CA2482138A1 - La1 - the genome of a lactobacillus strain - Google Patents
La1 - the genome of a lactobacillus strain Download PDFInfo
- Publication number
- CA2482138A1 CA2482138A1 CA002482138A CA2482138A CA2482138A1 CA 2482138 A1 CA2482138 A1 CA 2482138A1 CA 002482138 A CA002482138 A CA 002482138A CA 2482138 A CA2482138 A CA 2482138A CA 2482138 A1 CA2482138 A1 CA 2482138A1
- Authority
- CA
- Canada
- Prior art keywords
- seq
- sequence
- sample
- identified
- protein
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- 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
- C07K14/335—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Lactobacillus (G)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
Abstract
The present invention pertains to the use of the DNA sequence of a Lactobacillus johnsonii strain, in particular to its genomic sequence for elucidating interactions of micro-organism with hosts they colonize, and moreover for elucidating the basis of probiotic properties exhibited by such strain. In addition, the present invention also relates to methods of detecting nucleic acids or polypeptides of Lactobacilli and related species, respectively. A data carrier is provided comprising nucleotide sequences and/or polypeptide sequences of La1.
Description
Lal - The genome of a Lactobacillus strain The present invention pertains to the use of the DNA sequence of a Lactobacillus johnsonii strain, in particular to its genomic sequence for elucidating interactions of micro-organism with hosts they colonize, and moreover for elucidating the basis of probiotic properties exhibited by such strain. In addition, the present invention also relates to methods of detecting nucleic acids or polypeptides of Lactobacilli and related species, respectively. A data Garner is provided comprising nucleotide sequences and/or polypeptide sequences of Lal .
Lactic acid bacteria, i.e. micro-organisms that produce lactic acid during their (fermentative) activity, are known for a long time and comprise e.g. the genera Lactococcus, Lactobacillus, Streptococcus, Bifidobacterium and Pediococcus. These bacteria are usually prominent in milk and also in milk processing factories, respectively, living or decaying plants and represent a constituent of the intestinal micro-flora in mankind and animals.
Lactic acid bacteria have been utilized as agents for the preservation of food taking benefit of a lowering of the pH and the action of products generated during the fermentative activity thereof to e.g. inhibit the growth of spoilage bacteria. In addition, lactic acid bacteria have also been used for preparing a variety of different foodstuff such as cheese, yogurt and other fermented dairy products from milk.
Lately, lactic acid bacteria have~attracted a great deal of attention in that some strains have been found to exhibit valuable properties to man and animals upon ingestion. In particular, specific strains of the genus Lactobacillus and Bifidobacterium have been found to pass the gastro-intestinal tract in a viable and live form without getting destroyed in the upper part thereof, especially by the impact of the low pH prevailing in the stomach. Moreover, they were found to be able to colonize the intestinal mucosa, with their temporary or sustained presence in the gut being postulated to bring about numerous positive effects on the health of the living beings.
These strains are generically termed probiotics.
Lactic acid bacteria, i.e. micro-organisms that produce lactic acid during their (fermentative) activity, are known for a long time and comprise e.g. the genera Lactococcus, Lactobacillus, Streptococcus, Bifidobacterium and Pediococcus. These bacteria are usually prominent in milk and also in milk processing factories, respectively, living or decaying plants and represent a constituent of the intestinal micro-flora in mankind and animals.
Lactic acid bacteria have been utilized as agents for the preservation of food taking benefit of a lowering of the pH and the action of products generated during the fermentative activity thereof to e.g. inhibit the growth of spoilage bacteria. In addition, lactic acid bacteria have also been used for preparing a variety of different foodstuff such as cheese, yogurt and other fermented dairy products from milk.
Lately, lactic acid bacteria have~attracted a great deal of attention in that some strains have been found to exhibit valuable properties to man and animals upon ingestion. In particular, specific strains of the genus Lactobacillus and Bifidobacterium have been found to pass the gastro-intestinal tract in a viable and live form without getting destroyed in the upper part thereof, especially by the impact of the low pH prevailing in the stomach. Moreover, they were found to be able to colonize the intestinal mucosa, with their temporary or sustained presence in the gut being postulated to bring about numerous positive effects on the health of the living beings.
These strains are generically termed probiotics.
EP 0 768 375 discloses such a specific strain of the genus Bifidobacterium, that is capable to become implanted in the intestinal flora. This Bifidobacterium strain is reported to assist in immuno-modulation, being capable to competitively exclude adhesion of pathogenic bacteria to intestinal cells, thus supporting the maintenance of the individual's health.
Apart from Bifidobacteria, also some strains of Lactobacilli have been found to exert favourable properties to humans, such as preventing colonization of the gut by pathogenic bacteria or obstructing rotaviral infection. In particular, PCT/EP02/00958 discloses such a strain having both of said properties.
In the last few years the food industry has applied such strains in products, such as milk drinks or fermented acidified milk products. Clinical studies performed with these products and/or the bacterial strains confirmed the notion that these kind of bacteria account for health promoting traits in vivo and may even be utilized for contending diseases, such as ulcers. In particular, a strain of the genus Lactobacillus johnsonii has proven to be capable to combat Helicobacter, an acknowledged cause of ulcer in man.
In view of these valuable properties particular strains of lactic acid bacteria may provide, there is a strong desire in the art for elucidating the molecular basics of these health promoting properties. In particular it would be of great interest to determine the substance or substances responsible for these effect(s). To this end, tools are required to study these micro-organisms in more detail, so as to clarify the molecular principles underlying the probiotic properties, such as interaction with the hosts, the phenomena of passing (survive in) different environmental conditions in the .gut as well as having the capability to adhere to the intestine's mucosa and eventually the involvement in the enhancement of the immune system and defense against pathogens, which information will allow a better understanding of these mechanisms.
Consequently, a problem of the present invention is to provide substantial data about bacterial strains that exhibit properties.beneficial for man andlor animals.
Apart from Bifidobacteria, also some strains of Lactobacilli have been found to exert favourable properties to humans, such as preventing colonization of the gut by pathogenic bacteria or obstructing rotaviral infection. In particular, PCT/EP02/00958 discloses such a strain having both of said properties.
In the last few years the food industry has applied such strains in products, such as milk drinks or fermented acidified milk products. Clinical studies performed with these products and/or the bacterial strains confirmed the notion that these kind of bacteria account for health promoting traits in vivo and may even be utilized for contending diseases, such as ulcers. In particular, a strain of the genus Lactobacillus johnsonii has proven to be capable to combat Helicobacter, an acknowledged cause of ulcer in man.
In view of these valuable properties particular strains of lactic acid bacteria may provide, there is a strong desire in the art for elucidating the molecular basics of these health promoting properties. In particular it would be of great interest to determine the substance or substances responsible for these effect(s). To this end, tools are required to study these micro-organisms in more detail, so as to clarify the molecular principles underlying the probiotic properties, such as interaction with the hosts, the phenomena of passing (survive in) different environmental conditions in the .gut as well as having the capability to adhere to the intestine's mucosa and eventually the involvement in the enhancement of the immune system and defense against pathogens, which information will allow a better understanding of these mechanisms.
Consequently, a problem of the present invention is to provide substantial data about bacterial strains that exhibit properties.beneficial for man andlor animals.
The above problem has been solved by providing the DNA sequence making up the probiotic strain Lactobacillus johnsonii Lal.
In one aspect the present invention relates to the use of a nucleotide sequence of the lactic acid bacterium Lactobacillus johnsonii Lal genome having the sequence SEQ. m. NO.
l, parts thereof or sequences homologous thereto for elucidating interactions between bacteria and a host, preferably lactic acid bacteria and a host, more preferably lactobacilli and a host, in particular for determining factors accounting for the probiotic properties of such strains.
In the context of this application the terms genome or genomic sequence shall be understood to mean the sequence of the chromosome of Lactobacillus johnsonii. The terms nucleotide sequence, polynucleotide or nucleic acid shall designate a double-stranded DNA, a single-stranded DNA or transcriptional products of the said DNAs of various length including oligo-nucleotides of,about 5 to 200, preferably 10 to 100 nucleotides in length.
According to the present invention a homologous nucleotide sequence is understood to mean a nucleotide sequence having a percentual identity with the sequence of SEQ ID.
