WO2001011008A2 - Methode d'obtention et de detection de bacteries gram positif fluorescentes - Google Patents

Methode d'obtention et de detection de bacteries gram positif fluorescentes Download PDF

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WO2001011008A2
WO2001011008A2 PCT/ES2000/000305 ES0000305W WO0111008A2 WO 2001011008 A2 WO2001011008 A2 WO 2001011008A2 ES 0000305 W ES0000305 W ES 0000305W WO 0111008 A2 WO0111008 A2 WO 0111008A2
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gfp
gene
expression
fluorescence
cells
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WO2001011008A3 (fr
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Concepción NIETO MAZARRON
Paloma Acebo Pais
Pilar Fernandez De Palencia
Mª de los Angeles CORRALES GONZALEZ
Manuel Espinosa Padron
Paloma Lopez Garcia
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Consejo Superior De Investigaciones Cientificas
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Publication of WO2001011008A3 publication Critical patent/WO2001011008A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C12N15/746—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for lactic acid bacteria (Streptococcus; Lactococcus; Lactobacillus; Pediococcus; Enterococcus; Leuconostoc; Propionibacterium; Bifidobacterium; Sporolactobacillus)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/315—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Streptococcus (G), e.g. Enterococci
    • C07K14/3156—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Streptococcus (G), e.g. Enterococci from Streptococcus pneumoniae (Pneumococcus)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43595—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from coelenteratae, e.g. medusae

