many details have been set forth for purposes of illustration, it will be apparent to those skilled in the art that the invention is susceptible to additional embodiments and that certain of the details described herein may be varied considerably without departing from the basic principles of the invention.
Claims (38)
1. An isolated polynucleotide comprising a sequence selected from the group consisting of: SEQ ID NO: 1-33.
2. An isolated polynucleotide comprising a sequence selected from the group consisting of:
(a) complements of SEQ ID NO: 1-33;
(b) reverse complements of SEQ ID NO: 1-33; and
(c) reverse sequences of SEQ ID NO: 1-33.
3. An isolated polynucleotide comprising a sequence selected from the group consisting of:
(a) sequences having at least 75%, identity to a sequence of SEQ ID NO: 1-33;
(b) sequences having at least 90% identity to a sequence of SEQ ID NO: 1-33; and
(c) sequences having at least 95% identity to a sequence of SEQ ID NO: 1-33, wherein the polynucleotide encodes a polypeptide having substantially the same functional properties as a polypeptide encoded by SEQ ID NO: 1-33.
4. An isolated polynucleotide comprising a sequence selected from the group consisting of:
(a) nucleotide sequences that are 200-mers of a sequence recited in SEQ ID NO: 1-33;
(b) nucleotide sequences that are 100-mers of a sequence recited in SEQ ID NO: 1-33;
(c) nucleotide sequences that are 40-mers of a sequence recited in SEQ ID NO: 1-33; and
(d) nucleotide sequences that are 20-mers of a sequence recited in SEQ ID NO: 1-33;
5. An isolated oligonucleotide probe or primer comprising at least 10 contiguous residues complementary to 10 contiguous residues of a nucleotide sequence recited in any one of claims 1-3.
6. A kit comprising a plurality of oligonucleotide probes or primers of claim 5 .
7. A genetic construct comprising a polynucleotide of any one of claims 1-3.
8. A transgenic host cell comprising a genetic construct according to claim 7 .
9. A transgenic non-human organism comprising a transgenic host cell of claim 8 .
10. The transgenic organism of claim 9 , wherein the organism is selected from the group consisting of Lactobacillus species.
11. An isolated polynucleotide comprising a nucleotide sequence that differs from a nucleotide sequence recited in SEQ ID NO: 1-33 as a result of deletions and/or insertions totalling less than 15% of the total sequence length.
12. The isolated polynucleotide of claim 11 , wherein the nucleotide sequence differs from a nucleotide sequence recited in SEQ ID NO: 1-33 as a result of substitutions, insertions, and/or deletions totalling less than 10% of the total sequence length.
13. A genetic construct comprising, in the 5′-3′ direction:
(a) a gene promoter sequence; and
(b) a polynucleotide sequence comprising at least one of the following: (1) a polynucleotide coding for at least a functional portion of a polypeptide of SEQ ID NO: 42-75; and (2) a polynucleotide comprising a non-coding region of a polynucleotide of any one of claims 1-3.
14. The genetic construct of claim 13 , wherein the polynucleotide is in a sense orientation.
15. The genetic construct of claim 13 , wherein the polynucleotide is in an anti-sense orientation.
16. The genetic construct of claim 13 , wherein the gene promoter sequence is functional in a prokaryote or eukaryote.
17. A transgenic host cell comprising a construct of claim 13 .
18. A transgenic organism comprising a transgenic host cell according to claim 17 , or progeny thereof.
19. The transgenic organism of claim 18 , wherein the organism is selected from the group consisting of Lactobacillus species.
20. A method for modulating the activity of a polypeptide in an organism, comprising stably incorporating into the genome of the organism a polynucleotide of any one of claims 1-3.
21. The method of claim 20 , wherein the organism is a microbe.
22. An isolated polypeptide comprising an amino acid sequence selected from the group consisting of: SEQ ID NO: 42-75.
23. An isolated polypeptide comprising an amino acid sequence selected from the group consisting of:
(a) sequences having at least 75% identity to a sequence of SEQ ID NO: 42-75;
(b) sequences having at least 90% identity to a sequence of SEQ ID NO: 42-75; and
(c) sequences having at least 95% identity to a sequence of SEQ ID NO: 42-75, wherein the polypeptide has substantially the same functional properties as a polypeptide of SEQ ID NO: 42-75.
24. An isolated polypeptide encoded by a polynucleotide of any one of claims 1-3.
25. An isolated polynucleotide that encodes a polypeptide of any one of claims 22 and 23.
26. A fusion protein comprising at least one polypeptide according to any one of claims 22 and 23.
27. A composition comprising a polypeptide according to any one of claims 22 and 23 and at least one component selected from the group consisting of: physiologically acceptable carriers and immunostimulants.
28. A composition comprising a polynucleotide according to any one of claims 1-3 and at least one component selected from the group consisting of: physiologically acceptable carriers and immunostimulants.