No. 1 (or selected parts thereof) of at least 90 %, preferably at least 95 %, more preferably 96 % and even more preferably at least 98%. The said homologous rnay comprise, e.g., sequences corresponding to the genomic sequence or to the sequences of fragments thereof belonging to the species Lactobacillus, more preferably to the species Lactobacillus johnsonii, as well as the sequences corresponding to the genomic sequence or to the sequences of its representative fragments of a bacterium belonging to related species. In the present invention, the terms species and genus are mutually interchangeable.
These homologous sequences may thus correspond to variations linked to mutations within the same species or between species and may correspond in particular to truncations, substitutions, deletions and/or additions of at least one nucleotide. The said homologous sequences may also correspond to variations linked to the degeneracy of the genetic code or to a bias in the genetic code which is specific to the family, to the species or to the variant and which are likely to be present in Lactobacillus.
Protein and/or nucleic acid sequence homologies may be evaluated using any of the variety of sequence comparison algorithms and programs known in the art. Such algorithms and programs include, but are by no means limited to, TBLASTN, BLASTP, FASTA, TFASTA, and CLUSTALW (see e.g. Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85 (8): 2444-2448; Altschul et al., 1990, J. Mol. Biol. 215 (3) : 403-410; Thompson et al., 1994, Nucleic Acids Res. 22 (2): 4673-4680; Higgins et al., 1996, Methods Enzymol. 266: 383-402; Altschul et al., 1990, J. Mol. Biol: 215 (3) : 403 - 410; Altschul et al., 1993, Nature Genetics 3: 266-272).
In a particularly preferred embodiment, protein and nucleic acid sequence homologies are evaluated using the Basic Local Alignment Search Tool ("BLAST") which is well_known in the art (supra). In particular, four specific BLAST programs have been used to perform the following task:
(1) BLASTP: Compares an amino acid query sequence against a protein sequence database (2) BLASTN: Compares a nucleotide query sequence against a nucleotide sequence database (3) BLASTX: Compares a nucleotide query sequence translated in all reading frames against a protein sequence database (4) TBLASTN: Compares a protein query sequence against a nucleotide sequence database dynamically translated in all reading frames Among these representative fragments, those capable of hybridizing under stringent conditions with a nucleotide sequence disclosed in the present invention are preferred.
Hybridization under stringent conditions means that the temperature and ionic stiength conditions are chosen such that they allow hybridization to be maintained between two complementary DNA
fragments. Such conditions off, high stringency may e.g. be achieved, by carrying out the hybridisation at a preferred temperature of 65 °C in the presence of SSC buffer, e.g. 1 x SSC
corresponding to 0.15 M NaCI and 0.05 M Na-citrate. The washing steps may be, for example, the following: 2 x SSC, 0.1 ~o SDS at room ,temperature followed by three washes with 1 x SSC, 0.1 % SDS; 0.5 x SSC, 0.1 % SDS; 0.1 x SSC, 0.1 % SDS at 68 C
for 15 minutes.
The nucleotide sequences SEQ. m. NO. 1 has been obtained by sequencing the genome of Lactobacillus johnsonii Lal by the method of directed sequencing after fluorescent automated se-quencing of the inserts of clones and assembling of these sequences of nucleotide fragments (inserts) by means of softwares. To this end, fragments of the genome were created, ligated into suitable vectors for amplification and propagation and the corresponding fragments were sequenced. Overlaps and the final arrangement of the fragments, the nucleotide sequence thereof, were assessed by the aid of appropriate softwares.
Clones for sequencing also included 10'000 by plus fragments as BAC clones that were used to provide a larger scale framework to the assembly. Due to the presence of several repeated regions a correct assembly proved extremely difficult. These included especially the repeated regions such as IS elements, the ribosomal operons and specifically the genes for two large cell surface proteins that contain between 100 and 200 almost perfect 1.0 amino acid repeats. In this case the exact sequence of these regions could not be determined due to the inability of current DNA
sequencing techniques to cover the region in one run. Internal sequencing primers aie excluded as they prime at multiple sites within the gene. Also, the relative orientation of these two genes, their long and very high sequence similarity makes them potential targets for host recombination.
While the topology-presented here has been confirmed by PCR with appropriate primers, the genome is very probably a product of such a recombination event as implied by 'the relative positions of the origin and termination of replication. A second problem encountered with the .ribosomal operon repeats is that the presence of 6 operons at only 4 loci had been identified, and the exact location of their positions of the extra loci was noly difficult to achieve. Finally, two of the IS elements are present in multiple copies, and depending on their replative orientations, they may be .targets for host recombination. Such an event has been identified by studying the sequences flanking the IS element, and specifically the chromosomal target sequence that is duplicated on transposition, and hence each IS element should be flanked by identical direct repeats. We have identified twa, IS elements where the direct repeats have.
been switched due to host recombination within the IS elements. This produces an approximately 600'000 by inversion that has been confirmed by PCR with specific primers. This IS element specific recombination may be a dynamic event that is taking place within a growing culture, leading to a major species plus a small presence of the recombined genome (seen as a faint PCR band).
Finally we have the case of the prophage L771 (approximately 40'000 bp) that is constantly being excissed by a site-specific recombinase. We have developed a quantitative PCR technique to detect the presence and measure the relative abundance of each variant. No pure cultures have been prepared to date.
Particularly preferred fragments of the nucleic acid sequence as identified by SEQ. ID. No. 1 are from 1 - 54596, from 56070 - 77430, from 81302- 308537, from 309588 - 342757, from 378458 - 389217, from 389779 - 404510, from 405561 - 501116, from 503873 -558194, from 563262 - 696518, from 697569 - 721736, from 722787 - 756845, from 761682 -860446, from 860723 - 865550, from 867260 - 867490, from 868541 - 1448288, from 1463851 - 1526077, from 1527278 - 1552024, from 1563147 - 1809115, from 1858190, from 1863258 - 1872871, from 1877939 - 1930430, from 1932063 -1983043, based on the numbering of SEQ ID. No. 1, each.
The present invention may also be utilized for producing polypeptides by using the knowledge of open reading frames (ORFs) as derived from SEQ. ID. NO. 1 and expressing the polypeptide desired according to well known techniques. In this respect, a nucleic acid corresponding to an open reading frame may be selected and inserted into an expression vector. The vector may then be introduced into a host, that enables transcription and translation of the open reading frame into the polypeptide under suitable conditions.
Nucleic acid molecules derived from the genomic sequence as identified by SEQ.
ID. NO. 1 may easily be obtained, by e.g. specific amplification of the corresponding sequence using the polymerase chain reaction. Due to the sequence information provided herein the skilled person may design and synthesize any suitable primer nucleotide and amplify a fragment of interest using the polymerase chain reaction. Therefore, the present invention also comprises nucleotide sequences selected from sequence SEQ. ID. NO. 1 which~can be used as a primer for the amplification of nucleic acid sequences. Other techniques for amplifying the target nucleic acid may of course also be used, such as e.g. the TAS (Transcription-based Amplifi .., cation System) technique, the 3SR (Self Sustained Sequence Replication) technique, the NASBA (Nucleic Acid Sequence Based Amplification) technique, the SDA (Strand Dis-placement Amplification) technique or the TMA (Transcription Mediated Amplification) technique etc..
The (poly)nucleotides may be used as probes and techniques for amplifying or modifying a nucleic acid serving as a probe, such as e.g. the LCR (Ligase Chain Reaction) technique, the RCR (Repair Chain Reaction) technique, the CPR (Cycling Probe Reaction) technique or the Q-beta-replicase amplification technique may well be applied.
The present invention, therefore, envisages both hybridization (detection) probes and primers for detecting a nucleotide sequence (target nucleotide) of the present invention. In the case of the target being a RNA molecule, e.g. a mRNA, said mRNA may be directly detected or transformed to a cDNA prior to detection.
Alternatively, in order to obtain fragments of the nucleic acid represented by SEQ. ID. NO. l the Lactobacillus johnsonii genomic DNA may be subjected to digestion with selected restriction enzymes, with the fragments being separated by e.g.
electrophoresis or another suitable separation technique. Such techniques are well known in the art and are inter alia disclosed in Sambrook et al. A Laboratory Manual, Cold Spring Harbor, 1992.