Definitions

  • Biotechnology Genetic engineering of microorganisms. Gene expression. Streptococcus pneumoniae. Lactococcus aclis. Cell mareaje.
  • the green fluorescent protein (GFP) encoded by the gfp gene of the jellyfish Aequorea victoria has unique properties to be used as a non-isotopic marker and real-time biomolecules in living systems. In fact it has already been widely used in eukaryotic organisms [Sullivan and Kay Eds. (1999) Green fluorescent proteins, Methods in Cell Biology vol.
  • the wild GFP protein and its modified versions have the characteristic of being autofluorescent (their detection does not require the use of a substrate), they have a very long half-life, do not cause toxic effects in the organisms tested so far and are active in all cells and tissues, live or fixed, tested so far.
  • Streptococcus pneumomae colonizes approximately 1 in 2.5 individuals of the human species, but only 1 in 200 develops pneumonia, otitis media or septicemia during their life cycle (Deitsch and cois (1997) Microbiol Mol Biol Rev 61 281-293] At present, the mechanisms involved in ecological bases and the spread of S pneumoniae are unknown.
  • pneumomae and Eschenchia coh [Schuster and cois (1998) FEMS Microbiol Lett 164 427-431] Gene cloning pneumococcal in S. pneumomae was performed for the first time [Stassi and cois (1982) Proc Nati Acad Sci USA 78 7028-7032] using plasmid pMV158 from S.
  • Gene expression requires the use of promoters that are recognized by the host RNA polymerase.
  • the expression of the pneumococcal gene malM is regulated at the transcriptional level in S. pneumomae and the levels of its expression depend on the sugar present in the culture medium. This regulation is the MalR repressor, product of the malR chromosomal gene [Puyet et cois (1993) J Biol Chem 268 25402-25408], which inhibits the expression of the malM gene when S.
  • pneumomae is grown in medium containing sucrose [Lacks (1968) Genetics 60 685-706) by binding to the promoter P M (Nieto and cois (1997) J Biol Chem 272 30860-30865] Repression does not take place, or is partial, when S. pneumomae is grown in media containing maltose, glucose or sucrose and maltose (Lacks (1968) Genetics 60 685-706] This regulation only takes place in S.
  • the malM gene, its promoter and the alR gene encoding the MalR repressor as discussed in the background are part of the ale region and will allow the development of different gene constructs for the regulation of gene expression in Bacteria, among others S. pneumomae and L. Lactis
  • a method of subcloning a gene of interest is described, taking as example the gfp gene, under the control of the promoters of the malM gene, wild and mutant, S.
  • plasmids pLS70GFP and pLS69GFP ( Figure 1 and Example 1 and 2) have been constructed and established in S. pneumomae, and transferred the gfp gene to the S. pneumomae chromosome ( Figure 1 and Example 3), whereby these gene constructs, expression vectors and transformed cells form part of the present invention.
  • the plasmids mentioned above and the chromosomal insert encode the GFP protein and their presence in the bacteria confers them fluorescence which has been determined by a fluorescence spectrophotometer (Table I and Example 5).
  • the malM gene promoter is functional in L. lactis and has allowed the expression of gfp and the synthesis of GFP actively in this microorganism (Table I) from plasmid pLSIGFP built in E. coh and subsequently transferred to L. lactis ( Figure 2 and Example 4)
  • Overexpression of the GFP protein has allowed the detection of S. pneumomae and L. lactis lines bearing GFP in mixed cultures with non-carrier isogenic strains ( Figure 3 and Example 6) by Fluorescence microscopy and its use is part of the present invention.
  • pneumomae RNA polymerase is regulated by the MalR repressor so that in the present invention the development of new gene constructs and cellular transformations (Example 7) that have allowed the expression of a gene of interest, gfp, in monocopy or multicopy in a constitutive or inducible way in S. pneumomae depending on the bacterial strain used ( Figure 4 and Example 8) assessed by determining autofluorescence of your GFP product For all this, these new gene constructs, expression vectors and transformed cells and their use in the manipulation of S. pneumomae gene expression form part of the present invention.
  • This invention has the advantage of allowing the detection of strains of S. pneumo ae and L. lactis culture medium for fluorescence techniques due to overproduction of GFP This can p'llow in a future use for the detection of 1) the colonizing capacity of the pathogenic microorganism S. pneumomae in cultures of human tissue and in animal models used by the pharmaceutical industries and 2) the colonizing capacity of the microorganism that integrates the starter cultures, L. lactis, during cheese maturation, which is of interest to the dairy industries So far in the present invention a gene expression system of the gfp gene has been described constitutively in the two aforementioned hosts and also partially controlled in S.