29. A method for treating a disorder in a mammal, comprising administering a composition according to claim 27 .
30. A method for treating a disorder in a mammal, comprising administering a composition according to claim 28 .
31. A method for modifying a property of a microbe, comprising modulating the polynucleotide content or composition of the microbe by transforming the microbe with a polynucleotide of any one of claims 1-3.
32. The method of claim 31 , wherein the microbe is used in the manufacture of a milk-derived product, food product, food additive, nutritional supplement, bioactive substance or probiotic supplement.
33. A method for modifying at least one property of a product, food, food additive, nutritional supplement or probiotic supplement, wherein the product, food, food additive, nutritional supplement or probiotic supplement is prepared from milk, the method comprising adding a polypeptide of any one of claims 22-24 to the milk.
34. The method of claim 33 , wherein the property is selected from the group consisting of: flavor; aroma; texture; nutritional benefits; immune system modulation; and health benefits.
35. A food product comprising an isolated polypeptide of any one of claims 22 and 23.
36. The food product of claim 35 , wherein the food product is derived from milk.
37. The food product of claim 36 , wherein the food product is selected from the group consisting of: cheese; and yoghurt.
38. The food product of claim 35 , wherein the food product has at least one modified property selected from the group consisting of: flavor; aroma; texture; nutritional benefits; immune system modulation; and health benefits.
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US11/314,869 US20060251635A1 (en) | 2000-08-08 | 2005-12-20 | Lactobacillus rhamnosus polynucleotides, polypeptides and methods for using them |
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Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6379663B1 (en) * | 1997-08-21 | 2002-04-30 | New Zealand Dairy Board | Immunity enhancing lactic acid bacteria |
US6476209B1 (en) * | 2000-11-28 | 2002-11-05 | Genesis Research & Development Corporation Ltd. | Polynucleotides, materials incorporating them, and methods for using them |
US6544772B1 (en) * | 2000-08-08 | 2003-04-08 | Genesis Research & Development Corp. Ltd | Polynucleotides, materials incorporating them, and methods for using them |
Family Cites Families (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
DE60128742T2 (en) * | 2000-03-03 | 2008-02-07 | Fujifilm Manufacturing Europe B.V. | Process for the preparation of polymer-laminated paper supports and polymer-laminated photographic paper supports prepared by this process |
WO2001077335A2 (en) | 2000-04-11 | 2001-10-18 | Institut Pasteur | Listeria monocytogenes genome, polypeptides and uses |
-
2001
- 2001-08-08 AU AU8455801A patent/AU8455801A/en active Pending
- 2001-08-08 NZ NZ523995A patent/NZ523995A/en unknown
- 2001-08-08 NZ NZ536894A patent/NZ536894A/en unknown
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- 2005-12-20 US US11/314,869 patent/US20060251635A1/en not_active Abandoned
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6379663B1 (en) * | 1997-08-21 | 2002-04-30 | New Zealand Dairy Board | Immunity enhancing lactic acid bacteria |
US6544772B1 (en) * | 2000-08-08 | 2003-04-08 | Genesis Research & Development Corp. Ltd | Polynucleotides, materials incorporating them, and methods for using them |
US6476209B1 (en) * | 2000-11-28 | 2002-11-05 | Genesis Research & Development Corporation Ltd. | Polynucleotides, materials incorporating them, and methods for using them |
Cited By (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20080187525A1 (en) * | 2007-02-01 | 2008-08-07 | Porubcan Randolph S | Formulations Including Digestive Enzymes and Polysorbate Surfactants that Enhance the Colonization of Administered Probiotics Microoganisms |
US8066986B2 (en) * | 2007-02-01 | 2011-11-29 | Master Supplements, Inc. | Formulations including digestive enzymes and polysorbate surfactants that enhance the colonization of administered probiotics microoganisms |
US20120034200A1 (en) * | 2007-02-01 | 2012-02-09 | Porubcan Randolph S | Formulations Including Digestive Enzymes, Polysorbate-Based Surfactants and Probiotics |
US8722035B2 (en) * | 2007-02-01 | 2014-05-13 | Master Supplements, Inc. | Formulations including digestive enzymes, polysorbate-based surfactants and probiotics |
WO2011099875A1 (en) * | 2010-02-15 | 2011-08-18 | Salmon, Bernadette | Use of lactic acid bacteria to treat or prevent rhinitis |
US9011909B2 (en) | 2010-09-03 | 2015-04-21 | Wisconsin Pharmacal Company, Llc | Prebiotic suppositories |
US9730969B2 (en) | 2015-11-06 | 2017-08-15 | Mead Johnson Nutrition Company | Nutritional compositions for promoting gut barrier function and ameliorating visceral pain |
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EP1311684A4 (en) | 2005-10-19 |
CA2416982A1 (en) | 2002-02-14 |
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