Such fragments may easily be obtained by isolating the genomic DNA of Lactobacillus johnsonii Lal and performing the necessary steps.
In an alternative form the nucleic acids may also be obtamect by cnerrucat synthesis when they are not too large in size according to methods well known to a person skilled in the art.
Modified nucleotide sequences shall be understood to mean any nucleotide sequence ob-tained by mutagenesis according to techniques well known to a skilled person and exhibiting modifications in relation to the normal sequences, for example mutations in the regulatory and/or promoter sequences for the expression of a polypeptide, in particular leading to a modification of the level of expression of the said polypeptide or to a modulation of the replicative cycle. Modified nucleotide sequence will also be understood to mean any nucleotide sequence encoding a modified polypeptide as defined below.
During the study of the Lactobacillus johnsonii genome the following open reading frames could be determined with an annotation of the function of the resulting polypeptide being possible on the basis of homology to known proteins.
Table I
Gene StartStop Complement*% Function LJ_0008 9756 9992 84,8Ribosomal protein S18 (78 as*) LJ 0043 5284854002 complement76,4INOSINE-5'-MONOPHOSPHATE DEHYDROGENASE
(384 aa) (EC 1.1.1.205) LJ_0045 5472855741 complement96,7D-lactate dehydrogenase (EC 1.1.1.28) (337 aa) LJ 0054 7567176585 93 Prolinase prolyl aminopeptidase (EC
(304 aa) 3.4.11.5) LJ_0056 7746578415 complement99 Conjugated bile salt hydrolase (EC
(316 aa) 3.5.1.24) LJ 0057 7843179786 complement88 Putative bile salt transporter (451 aa) LJ 0058 7981081168 complement81,4Putative bile salt transporter (452 aa) LJ 0065 8781688523 74,6Response regulator (235 aa) LJ 0124 146032146481complement80,4Nucleoside deoxyribosyltransferase-II
(149 aa) (EC 2.4.2.6) LJ 0178 211269212579complement71,1Aminopeptidase G (EC 3.4.22.-) (436 aa) LJ 0182 214451215899complement98,76-PHOSPHO-BETA-GLUCOSIDASE (EC 3.2.1.86) (482 aa) LJ 0215 248929250032 70,2Multiple sugar-binding transport ATP-binding (367 aa) protein msn~.
(EC 2.7.1.69) LJ 0229 264155265708 ~ GMP synthase [glutamine-hydrolyzing (51? aa) 75,2(EG 6.3.5.2) LJ 0258 287474288889complement85,9Dipeptidase A (EC 3.4.*.*) (471 aa) LJ 0260 290018291979 75 Raffinose carrier protein (RAFFINOSE
(653 aa) PERMEASE) LJ_0262 294170295612 71,6sucrose phosphorylase (EC 2.4.1.7) (480 aa) LJ 0274 307454308425complement84,4L-lactate dehydrogenase (EC 1.1.1.27) (323 aa) LJ_0295 332973333722 79,7ORF 169a (prophage protein) (249 aa) LJ 0307 343364344218 100temunase small subunit (prophage protein) (284 aa) LJ 0308 344205345479 100orf345; terniinase large subunit (prophage (424 aa) protein) LJ_0309 345495346994 99,7orf500; putative portal protein (prophage (499 aa) protein) LJ_0311 347218348300 100orf360; putative minor head protein (360 aa) (prophage protein) LJ 0312 348455349099; 100orfzl4; scaffold protein (prophage (214 aa) protein) LJ_0313 349112349477 100Orf121 (prophage protein) (121 aa) LJ_0314 349498350547 100orf349; major head protein (prophage (349 aa) protein) LJ 0315 350557350874 99 Orf105 (prophage protein) (105 aa) LJ 0316 350871351224 100Orf117 (prophage protein) (117 aa) LJ 0317 351217351765 99 Orf106 (prophage protein) (182 aa) LJ_0318 351766352134 100Orf122 (prophage protein) (122 aa) LJ 0319 352137352616 100orf159; putative major tail protein (159 aa) (prophage protein) LJ 0320 352694353104 93,3Orf136 (prophage protein) (136 aa) LJ 0321 353197353490 100Orf109 (prophage protein) (97 aa) LJ 0322 353490359555 92,6orf1434; putative minor tail protein (2021 aa) (prophage protein) LJ 0323 359573359929 99 Orf109a (prophage protein) (118 aa) LJ_0324 359943364817 100Orf977 (prophage protein) (1624 aa) LJ 0325 364949365209 100Orf86 (prophage protein) (86 aa) LJ 0326 365209365616 100Orf135 (prophage protein) (135 aa) LJ 0327 365626365883 88,2Orf85 (prophage protein) (85 aa) LJ 0328 365876366223 100orfl 15; putative holin (prophage (115 aa) protein) LJ 0329 366216367148 99,6orf376; lysin (prophage protein) (310 aa) LJ 0332 370820374449 70,9rpoB; RNA polymerase (beta subunit) (1209 aa) (EC 2.7.7.6) LJ_0333 374470378144 70 rpoC; RNA polymerase (beta subunit) (1224 aa) (EC 2.7.7.6) LJ 0335 379054379461 85,8RS12; ribosomal protein 512 (135 aa) LJ 0336 379485379955 76,7RS7; 30S ribosomal protein S7 (156 aa) LJ_0337 379985382081 70,7translation elongation factor G, EF-G
(698 aa) LJ 0339 382686383315 73,4rplC; 505 ribosomal' protein L3 (209 aa) LJ 0342 384263385099 75,1rplB; 505 ribosomal protein L2 (278 aa) LJ 0343 385121385408 81,1rpsS; 30S ribosomal protein 519 (95 aa) LJ 0344 385429385782 75,4rplV; ribosomal protein L22 (117 aa) LJ_0345 385800386468 70 30S ribosomal protein S3 (222 aa) LJ 0346 386468386905 84,8ribosomal protein L16 (145 aa) LJ_0347 387118387384 72 30S RIBOSOMAL PROTEIN S17 (88 aa) LJ 0348 387415387783 72;9rplN; ribosomal protein LI4 (122 aa) LJ 0350 388058388600 79,7RLS; ribosomal protein L5 (BL6) (180 aa) LJ 0351 388825389223 70,4rpsH; 30S Ribosomal protein S8 (132 aa) LJ 0352 389248389778 98,2lecLA2-20; lectin-like protein LA2-20 (176 aa) LJ 0353 389806390165 72,2rplR; SOS ribosomal protein LI8 (119 aa) LJ_0358 393402393623 84,5Translation initiation factor IF-1 (73 aa) LJ 0359 393788394135 73,6rpsM; ribosomal protein 513 (115 aa) LJ 0360 394160394549 73 rpsK; 305 Ribosomal protein S11 (129 aa) LJ 0362 395560395943 73,2rplQ; 50S Ribosomal protein L17 (127 aa) (131 aa) LJ_03.95 441168442517complement81,5Aminopeptidase C (EC 3.4.22.40) (449 aa) LJ 0399 445878447377 73,5Glutamyl-tRNA synthetase (EC 6.1.1.17) (499 aa) LJ_0410 458506458931 77 505 ribosomal protein LI 1 LJ 0441 483735484727complement70,6GMP reductase (EC 1.6.6.8) (330 aa) LJ 0460 501772502056 99 GroES chaperone (94 aa) LJ 0461 502087503718 99 GroEL chaperone (543 aa) LJ 0490 543751544857complement75,2pepQ; 3Caa-Pro dipeptidase (EC 3.4.13.9) (368 aa) LJ 0493 548984550381complement71,6pepV; Xaa-His dipeptidase (EC 3.4.13.3) (465 aa)' LJ_0505 565393566322 73,1Mannose-specific phosphotransferase (309 aa) system comp. IID (EC 2.7.1.69) LJ_0521 583645585255 79 Putative ABC transporter (536 aa) L7 0563 623532624218 83,7putative response regulator (228 aa) L,J 0631 705767706243complement74 Autoinducer protein luxS