  • Plasmid pLSIRGFP like pLSIGFP ( Figure 2), encodes GFP and its presence in bacteria gives them fluorescence (Table II and Example 12)
  • the use of the malM gene promoter by S. pneumomae RNA polymerase is regulated by the repressor MalR and the presence of the alR gene in multicopy in the plasmid pLSIRGFP allows controlling the expression of the gfp gene by using different sugars in the culture medium (Table II, Table III and Figure 6; Example 10 and 1 1). It also allows a controlled induction of the gfp gene dependent on the concentration of maltose added to the culture media ( Figure 7 and Example 13).
  • the regulation of the gfp gene in these microorganisms is an example of the possibility of expressing genes of pharmaceutical industrial interest and assessing their effect on these transformed cells and forms part of the present invention.
  • its use would allow 1) the cloning and controlled expression of genes coding for toxic products in the human pathogen S. pneumoniae by substitution of the gfp gene with genes of interest, and 2) the overproduction of products of interest in said microorganism,
  • This is of interest to the pharmaceutical industries, as it may allow them to develop new bactericidal drugs and vaccines against S. pneumoniae, as well as increase knowledge of the factors involved in the pathogenicity of pneumococcus.
  • Figure 1 Construction of plasmids pLS70GFP, pLS69GFP, pJS3 and the malM .gfpcat chromosomal insert containing the gfp gene under the control of the promoters of wild S. pneumomae P M (pLS70GFP and chromosomal insert) or mutant P ' M ( pLS69GFP). It contains the physical maps of pLS70, pLS69, pGreenTIR, pLS70GFP, pLS69GFP and the malMv.gfpcat chromosomal insert.
  • gfp codes for the green fluorescent protein
  • malM encodes for the enzyme amylomaltase
  • malX encodes a permease of maltosaccharides
  • malP encodes the enzyme maltose phosphorylase
  • lime, tetL and bla code for the proteins responsible for resistance to chloramphenicol, tetracycline and ampicillin respectively
  • copG and repB encode proteins involved in the replication of plasmids based on pMV158
  • ori region of origin of replication of plasmid pGreenTIR
  • P M and P ' M wild and mutant promoters of malM
  • P ' ⁇ wild and mutant promoters of alX
  • Piac gfp gene promoter in pGreenTIR, TIR, region required for translation of the g / gene
  • ENH sequence that increases the translation of gfp, SD, ribosome binding site
  • Figure 2. Construction of plasmid pLSIGFP containing the gfp gene under the conlrol of the promolor wild Contains the physical maps of plasmids pJDC9, pGreenTIR, pJDC9XM, pJDC9GFP, pGreenTIR, pLSl and pLS IGFP
  • the ulilized genetic symbols are gfp, encode for prolein fluorescenle green, copG and repB, encode for plasmid replication proleins based pMV158, tetL, bla and erm, code for the proleins responsible for resistance to tetracycline, ampicillin and erythromycin respectively, or ⁇ , identify the regions necessary for the replication of the different plasmids, ter, transcriptional terminator of the plasmid mob gene pMV158, P M and P ⁇ , wild promoters of the malX and malM gene respectively, lacZ ' , fragment MI 5 of the lacZ gene
  • Figure 3 Deletion of the expression of GFP in S. pneumomae and L. lactis by microscopy. It contains photographs of cellular fluxes detected by phase contrast microscopy (left) and fluorescence (right)
  • A Mixed culture of the sipirpes of S. pneumomae R61 and R61 [pLS70GFP]
  • B Mixed mixture of the S. pneumomae R61 and R61 malM gfpcat lines
  • C Mixed cul ⁇ ivo of the L. lactis lineages MG1363 [pLS l] and MG1363 [pLSlGFP]
  • the arrows indicate some of the non-fluorescent cells
  • Figure 4. Deletion of the constitutive or inducible expression of gfp in monocopy and multicopy in S. pneumoniae Contains the fluorescence levels of cultures of (A) Rcl9 [pLS70GFP], (B) Rcl9 malM gfpcat, (C) R61 [ pLS70GFP], (D) R61 malM: gfpcat Symbols S, cultures grown in sucrose, G, cultures grown in glucose, M, crops grown in maltose, SM, crops grown in sucrose and malignant The fluorescence of 1 ml of sample was measured in cuvette using an LS-50B fluorescence spectrofolomer (Perkin Elmer) measured an excitation at a wavelength of 488 nm with an aperture of 2.5 and fluorescence emission detection was performed at a wavelength of 51 1 nm with an opening of 5 Figure 5.
  • A Rcl9 [pLS70GFP]
  • B Rcl9 malM gfpcat
  • C R61 [ pLS
  • Plasmid pLS IRGFP which contain the g / p gene under the control of the S. pneumomae P M promoter and the malR gene under the S. agalactiae PtetL promoter. Contains the physical maps of pJDC9GFP, pAMP22 and pLSIRGFP.