(158 aa) LJ 0677 762315763523 70 metK; S-adenosylmethionine synthetase (402 aa) (EC 2.5.1.6) LJ 0764 859739861046 100 putative sensor histidine kinase (435 aa) LJ 0767 863354865513 77,9Sequence from patent (719 aa) LJ_0768 865524866117 91,4Lacticin F transporter accesory protein {197 aa) LJ_0769 866244866471 98,6Bacteriocin lactacin F, subunit IafA
(75 aa) precursor LJ_770 (62 866485866671 100 Bacteriocin lacticin F, subunit lafX
aa) precursor LJ_0771 866757867131 90,3Bacteriocin lacticin F immunity protein, (124 aa) IatI
LJ 0775 869095871254 77,9Sequence from patent (719 aa) LJ 0776 871265871858 77,1Hypothetical protein (197 aa) LJ 0817 910392910658 79,5Phosphocarrier protein HPr (88 aa) LJ 0827 918499920274 71 Sequence from patent (591 aa) LJ 0840 932899933981 72 reeA; Recombinase A
(360 aa) LJ_0846 939718940263 80,1Hypothetical protein (181 aa) LJ_0847 940421942820 81,9preprotein translocase SecA subunit (799 aa) LJ 0848 943020944018 72,5peptide chain release factor 2 (332 aa) LJ_0853 947228948163 71,8trxB; THIOREDOXIN REDUCTASE (EC 1.6.4.5) (311 aa) LJ 0855 949277950230complement71 lacM; Beta-galactosidase small subunit (317 aa) (EC 3.2.1.23,) LJ 0856 950211952091complement75,2lacL; Beta-galactosidase large subunit (626 aa) (EC 3.2.1.23) LJ_0860 957611958780 76,8galK; Galactokinase (EC 2.7.1.6) (Galactose (389 aa) kinase) LJ 0861 958799960286 74,7gall; Galactose-1-phosphate uridylyltransferase (495 aa) (EC 2.7.7.10) LJ 0864 963226965241 76,9uvrB; EXCINUCLEASE ABC SUBUNIT B
(671 aa) LJ_0870 971745972332complement70 clpP, ATP-dependent Clp protease proteolytic (195 aa) subunit (EC 3.4.21.92) LJ 0873 975442976458~ 87,8gapdh; Glyceraldehyde 3-phosphate (338 aa) dehydrogenase(EC 1.2.1.12) LJ 0874 976565977776 84,3pgk; Phosphoglycerate kinase (EC 2.7.2.3) (403 aa) LJ 0875 977795978550 84,8tim; Triosephosphate isomerase (EC
(251 aa) 5.3.1.1) LJ_0876 97860097989$ 71 Enolase (EC 4.2.1.11) (2-phosphoglycerate dehydratase) LJ_0925 10230891024432 72,5Glucose-6-phosphate isomerase (EC
(447 aa) 5.3.1.9) LJ 0934 10329681033579 73,7uracil phosphoribosyltransferase (EC
(203 aa) 2.4.2.9) LJ 0936 10344251034637 73,9atpE; F1FO~ATPase subunit c (EC 3.6.1.34) {70 aa) LJ 0937 10346901035190 70 atpF; F1F0-ATPase subunit b (EC 3.6.1.34) (166 aa) LJ 0939 10357501037261 84,8atpA; F1F0-ATPase subunit alpha (EC
(503 aa) 3.6.1.34) I
LJ_0941 10382581039700 84,7atpD; F1F0-ATPase subunit beta (EC
(480 aa) 3.6.1.34) LJ 0954 10492121050366 70,8nifS; pyridoxal-phosphate dependent (384 aa) aminotransferase (EC 4.4.1.- ).
(458 aa) LJ 0996 10939851095841 70 elongation factor Tu family protein (618 aa) LJ 1007 (89 11039991104268 71,9rps0; 30S ribosomal protein S15 aa) LJ_1010 (39611072391108429 '74EF-Tu; Elongation factor Tu aa) LJ 1033 (37211292761130394 85,8Sequence from patent aa) LJ_1079 (31911813841182343 76,1K6PF; 6-phosphofructokinase (EC
2.7.1.11) aa) LJ_1080 (58911823781184147 83 pyk; Pyruvate kinase (EC 2.7.1.40) aa) LJ 1092 (91 11937101193985 77,5hu; DNA-binding protein II
aa) LJ 1111 (17412153821215906 83,9hslU;heatshockinduced protein HtpI
aa) LJ 1112 (46412159171217311. 76,1HSLU; ATP-dependent hsl protease ATP-binding aa) subunit hslU.
LJ 1138 264 12555821256376 70 ABC transporter ATP-binding protein aa).
LJ_1170 (66112878171289802 71 topoisomerase IV B subunit (EC 5.99.1.*) aa) -LJ_1200 (43213242751325573complement83,5asnAl; Asparaginyl-tRNA synthetase aa) (EC 6.1.1.22) LJ 1207 (35713380651339138 70,1pmk; phosphomevalonate kinase (EC
2.7.1.36) aa) LJ 1298 (75 14252441425471 71,8tpnA; transposase, fragment only aa) LJ 1303 (41.514285751429822complement85,4pepT, PEPTIDASE T (EC 3.4.11: ) (aminotripeptidase) aa) (tripeptidase) LJ 1304 (26514298341430631complement81,1Hypothetical protein aa) LJ 1317 (37214410161442134complement81,4rpoD; RNA polymerase sigma factor rpoD
aa) (Sigma-42) LJ_1320 (30514460501446967complement70,4glyQ; Glycyl-tRNA synthetase alpha aa) chain (EC 6.1.1.14) LJ_1389 (14214571281457556complement71,7Peptide methionine sulfoxide reductase aa) (EC 1.8.4.6) LJ 1356 (32614849031485883complement99 conjugated bile salt hydrolase bile aa) (EC 3.5.1.24) LJ_1384 (47015103221511734complement100orf338; putative portal protein (prophage aa) protein) LJ 1385 (42215117461513014complement100orf42; terminase large subunit (prophage aa) protein) LJ 1386 (15115130071513462complement100orf155; terminase sriiall subunit (prophage aa) protein) LJ 1387 (21815135191514175complement100Orf221 (prophage protein) aa) LJ 1388 (17415143571514881complement90,5Orf174 (prophage protein) aa) LJ_1389 (14615159251516365complement100Orf154 (prophage protein) aa) LJ 1390 (73 15164541516675complement100OrfbS (prophage protein) ' aa) LJ 1391 (18415166951517249complement93,4Orf197 (prophage protein) aa) LJ 1392 (13215172511517649complement82,6Orf79 (prophage protein) aa) LJ 1393 (71 15176501517865complement96,5Orf78a (prophage protein) aa) LJ_1394 (29615180251518915complement98,1Ort212 (prophage protein) aa) . , LJ 1395 (26115189281519713complement.93,9Orf223 (prophage protein) aa) LJ_1396 (29715197151520608complement100Orf309 (prophage protein) aa) LJ 1397 (71 15212851521500complement100Orf73 (prophage protein) aa) LJ 1415 (31815340641535020complement71,4thyA; thymidylate synthase (EC
2.1.1.45) aa) LJ_1423 (62415454101547284complement85,3dnaK; heat shock protein DnaK
aa) LJ_1431 (88015537961556438complement70,3IF2; Translation initiation factor aa) IF-2.
LJ 1442 (24115682881569013complement70,5pyres; UMP-kinase (EC 2.7.4.-) aa) LJ_1444 (26115701861570971complement75,6RS2; 30S ribosomal protein S2.
aa) LJ 1446 (12515810601581437complement76,1Khl9; 505 ribosomal protein L19.
aa) LJ 1447 16057461606000 80 RL28; 50S ribosomal protein L28.
(84 aa) LJ 1429 16567251659109 71,5pepX; Xaa-Pro dipeptidyl-peptidase (794 aa) (EC 3.4.14.11) LJ~,1537 16661621667067complement71,2galU; UDP-glucose pyrophosphorylase, (301 aa) (EC 2.7.7.9) LJ 1558 16868631688200complement72,8Glutamine synthetase (EC 6.3.1.2) (445 aa) (Glutamate--ammonia ligase) LJ 1584 17133301713686complement70,7RL20; 50S ribosomal protein L20 (118 aa) LJ_1681 18269011827287 72,6tagD; Glycerol-3-phosphate cytidylyltransferase (128 aa) (EC 2.7.7.39) LJ_1741 19029451903592complement72,5Pyrrolidone-carboxylate peptidase (215 aa) (EC 3.4.19.3) I
LJ 1767 19306101931257 100deoxyadenosine ltinase (EC 2.7.1.76) (215 aa) j LJ 1768 19312791931953 99,1deoxyguanosine kinase (EC 2.7.1.113) (224 aa) * complement = on the reverse strand * as = amino acids The ORFs corresponding to various poplypeptides are. shown in table 1, supra, and are represented by their position in the genomic sequence as identified by SEQ.
ID. NO. 1.
The open reading frames.have been identified via homology analyses as well as via analyses of potential ORF start sites. It is to be understood that each identified ORF
comprises a nucleotide sequence that spans the contiguous nucleotide sequence from the codon immediately 3' to the stop codon of the preceding ORF and through the 5' codon to the next stop codon of SEQ. ID. NO. 1 in frame to the ORF nucleotide sequence.
Table 1 also depicts the results of homology searches that compared the sequences of the polypeptides encoded by each of the ORFs to sequences present in databases.
The sequence information disclosed in the present application may be utilized for selecting a r polynucleotide of interest, i.e. a nucleic acid containing an open reading frame encoding a known or an unknown, putative polypeptide and transforming micro-organisms with the selected polynucleotide. As transformation vehicles the well known plasmids, phage vectors (trans-fection) or F-vectors (conjugation) may be utilized. The nucleic acid introduced into the micro-organism selected may be expressed and its biological function may be either utilized as such, if known, or elucidated, in case a so far unknown polypeptide is expressed. The micro-organism selected may be a Lactobacillus itself or other well known micro-organisms, such as bacteria, e.g.
E.coli, Streptococci or yeast, insect cells or even animal and plant cells.
It will be understood that the polypeptides may be expressed as such or as a fusion polypeptide.
The skilled person is well aquatinted with techniques performing such a ligation and expressing the corresponding fusion-polypeptide in an appropriate cell.
In view of the present invention also new recombinant vectors for the cloning and/or the expression of a nucleotide sequence according to the present invention may be devised. The vectors comprise elements necessary to enable expression and/or secretion of the nucleotide sequences 'in a given host cell, such as a promoter, signals for initiation and for termination of translation, as well as appropriate regions for regulation of transcription.
For example, expression of a protein or peptide may be controlled by any promoter/enhancer element known. in the art.
Exemplary promotors are the CMV promoter, the SV40 early promoter region, the promoter contained in the 3' long terminal repeat of the roux sarcoma virus, the herpes thymidine kinase promoter, the regulatory sequences of the metallothionein gene, or, for prokaryotic expression systems, the 13-lactamase promoter, the tac promoter or the T7 promoter.
The vector should be capable of being stably maintained in the host cell and may optionally possess particular signals specifying the secretion of the translated protein.
These different elements are chosen according to the host cell utilized. To this effect the nucleotide sequences according to the invention may be inserted into autonomously-replicating vectors within the chosen host, or integrative vectors in the chosen host, such as e.g. yeast artificial chromosomes, plasmids or viral vectors.
Any of the standard methods known to those skilled in the art for inserting DNA fragments into a vector may be used to construct expression vectors containing a chimeric gene consisting of appropriate transcriptional/trans'lational control signals and the protein coding sequences. These methods may include in vitro recombinant DNA and synthetic techniques and in vivo recombinants (genetic recombination).
The vector may be used for transcription and/or translation of a nucleic acid comprised in SEQ.
ID. NO. l, to produce RNA or antisense RNA, respectively. Such a vector can remain episomal or become chromosomally integrated, as long as it can be transcribed to produce the desired transcript.
The antisense nucleic acids of the invention comprise a sequence complementary to at least a portion of a RNA transcript of a polynucleotide sequence in SEQ. >D. NO. 1, designating a sequence having sufficient complementarity to be able to hybridize with the RNA, forming a stable~duplex. In the case of double-stranded antisense nucleic acid sequence, a single strand of the duplex DNA may thus be tested, or triplex formation may be assayed.
In knowledge of the present invention also host cells may be obtained transformed with a nucleic acid or a vector according described herein. These cells may be attained by introducing into an appropriate cell a nucleotide sequence or a vector as defined above, and then culturing the said cell under conditions allowing the replication andlor the expression of the transformed/trans-fected nucleotide sequence.
The host cell may be chosen from eukaryotic or prokaryotic system, such as for example bacterial cells, yeast cells, animal cells as well as plant cells. In the context of this invention a cell shall be understood to comprise higher biological systems. Such as animals, whole plants or parts thereof.
Furthermore, a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired.
A preferred host cell for the expression of the proteins of the invention consists of prokaryotic cells, such as gram negative or gram positive bacteria. A further preferred host cell .according to the invention is a bacterium belonging to the Lactobacillus family, more preferably belonging to the species Lactobacillus johnsonii or chosen from a microorganism associated with the species Lactobacillus.
The transformed/transfected cells according to the invention may advantageously serve as a model and may be used in methods for studying, identifying and/or selecting compounds capable of being responsible for any of the beneficial effects brought about by the present Lactobacillus strain.
The invention further enables the synthesis of polypeptides encoded by the Lactobacillus johnsonii ORFs, in particular those listed in table 1. In the present description, the terms polypeptide, peptide and protein are used interchangeably. Furthermore the present invention also enables to carry out method for preparing such polypeptides by recombinant means comprising the steps of , (a) culturing a host cell according to the present invention under conditions suitable to produce the polypeptide encoded by the polynucleotide;
and (b) recovering the polypeptide from the culture.
It will be appreciated that the above polypeptides may also be obtained using combinatory chemistry, wherein the polypeptide is modified at some locations before testing them in model systems, so as to select the compounds which are the most active or which exhibit the desired properties.
In this context, chemical synthesis has the advantage of being able to use non-natural amino acids or non-peptide bonds. Accordingly, in order to e.g. extend the life of the polypeptides according to the invention, it may be advantageous to use such non-natural amino acids, for example in the D form, or alternatively amino acid analogues, preferably sulphur-containing forms.
Finally, the structure of the polypeptides according to the invention, its homologous or modified forms, as well as the corresponding fragments may be integrated into chemical structures of the polypeptide type and the like. Accordingly, in order to preserve the polypeptide in an in vivo environment it will be preferred to provide at the N- and C-terminal ends compounds which convey a resistance to degradation to proteases.
It will also be appreciated that the different polypeptides according to the present invention and produced by the above method may represent antigens to the immune system of a host animal, so that antibodies may be produced directed against said polypeptides. These antibodies may be used for the detection of a polypeptide of interest in a mixture or generically of a strain of Lactobacillus in a sample. In addition they may be used as research tools by e.g. producing antibodies against cellular surface epitopes and determining the effect of blocking certain polypeptides on the bacterial cell wall.
According to another aspect the present invention also provides a method for the detection and/or identification of Lactobacilli, preferably Lactobacillus johnsonii in a biological sample. This method may comprise several techniques known in the art, such as PCR or simply hybridization with a suitable probe. Alternatively, an antibody raised against a cell wall epitope of Lactobacillus, preferably Lactobacillus johnsonii may be used for said purpose. It will be appreciated that the above method may also be reversed and the presence of antibodies against Lactobacillus may be determined by contacting the sample to be tested with a polypeptide of Lactobacillus under conditions to allow formation of immune complexes.
The polypeptides and antibodies obtainable in knowledge of the present invention and the nucleotide sequences described herein may be used in in vitro and/or in vivo methods for the detection and/or the identification of bacteria belonging to the species Lactobacillus in a biological sample (biological tissue or fluid) which is likely to contain them. These methods, depending on the specificity of the polypeptides, of the antibodies and of the nucleotide sequences described herein, which will be used, may detect and/br identify the bacterial variants belonging to the species Lactobacillus as well as associated microorganisms capable of being detected by the polypeptides, the antibodies and the nucleotide sequences according to the invention which will be chosen. It may, for example, be advantageous to choose a polypeptide, an antibody or a nucleotide sequence according to the invention, which is capable of detecting any bacterium of the Lactobacillus family by choosing a polypeptide, an antibody and/or a nucleotide sequence according to the invention which is specific to the family.
The sequences referred to herein SEQ ID. NO. 1 is listed in the attached sequence listings which is to be considered as part of the specification.