  • gfp codes for the green fluorescent protein
  • malR codes for the repressor MalR
  • erm codes for the protein responsible for resistance to erythromycin, copG and repB, encodes for proteins involved in plasmid replication based on pMV158, or ⁇ , region of plasmid replication origin pJDC9GFP, P M; wild malM promoter, P x , wild malX promoter, P ⁇ L malR gene promoter in pAPM22 and pLSIGFP, TIR, region required for efficient translation of the gfp gene, ENH, sequence that increases the translation of gfp, SD, site ribosome binding
  • FIG. 6 Detection of GFP synthesis in S. pneumomae strains carrying plasmids
  • Plasmid pLS70 ( Figure 1 and Stassi et al (1981) Proc Nati Acad Sci USA 78 7028-7032) composed of the plasmid of S. agalactiae pMV158 and a S. pneumoniae chromosomal insert containing ⁇ malXmalK ⁇ malP and the wild promoters P ⁇ and P M (Stassi et al. (1982) Gene 20: 359-366) was linearized with the restriction enzyme Sacl. Plasmid pGreenTIR ( Figure 1 and Miller and Lindow (1997) Gene 191: 149-153), composed of the E.
  • coli vector pUC 1813 and the mutant gfp gene encoding the GFP-F64LS65T protein was digested with the restriction enzyme Sacl .
  • One of the generated Sacl fragments, 0.8 kb including the gfp gene, was purified, ligated to plasmid pLS70 using the T4 coliphage DNA ligase and said ligation mixture was used to transform S. pneumomae.
  • Tetracycline resistant transformants (plasmid marker) were analyzed for plasmid content in 0.8% agarose gels.
  • the recombinant plasmid obtained was designated as part of this invention pLS70GFP ( Figure 1) and contains the gfp gene under the control of the wild P promoter.
  • the line of S. pneumoniae R61 [pLS70GFP] is deposited in the Spanish Type Culture Collection and has all the characteristics of CECT No. 5162.
  • Plasmid pLS69 ( Figure 1 and Stassi et al. (1981) Proc. Nati. Acad. Sci USA 78: 7028-7032) composed of plasmid pMV158 and a chromosomal insert of S. pneumoniae containing ⁇ malXmalM ⁇ malP and weak mutant promoters P ' ⁇ and P ' M (Stassi et al.
  • the recombinant plasmid obtained was designated as part of this invention pLS69GFP ( Figure 1) and contains the gfp gene under the control of the malM mutant promoter (P ' M ) -
  • the S. pneumoniae T4 line [pLS69GFP] is deposited in the Spanish Collection Type Crop and presents all the characteristics of the CECT n ° 5160.
  • Example 3.- Construction of the R61 malM :: gfpcat strain of S. pneumoniae.
  • Plasmid pLS70GFP ( Figure 1) was partially digested with the restriction enzyme Sacl. The linearized plasmid was purified and its 3 ' protruding ends (generated by digestion) converted to blunts by digestion with the Mung bean nuclease and polymerization with the Klenow fragment of E. coli DNA polymerase I (Klenow fragment). Plasmid pJS3 [ Figure 1 and Ballester and cois. (1986) Gene 41: 153-163] was digested with the restriction enzyme Sau3M. One of the generated fragments, of 1.1 kb including the cat gene, was purified, its 5 ' protuberant ends filled and the fragment linked to the linearized plasmid pLS70. Said ligation mixture was used to transform S.
  • R61 Chloramphenicol resistant transformants were analyzed to determine their plasmid content in 0.8% agarose gels. The absence of plasmid indicated that the cat gene had been introduced into the chromosome by disruption of the malM gene. The identity of the chromosomal insertion gene structure of one of the transformants, called R61 malMr.gfpcat, was established by determining the nucleotide sequence of its mutated malM gene.
  • said gene was amplified by the technique of the chain polymerization reaction (PCR) using Dynazyme DNA polymerase and using as substrates: chromosomal DNA and oligonucleotides bad ⁇ (5 -CGAGCACGAGCATCTGCATCTGG-3) and mal2 (5 -GCGGTGAAACGTTTCTTTGAAGTCTTTGAAG 3 ).
  • the 2.0 kb amplified fragment was subsequently sequenced.
  • the DNA sequence data (SEQ ID NO: 1) showed that the chromosomal insert contains a gfp gene and a cat gene under the control of the wild P M promoter ( Figure 1).
  • the S. pneumoniae R61 malM :: gfpcat line is deposited in the Spanish Type Crops Collection and has all the characteristics of CECT No. 5158.
  • plasmids pLS70GFP and pLS69GFP were transferred to the MG1363 strain by electroporation, using plasmid DNA obtained from the R61 [pLS70GFP] and T4 [pLS69GFP] strains and selecting the resistance transformants to tetracycline. Both plasmids, although expressing GFP, presented structural instability and the generation of deleted plasmids was detected (results not shown).
  • pneumomae chromosomal insert present in pLS69 and containing weak P promoters ' ⁇ and P ' M had not been previously observed either in B. subtihs [Espinosa and cois (1984) Gene 28 301-310] or in E. coh [Stassi and cois (1982) Gene 18 319-328]
  • the region that includes the P and Px promoters was amplified by the PCR technique using the DNA polymerase Pfii and using as DNA substrates the plasmid pLS70 and the oligonucleotides mal3 (5 -GCAGAATTCAAGTTTTATTGATAAGGAAAC-3) and mal4 (5 -CGCGGATCCATCTCTAGAGTATTTTGCAGACGCAAACG-3) which include recognition sequences for restriction enzymes EcoRI and BamHl respectively
  • the amplified fragment of 0.5 kb was purified, digested with the e EcoRI-Bamr restriction enzymes and plasmid bound pJDC9 ( Figure 2 and (Chen and Morrison (1988) Gene 155-164) previously linearized by digestion with EcoRI and BamY.
  • Said ligation mixture was used to transform E. coh JM109.
  • the resistant erythromycin transformants were analyzed to determine their plasmid content in 0.8% agarose gels.
  • the resulting plasmid pJDC9XM ( Figure 2) was digested with the restriction enzyme San. and the 5 ' protruding ends generated by the digestion were filled with the Klenow fragment.
  • the plasmid pGreenTIR was digested with the restriction enzyme EcoKl.
  • One of the EcoRI fragments generated, from 0.8 kb including the gfp gene was purified, its protuberant ends filled and bound to the linearized plasmid pJDC9XM.
  • Said ligation mixture was used to transform E. coli JM109.
  • Transformants selected for resistance to erythromycin contained plasmid pJDC9GFP ( Figure 2) and expressed the GFP protein (result not shown).
  • This recombinant plasmid was digested with the restriction enzymes Clal and Eco J and subsequently the 5 ' protruding ends generated by the filled digestion.
  • One of the restriction fragments obtained from 3.1 kb contains the P M and Px promoters and the gfp and erm genes.
  • Plasmid pLSl was digested with the restriction enzymes Hindlll and EcoRI and the 5 ' protruding ends generated by the filled digestion.
  • One of the 4.0 kb fragments was purified, ligated to the 3.1 kb fragment from plasmid pJDC9GFP and the ligation mixture was used to transform E. coli C600.
  • the transformants were selected for resistance to erythromycin and their plasmid content analyzed in 0.8% agarose gels Fantasy Of the recombinant plasmids analyzed, one containing the cloned fragment in the correct orientation was selected so that transcription from Px stopped at the transcriptional terminator of the vector mob gene ( Figure 2).
  • This plasmid was designated as part of this invention pLSIGFP and contains the gfp gene under the control of the wild P M promoter.
  • coli C600 line [pLSIGFP] is deposited in the Spanish Type Culture Collection and has all the characteristics of CECT No. 5157.
  • the plasmid pLSIGFP was transferred to L. aclis MG1363 by electroporation with plasmid DNA obtained from the lineage C600 [pLSlGFP] and selection for erythromycin resistance.
  • the plasmid content of the transformants and the structural stability of pLSIGFP after 100 generations of growth was checked by analysis on 0.8% agarose gels.
  • the L. aclis line MG1363 [pLS lGFP] is deposited in the Spanish Type Culture Collection and has all the characteristics of CECT n ° 5163.
  • Example 5 Detection of GFP expression in S. pneumoniae and L. lactis by determination of fluorescence levels.
  • Cells of the S. pneumoniae R61, R61 [pLS70GFP], T4 [pLS69GFP] and R61 ma ⁇ M :: gfpcat lines were grown in AGCH medium supplemented with 0.8% glucose at an Aeso of 0.4.
  • Cells from the L. lactis MG1363 line containing the plasmid vector pLSl or pLS IGFP were grown to an O6O of 0.4 in MI 7 medium supplemented with 1% glucose and 1 ⁇ g / ml tetracycline or 5 ⁇ g / ml erythromycin.
  • Example 6 Detection of GFP expression in S. pneumoniae and L. lactis by fluorescence microscopy.
  • Example 7 -Characterization of the Rcl9 strain of S. pneumoniae and construction of the Rcl9 [pLS70GFP] and Rcl9 malM :: gfpcat lines.
  • the expression of the malM gene in this strain is constitutive due to a mutation, which had previously been located in the malR gene [Lacks (1968) Genetics 60 685- 706].
  • the identity of the mutation was established by determining the nucleotide sequence of the mutated malR gene of the Re 19 strain. To that end, said gene was amplified by the PCR technique using Dynazyme DNA polymerase and using as substrates: chromosomal DNA and oligonucleotides malrl (5- ACGCAGAATTCGCCCGTTACGATTAAAGACGTG 3) and malr2 (5 ' - ACGTCAAGCTTTTATTTTTCGATAATTTTGTGGGC 3), which respectively contain the cut sites for the restriction enzymes EcoRI and Hindlll.
  • the 1010 bp amplified fragment was subsequently sequenced.
  • the DNA sequence data (SEQ ID NO. 2) showed that the MalR protein, encoded by the Re 19 line, carried a 4 amino acid deletion in a region of the protein supposedly involved in DNA binding, a condition necessary to lead to Perform its repressive function.