The invention also comprises, the nucleotide sequences or polypeptides according to the invention covalently or non-covalently immobilized on a solid support. In the first case such a support may serve to capture, through specific hybridization, the target nucleic acid obtained from a biological sample to be tested. If necessary, the solid support is separated from the sample and the hybridization complex formed between the capture probe and the target nucleic acid is then detected by means of a second probe, called detection probe, labeled with an easily detectable element.
Such support may take the form of so-called DNA array or DNA chips, a multitude of molecular probes precisely organized or arrayed on a solid support, which will allow sequencing genes, studies of mutations contained therein and the expression of genes, and which are currently of interest given their very small size and their high capacity in terms of number of analyses.
The function of these arrays/chips is based on molecular probes, mainly oligonucleotides which are attached to a carrier having a size of generally a few square centimetres or more as desired. For an analysis the carrier (DNA array/chip) is coated with probes that are arranged at a predetermined location of the carrier. A sample containing fragments of a target nucleic acid to be analyzed, for example DNA or RNA or cDNA, that has been labeled beforehand, is subsequently contacted with the DNA array/chip leading to the formation;
through hybridization, of a duplex. After a washing step, analysis of the surface of the chip allows the effective hybridization to be located by means of the signals emitted by the labels tagging the target. A hybridization fingerprint results from this analysis which, by appropriate computer processing, allows to retrieve information such as the expression of genes, the presence of specific fragments in the sample, the determination of sequences and the presence of mutations.
The hybridization between the probes of the invention, deposited or synthesized in situ on the DNA chips, and the sample to be analyzed, may, e.g. be determined by means of fluorescence, radioactivity or by electronic detection.
The nucleotide sequences according to the invention may be used in DNA
arrays/chips to carry out analyses of the expression of the Lactobacillus genes. This analysis is based on DNA arrays/chips on which probes, chosen for their specificity to characterize a given gene, are present. The target sequences to be analyzed are labeled before being hybridized onto the chip. After. washing the labeled compounds are detected and quantified, with the hybridization being carried out at least in duplicate. Comparative analyses of the signal intensities obtained with respect to the same probe for different samples and/or for different probes with the same sample, determine a differential transcription of RNA derived from the sample.
The DNA arrays/chips according to the present invention may also contain nucleotide probes specific for other microorganisms, which will enable a serial testing allowing rapid identification of the presence of a microorganism in a sample.
The principle of the DNA chip, as detailed above may also be used to produce-protein chips on which the support has been coated with a polypeptide or an antibody according to the invention, or arrays thereof, in place of the DNA. These protein chips make it possible to analyze the biomolecular interactions (BIA) induced by the affinity capture of target analytes onto a support coated e.g. with proteins, by surface plasma resonance (SPR).
The polypeptides or antibodies according to the invention, capable of specifically binding anti-bodies or polypeptides derived from the sample to be analyzed, may thus be used in protein chips for the detection and/or the identification of proteins in samples.
The present invention also relates to a computer readable medium having recorded thereon one or more nucleotide and/or a polypeptide sequences according to the invention. This medium may also comprise additional information extracted from the present invention, such as e.g. analogies with already known sequences and/or information relating to the nucleotide and/or polypeptide sequences of other microorganisms so as to facilitate the comparative analysis and the exploitation of the results obtained. Preferred media are e.g. magnetic, optical, electrical and hybrid media such as, for example, floppy disks, CD-ROMs or recording cassettes.
The invention also relates to kits or sets for the detection and/or the identifi-ration of bacteria belonging to the species Lactobacillus johnsonii or to associated microorganisms, which comprises, a polypeptide according to the invention, where appropriate, the reagents for constituting the medium appropriate for the immunological or specific reaction, the reagents allowing the detection of the antigen-antibody complexes produced by the immunological reaction between the polypeptide (s) of the invention and the antibodies which may be present in the biological sample, it being possible for these reagents also to carry a label, or to be capable of being recognized in turn by a labeled reagent, more particularly in the case where the polypeptide according to the invention is not labeled, a reference biological sample (negative control) free of antibodies recognized by a polypeptide according to the invention, a reference biological sample (positive control) containing a predetermined quantity of antibodies recognized by a polypeptide according to the invention.
The invention also relates to a kit or set for the detection and/or the identification of bacteria belonging to the species Lactobacillus johnsonii or to an associated microorganism; or for the detection and/or the identification of a microorganism, wherein the kit comprises a protein chip according to the invention.
In one aspect the present invention relates to the use of a nucleotide sequence of the lactic acid bacterium Lactobacillus johnsonii Lal genome having the sequence SEQ. m. NO.
l, parts thereof or sequences homologous thereto for elucidating interactions between bacteria and a host, preferably lactic acid bacteria and a host, more preferably lactobacilli and a host, in particular for determining factors accounting for the probiotic properties of such strains.
In the context of this application the terms genome or genomic sequence shall be understood to mean the sequence of the chromosome of Lactobacillus johnsonii. The terms nucleotide sequence, polynucleotide or nucleic acid shall designate a double-stranded DNA, a single-stranded DNA or transcriptional products of the said DNAs of various length including oligo-nucleotides of,about 5 to 200, preferably 10 to 100 nucleotides in length.
According to the present invention a homologous nucleotide sequence is understood to mean a nucleotide sequence having a percentual identity with the sequence of SEQ ID.
No. 1 (or selected parts thereof) of at least 90 %, preferably at least 95 %, more preferably 96 % and even more preferably at least 98%. The said homologous rnay comprise, e.g., sequences corresponding to the genomic sequence or to the sequences of fragments thereof belonging to the species Lactobacillus, more preferably to the species Lactobacillus johnsonii, as well as the sequences corresponding to the genomic sequence or to the sequences of its representative fragments of a bacterium belonging to related species. In the present invention, the terms species and genus are mutually interchangeable.
These homologous sequences may thus correspond to variations linked to mutations within the same species or between species and may correspond in particular to truncations, substitutions, deletions and/or additions of at least one nucleotide. The said homologous sequences may also correspond to variations linked to the degeneracy of the genetic code or to a bias in the genetic code which is specific to the family, to the species or to the variant and which are likely to be present in Lactobacillus.
Protein and/or nucleic acid sequence homologies may be evaluated using any of the variety of sequence comparison algorithms and programs known in the art. Such algorithms and programs include, but are by no means limited to, TBLASTN, BLASTP, FASTA, TFASTA, and CLUSTALW (see e.g. Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85 (8): 2444-2448; Altschul et al., 1990, J. Mol. Biol. 215 (3) : 403-410; Thompson et al., 1994, Nucleic Acids Res. 22 (2): 4673-4680; Higgins et al., 1996, Methods Enzymol. 266: 383-402; Altschul et al., 1990, J. Mol. Biol: 215 (3) : 403 - 410; Altschul et al., 1993, Nature Genetics 3: 266-272).
In a particularly preferred embodiment, protein and nucleic acid sequence homologies are evaluated using the Basic Local Alignment Search Tool ("BLAST") which is well_known in the art (supra). In particular, four specific BLAST programs have been used to perform the following task:
(1) BLASTP: Compares an amino acid query sequence against a protein sequence database (2) BLASTN: Compares a nucleotide query sequence against a nucleotide sequence database (3) BLASTX: Compares a nucleotide query sequence translated in all reading frames against a protein sequence database (4) TBLASTN: Compares a protein query sequence against a nucleotide sequence database dynamically translated in all reading frames Among these representative fragments, those capable of hybridizing under stringent conditions with a nucleotide sequence disclosed in the present invention are preferred.
Hybridization under stringent conditions means that the temperature and ionic stiength conditions are chosen such that they allow hybridization to be maintained between two complementary DNA
fragments. Such conditions off, high stringency may e.g. be achieved, by carrying out the hybridisation at a preferred temperature of 65 °C in the presence of SSC buffer, e.g. 1 x SSC
corresponding to 0.15 M NaCI and 0.05 M Na-citrate. The washing steps may be, for example, the following: 2 x SSC, 0.1 ~o SDS at room ,temperature followed by three washes with 1 x SSC, 0.1 % SDS; 0.5 x SSC, 0.1 % SDS; 0.1 x SSC, 0.1 % SDS at 68 C
for 15 minutes.
The nucleotide sequences SEQ. m. NO. 1 has been obtained by sequencing the genome of Lactobacillus johnsonii Lal by the method of directed sequencing after fluorescent automated se-quencing of the inserts of clones and assembling of these sequences of nucleotide fragments (inserts) by means of softwares. To this end, fragments of the genome were created, ligated into suitable vectors for amplification and propagation and the corresponding fragments were sequenced. Overlaps and the final arrangement of the fragments, the nucleotide sequence thereof, were assessed by the aid of appropriate softwares.
Clones for sequencing also included 10'000 by plus fragments as BAC clones that were used to provide a larger scale framework to the assembly. Due to the presence of several repeated regions a correct assembly proved extremely difficult. These included especially the repeated regions such as IS elements, the ribosomal operons and specifically the genes for two large cell surface proteins that contain between 100 and 200 almost perfect 1.0 amino acid repeats. In this case the exact sequence of these regions could not be determined due to the inability of current DNA
sequencing techniques to cover the region in one run. Internal sequencing primers aie excluded as they prime at multiple sites within the gene. Also, the relative orientation of these two genes, their long and very high sequence similarity makes them potential targets for host recombination.
While the topology-presented here has been confirmed by PCR with appropriate primers, the genome is very probably a product of such a recombination event as implied by 'the relative positions of the origin and termination of replication. A second problem encountered with the .ribosomal operon repeats is that the presence of 6 operons at only 4 loci had been identified, and the exact location of their positions of the extra loci was noly difficult to achieve. Finally, two of the IS elements are present in multiple copies, and depending on their replative orientations, they may be .targets for host recombination. Such an event has been identified by studying the sequences flanking the IS element, and specifically the chromosomal target sequence that is duplicated on transposition, and hence each IS element should be flanked by identical direct repeats. We have identified twa, IS elements where the direct repeats have.
been switched due to host recombination within the IS elements. This produces an approximately 600'000 by inversion that has been confirmed by PCR with specific primers. This IS element specific recombination may be a dynamic event that is taking place within a growing culture, leading to a major species plus a small presence of the recombined genome (seen as a faint PCR band).
Finally we have the case of the prophage L771 (approximately 40'000 bp) that is constantly being excissed by a site-specific recombinase. We have developed a quantitative PCR technique to detect the presence and measure the relative abundance of each variant. No pure cultures have been prepared to date.
Particularly preferred fragments of the nucleic acid sequence as identified by SEQ. ID. No. 1 are from 1 - 54596, from 56070 - 77430, from 81302- 308537, from 309588 - 342757, from 378458 - 389217, from 389779 - 404510, from 405561 - 501116, from 503873 -558194, from 563262 - 696518, from 697569 - 721736, from 722787 - 756845, from 761682 -860446, from 860723 - 865550, from 867260 - 867490, from 868541 - 1448288, from 1463851 - 1526077, from 1527278 - 1552024, from 1563147 - 1809115, from 1858190, from 1863258 - 1872871, from 1877939 - 1930430, from 1932063 -1983043, based on the numbering of SEQ ID. No. 1, each.
The present invention may also be utilized for producing polypeptides by using the knowledge of open reading frames (ORFs) as derived from SEQ. ID. NO. 1 and expressing the polypeptide desired according to well known techniques. In this respect, a nucleic acid corresponding to an open reading frame may be selected and inserted into an expression vector. The vector may then be introduced into a host, that enables transcription and translation of the open reading frame into the polypeptide under suitable conditions.
Nucleic acid molecules derived from the genomic sequence as identified by SEQ.
ID. NO. 1 may easily be obtained, by e.g. specific amplification of the corresponding sequence using the polymerase chain reaction. Due to the sequence information provided herein the skilled person may design and synthesize any suitable primer nucleotide and amplify a fragment of interest using the polymerase chain reaction. Therefore, the present invention also comprises nucleotide sequences selected from sequence SEQ. ID. NO. 1 which~can be used as a primer for the amplification of nucleic acid sequences. Other techniques for amplifying the target nucleic acid may of course also be used, such as e.g. the TAS (Transcription-based Amplifi .., cation System) technique, the 3SR (Self Sustained Sequence Replication) technique, the NASBA (Nucleic Acid Sequence Based Amplification) technique, the SDA (Strand Dis-placement Amplification) technique or the TMA (Transcription Mediated Amplification) technique etc..
The (poly)nucleotides may be used as probes and techniques for amplifying or modifying a nucleic acid serving as a probe, such as e.g. the LCR (Ligase Chain Reaction) technique, the RCR (Repair Chain Reaction) technique, the CPR (Cycling Probe Reaction) technique or the Q-beta-replicase amplification technique may well be applied.
The present invention, therefore, envisages both hybridization (detection) probes and primers for detecting a nucleotide sequence (target nucleotide) of the present invention. In the case of the target being a RNA molecule, e.g. a mRNA, said mRNA may be directly detected or transformed to a cDNA prior to detection.
Alternatively, in order to obtain fragments of the nucleic acid represented by SEQ. ID. NO. l the Lactobacillus johnsonii genomic DNA may be subjected to digestion with selected restriction enzymes, with the fragments being separated by e.g.
electrophoresis or another suitable separation technique. Such techniques are well known in the art and are inter alia disclosed in Sambrook et al. A Laboratory Manual, Cold Spring Harbor, 1992.
Such fragments may easily be obtained by isolating the genomic DNA of Lactobacillus johnsonii Lal and performing the necessary steps.
In an alternative form the nucleic acids may also be obtamect by cnerrucat synthesis when they are not too large in size according to methods well known to a person skilled in the art.
Modified nucleotide sequences shall be understood to mean any nucleotide sequence ob-tained by mutagenesis according to techniques well known to a skilled person and exhibiting modifications in relation to the normal sequences, for example mutations in the regulatory and/or promoter sequences for the expression of a polypeptide, in particular leading to a modification of the level of expression of the said polypeptide or to a modulation of the replicative cycle. Modified nucleotide sequence will also be understood to mean any nucleotide sequence encoding a modified polypeptide as defined below.
During the study of the Lactobacillus johnsonii genome the following open reading frames could be determined with an annotation of the function of the resulting polypeptide being possible on the basis of homology to known proteins.
Table I
Gene StartStop Complement*% Function LJ_0008 9756 9992 84,8Ribosomal protein S18 (78 as*) LJ 0043 5284854002 complement76,4INOSINE-5'-MONOPHOSPHATE DEHYDROGENASE
(384 aa) (EC 1.1.1.205) LJ_0045 5472855741 complement96,7D-lactate dehydrogenase (EC 1.1.1.28) (337 aa) LJ 0054 7567176585 93 Prolinase prolyl aminopeptidase (EC
(304 aa) 3.4.11.5) LJ_0056 7746578415 complement99 Conjugated bile salt hydrolase (EC
(316 aa) 3.5.1.24) LJ 0057 7843179786 complement88 Putative bile salt transporter (451 aa) LJ 0058 7981081168 complement81,4Putative bile salt transporter (452 aa) LJ 0065 8781688523 74,6Response regulator (235 aa) LJ 0124 146032146481complement80,4Nucleoside deoxyribosyltransferase-II
(149 aa) (EC 2.4.2.6) LJ 0178 211269212579complement71,1Aminopeptidase G (EC 3.4.22.-) (436 aa) LJ 0182 214451215899complement98,76-PHOSPHO-BETA-GLUCOSIDASE (EC 3.2.1.86) (482 aa) LJ 0215 248929250032 70,2Multiple sugar-binding transport ATP-binding (367 aa) protein msn~.