  • Plasmid pLS70GFP obtained from R61, was transferred to Rcl9 by selecting transformants for tetracycline resistance.
  • Esti Re Rcl9 [pLS70GFP] is deposited in the Spanish Type Culture Collection and presents all the characteristics of CECT n ° 5159.
  • the malMv.gfpcat insert was transferred to the Re 19 style by chromosomal transformation using genomic DNA from the R61 malMv.gfpcat line and selection for chloramphenicol resistance.
  • the lineage obtained was called Re 19 malMv.gfpcat, it is deposited in the Spanish Type Culture Collection and has all the characteristics of CECT n ° 5161.
  • Example 8 Detection of the constitutive and inducible expression of GFP in S. pneumoniae.
  • Stile cells of S. pneumoniae R61, R61 [pLS70GFP], R61 maiM :: gfpcat,
  • Rcl9, Rcl9 [pLS70GFP] and Rcl9 ma ⁇ M :: gfpcat were grown in AGCH medium supplemented with 0.8% (R61 and isogenic) or 0.4% (Rcl9 and isogenic) of the sugars indicated in Figure 4
  • the strains with the malM :: gfpcat insert cannot use maltose as a carbon source, because the enzyme amylomaltase (product of the malM gene) is necessary for maltose metabolism and is not synthesized by these estuaries.
  • Example 9 Construction of plasmid pLSIRGFP. Cloning was performed in the wild line of S. pneumomae R61 [Lacks (1970) J Bacteriol 101 373-383] Plasmid pJDC9GFP ( Figures 2 and 5) was linearized with the restriction enzyme Sal ⁇ and the protruding 5 ' ends filled with Klenow fragment of Escherichia coh DNA polymerase I This plasmid DNA was subsequently digested with EcóRI and one of fragments obtained 1.4 kb containing the gfp gene under the control of the promoter P M, was purified. The plasmid pAPM22 carrying the alR gene of S.
  • Plasmid marker Erythromycin-resistant transformants were analyzed for plasmid content in 0.8% agarose gels Fantasy
  • the recombinant plasmid obtained was named as part of this invention pLSIRGFP ( Figure 5) and containing the gfp gene (encoding GFP) under the control of the promoter P M inducible wild maltose and MaLR (encoding MaLR, repressor gene P M ) under the control of the S. agalactiae tetL promoter.
  • the plasmid gfp gene appears flanked by unique Xbal and Sphl sites.
  • Example 10 Detection of GFP synthesis in S. pneumoniae by fractionation of SDS-polyacrylamide gel proteins and gel staining.
  • S. pneumoniae R61 cells line that has a copy of alR in its chromosome
  • pLSIRGFP carrier of the gfp and malR genes
  • pLSIGFP carrier of the gfp gene, but not of the malR gene
  • pAMP22 lacking gfp and malR
  • the cells were pelleted by centrifugation and diluted 1/10 in fresh medium containing sucrose (P M repression condition by MalR) or maltose (P M induction condition) at a concentration of 0.8%.
  • the cultures were grown at 050 of 0.6 and sedimented by centrifugation.
  • Total protein extracts were prepared as previously described (Espinosa et al. (1984) Gene 28: 301-310), fractionated on a 15% SDS-polyacrylamide gel » the proteins were visualized by staining with blue from coomassie ( Figure 6A) and the bands detected were quantified with the Molecular Analyst system (Bio-Rad).
  • Example 11 Immunodetection of GFP synthesis in S. pneumoniae by Western hybridization.
  • S. pneumomae R61 [pAMP22], R61 [pLS lRGFP] and R61 [pLSlGFP] esthetic cells were grown in medium containing sucrose, maltose or both sugars (partial MalR repression condition) and total protein extracts were prepared.
  • the membrane was washed four times with the same buffer, incubated with the secondary anti-rabbit IgG (Sigma Immunochemicals, diluted 1: 30000) and washed with TBS buffer containing 0.2% Tween 20. Chemiluminescent GFP detection was performed with the Immun-Start kit (Bio-Rad) following the instructions of the suppliers.
  • Example 12- Detection of GFP activity in S. pneumoniae by determination of fluorescence levels Cells of the S. pneumomae estuaries R61 [pAMP22], R61 [pLSlRGFP] and R61 [pLSlGFP] were grown to an Aeso of 0.7, in AGCH medium supplemented with sugars (0.8%) indicated in the Table II
  • One milliliter of each of the cultures was centrifuged and the cells were resuspended in 1 ml of PBS buffer pH 7.2 (10 mM Na 2 HPO 4 , 1 mM KH 2 PO 4 , 140 mM NaCl, 3 mM KC1) Determination of the fluorescence levels of these samples was performed on an LS-50B fluorescence spectrophotometer (Perkin Elmer).
  • Example 13 -Detection of the influence of maltose concentration on the inducible expression of GFP in S. pneumoniae.
  • Cells of the S. pneumomae esti ⁇ e R61 [pAMP22], R61 [pLSlGFP] and R61 [pLS lRGFP] were grown in AGCH medium supplemented with 0.8% sucrose, sedimented by centrifugation, washed and resuspended in fresh medium The cultures were divided into aliquots and subjected to maltose treatment at the concentrations indicated in Figure 7, during a doubling time.

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Abstract

L'invention concerne une méthode de surproduction de la protéine fluorescente verte (GFP). Le gène gfp Aequorea victoria est sous-cloné, dans des plasmides à large spectre d'hôte, sous le contrôle du promoteur du gène malM de Streptococcus pneumoniae. On obtient ainsi une souche de S. pneumoniae contenant le gène gfp dans son chromosome et les plasmides pLS70GFP, pLS69GFP, pLS1GFP et pLS1RGFP. Les cellules pneumococciques porteuses desdits plasmides ou de l'insertion chromosomique synthétisent la GFP. Pls1gfp permet l'expression constitutive de gfp dans Lactococcus lactis. PLS1RGFP, qui contient le gène malR, confère à S. pneumoniae une expression entièrement régulée de gfp. L'insertion chromosomique et pLS70GFP permettent l'expression de gfp; en monocopie ou multicopie, de manière inductible ou constitutive, en fonction de la souche pneumococcique utilisée. La détection des cellules porteuses ou de l'expression quantitative de la protéine GFP s'effectue par microscopie de fluorescence ou par spectrophotométrie de fluorescence et immunodétection.
PCT/ES2000/000305 1999-08-06 2000-08-04 Methode d'obtention et de detection de bacteries gram positif fluorescentes WO2001011008A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU64445/00A AU6444500A (en) 1999-08-06 2000-08-04 Process for obtaining and detecting gram-positive fluorescent bacteria

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
ES9901813A ES2166671B1 (es) 1999-08-06 1999-08-06 Procedimiento de obtencion y deteccion de bacterias gram-positivas fluorescentes.
ESP9901813 1999-08-06
ESP200000244 2000-02-04
ES200000244 2000-02-04

Publications (2)

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WO2001011008A2 true WO2001011008A2 (fr) 2001-02-15
WO2001011008A3 WO2001011008A3 (fr) 2001-05-25

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PCT/ES2000/000305 WO2001011008A2 (fr) 1999-08-06 2000-08-04 Methode d'obtention et de detection de bacteries gram positif fluorescentes

Country Status (2)

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AU (1) AU6444500A (fr)
WO (1) WO2001011008A2 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3192873A1 (fr) * 2009-09-29 2017-07-19 Intrexon Actobiotics NV Promoteurs de lactobacillus et de streptococcus et leurs utilisations

Non-Patent Citations (8)

* Cited by examiner, † Cited by third party
Title
ACEBO P. ET AL.: 'Quantitative detection of streptococcus pneumoniae cells harboring single or multiple copies of the gene encoding the green fluorescent protein' MICROBIOLOGY vol. 146, 2000, pages 1267 - 1273 *
FERNANDEZ DE PALENCIA P. ET AL.: 'Expression of green fluorescent protein in lactococcus lactis' FEMS MICROBIOLOGY LETTERS vol. 183, no. 2, 2000, pages 229 - 234 *
KAIN S.R. ET AL.: 'Green fluorescent protein as a reported el gene expression and protein localization' BIOTECHNIQUES vol. 19, no. 4, 1995, pages 650 - 655 *
NIETO C. ET AL.: 'Construction of a tightly regulated plasmid vector for streptococcus pneumoniae: Controlled expression of the green fluorescent protein' PLASMID vol. 43, no. 3, 2000, pages 205 - 213 *
NIETO C. ET AL.: 'The maltose/maltodextrin regulon of streptococcus pneumoniae' THE JOURNAL OF BIOLOGICAL CHEMISTRY vol. 272, no. 49, 1997, pages 30860 - 30865 *
PUYET A. ET AL.: 'Characterization of the streptococcus pneumoniae maltosaccharide regulator Mal R, a member of the Lac I-Gal R family of repressors displaying distinctive genetic features' THE JOURNAL OF BIOLOGICAL CHEMISTRY vol. 268, no. 34, 1993, pages 2502 - 25408 *
SCOTT K.P. ET AL.: 'The green fluorescent protein as a visible marker for lactic acid bacteria in complex ecosystems' FEMS MICROBIOL. ECOL. vol. 26, no. 3, 1998, pages 219 - 230 *
VALDIVIA R.H. ET AL.: 'Applications for green fluorescent protein (GFP) in the study of host-pathogen interactions' GENE vol. 173, no. 1, 1996, pages 47 - 52 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3192873A1 (fr) * 2009-09-29 2017-07-19 Intrexon Actobiotics NV Promoteurs de lactobacillus et de streptococcus et leurs utilisations

Also Published As

Publication number Publication date
AU6444500A (en) 2001-03-05
WO2001011008A3 (fr) 2001-05-25

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