(EC 2.7.1.69) LJ 0229 264155265708 ~ GMP synthase [glutamine-hydrolyzing (51? aa) 75,2(EG 6.3.5.2) LJ 0258 287474288889complement85,9Dipeptidase A (EC 3.4.*.*) (471 aa) LJ 0260 290018291979 75 Raffinose carrier protein (RAFFINOSE
(653 aa) PERMEASE) LJ_0262 294170295612 71,6sucrose phosphorylase (EC 2.4.1.7) (480 aa) LJ 0274 307454308425complement84,4L-lactate dehydrogenase (EC 1.1.1.27) (323 aa) LJ_0295 332973333722 79,7ORF 169a (prophage protein) (249 aa) LJ 0307 343364344218 100temunase small subunit (prophage protein) (284 aa) LJ 0308 344205345479 100orf345; terniinase large subunit (prophage (424 aa) protein) LJ_0309 345495346994 99,7orf500; putative portal protein (prophage (499 aa) protein) LJ_0311 347218348300 100orf360; putative minor head protein (360 aa) (prophage protein) LJ 0312 348455349099; 100orfzl4; scaffold protein (prophage (214 aa) protein) LJ_0313 349112349477 100Orf121 (prophage protein) (121 aa) LJ_0314 349498350547 100orf349; major head protein (prophage (349 aa) protein) LJ 0315 350557350874 99 Orf105 (prophage protein) (105 aa) LJ 0316 350871351224 100Orf117 (prophage protein) (117 aa) LJ 0317 351217351765 99 Orf106 (prophage protein) (182 aa) LJ_0318 351766352134 100Orf122 (prophage protein) (122 aa) LJ 0319 352137352616 100orf159; putative major tail protein (159 aa) (prophage protein) LJ 0320 352694353104 93,3Orf136 (prophage protein) (136 aa) LJ 0321 353197353490 100Orf109 (prophage protein) (97 aa) LJ 0322 353490359555 92,6orf1434; putative minor tail protein (2021 aa) (prophage protein) LJ 0323 359573359929 99 Orf109a (prophage protein) (118 aa) LJ_0324 359943364817 100Orf977 (prophage protein) (1624 aa) LJ 0325 364949365209 100Orf86 (prophage protein) (86 aa) LJ 0326 365209365616 100Orf135 (prophage protein) (135 aa) LJ 0327 365626365883 88,2Orf85 (prophage protein) (85 aa) LJ 0328 365876366223 100orfl 15; putative holin (prophage (115 aa) protein) LJ 0329 366216367148 99,6orf376; lysin (prophage protein) (310 aa) LJ 0332 370820374449 70,9rpoB; RNA polymerase (beta subunit) (1209 aa) (EC 2.7.7.6) LJ_0333 374470378144 70 rpoC; RNA polymerase (beta subunit) (1224 aa) (EC 2.7.7.6) LJ 0335 379054379461 85,8RS12; ribosomal protein 512 (135 aa) LJ 0336 379485379955 76,7RS7; 30S ribosomal protein S7 (156 aa) LJ_0337 379985382081 70,7translation elongation factor G, EF-G
(698 aa) LJ 0339 382686383315 73,4rplC; 505 ribosomal' protein L3 (209 aa) LJ 0342 384263385099 75,1rplB; 505 ribosomal protein L2 (278 aa) LJ 0343 385121385408 81,1rpsS; 30S ribosomal protein 519 (95 aa) LJ 0344 385429385782 75,4rplV; ribosomal protein L22 (117 aa) LJ_0345 385800386468 70 30S ribosomal protein S3 (222 aa) LJ 0346 386468386905 84,8ribosomal protein L16 (145 aa) LJ_0347 387118387384 72 30S RIBOSOMAL PROTEIN S17 (88 aa) LJ 0348 387415387783 72;9rplN; ribosomal protein LI4 (122 aa) LJ 0350 388058388600 79,7RLS; ribosomal protein L5 (BL6) (180 aa) LJ 0351 388825389223 70,4rpsH; 30S Ribosomal protein S8 (132 aa) LJ 0352 389248389778 98,2lecLA2-20; lectin-like protein LA2-20 (176 aa) LJ 0353 389806390165 72,2rplR; SOS ribosomal protein LI8 (119 aa) LJ_0358 393402393623 84,5Translation initiation factor IF-1 (73 aa) LJ 0359 393788394135 73,6rpsM; ribosomal protein 513 (115 aa) LJ 0360 394160394549 73 rpsK; 305 Ribosomal protein S11 (129 aa) LJ 0362 395560395943 73,2rplQ; 50S Ribosomal protein L17 (127 aa) (131 aa) LJ_03.95 441168442517complement81,5Aminopeptidase C (EC 3.4.22.40) (449 aa) LJ 0399 445878447377 73,5Glutamyl-tRNA synthetase (EC 6.1.1.17) (499 aa) LJ_0410 458506458931 77 505 ribosomal protein LI 1 LJ 0441 483735484727complement70,6GMP reductase (EC 1.6.6.8) (330 aa) LJ 0460 501772502056 99 GroES chaperone (94 aa) LJ 0461 502087503718 99 GroEL chaperone (543 aa) LJ 0490 543751544857complement75,2pepQ; 3Caa-Pro dipeptidase (EC 3.4.13.9) (368 aa) LJ 0493 548984550381complement71,6pepV; Xaa-His dipeptidase (EC 3.4.13.3) (465 aa)' LJ_0505 565393566322 73,1Mannose-specific phosphotransferase (309 aa) system comp. IID (EC 2.7.1.69) LJ_0521 583645585255 79 Putative ABC transporter (536 aa) L7 0563 623532624218 83,7putative response regulator (228 aa) L,J 0631 705767706243complement74 Autoinducer protein luxS
(158 aa) LJ 0677 762315763523 70 metK; S-adenosylmethionine synthetase (402 aa) (EC 2.5.1.6) LJ 0764 859739861046 100 putative sensor histidine kinase (435 aa) LJ 0767 863354865513 77,9Sequence from patent (719 aa) LJ_0768 865524866117 91,4Lacticin F transporter accesory protein {197 aa) LJ_0769 866244866471 98,6Bacteriocin lactacin F, subunit IafA
(75 aa) precursor LJ_770 (62 866485866671 100 Bacteriocin lacticin F, subunit lafX
aa) precursor LJ_0771 866757867131 90,3Bacteriocin lacticin F immunity protein, (124 aa) IatI
LJ 0775 869095871254 77,9Sequence from patent (719 aa) LJ 0776 871265871858 77,1Hypothetical protein (197 aa) LJ 0817 910392910658 79,5Phosphocarrier protein HPr (88 aa) LJ 0827 918499920274 71 Sequence from patent (591 aa) LJ 0840 932899933981 72 reeA; Recombinase A
(360 aa) LJ_0846 939718940263 80,1Hypothetical protein (181 aa) LJ_0847 940421942820 81,9preprotein translocase SecA subunit (799 aa) LJ 0848 943020944018 72,5peptide chain release factor 2 (332 aa) LJ_0853 947228948163 71,8trxB; THIOREDOXIN REDUCTASE (EC 1.6.4.5) (311 aa) LJ 0855 949277950230complement71 lacM; Beta-galactosidase small subunit (317 aa) (EC 3.2.1.23,) LJ 0856 950211952091complement75,2lacL; Beta-galactosidase large subunit (626 aa) (EC 3.2.1.23) LJ_0860 957611958780 76,8galK; Galactokinase (EC 2.7.1.6) (Galactose (389 aa) kinase) LJ 0861 958799960286 74,7gall; Galactose-1-phosphate uridylyltransferase (495 aa) (EC 2.7.7.10) LJ 0864 963226965241 76,9uvrB; EXCINUCLEASE ABC SUBUNIT B
(671 aa) LJ_0870 971745972332complement70 clpP, ATP-dependent Clp protease proteolytic (195 aa) subunit (EC 3.4.21.92) LJ 0873 975442976458~ 87,8gapdh; Glyceraldehyde 3-phosphate (338 aa) dehydrogenase(EC 1.2.1.12) LJ 0874 976565977776 84,3pgk; Phosphoglycerate kinase (EC 2.7.2.3) (403 aa) LJ 0875 977795978550 84,8tim; Triosephosphate isomerase (EC
(251 aa) 5.3.1.1) LJ_0876 97860097989$ 71 Enolase (EC 4.2.1.11) (2-phosphoglycerate dehydratase) LJ_0925 10230891024432 72,5Glucose-6-phosphate isomerase (EC
(447 aa) 5.3.1.9) LJ 0934 10329681033579 73,7uracil phosphoribosyltransferase (EC
(203 aa) 2.4.2.9) LJ 0936 10344251034637 73,9atpE; F1FO~ATPase subunit c (EC 3.6.1.34) {70 aa) LJ 0937 10346901035190 70 atpF; F1F0-ATPase subunit b (EC 3.6.1.34) (166 aa) LJ 0939 10357501037261 84,8atpA; F1F0-ATPase subunit alpha (EC
(503 aa) 3.6.1.34) I
LJ_0941 10382581039700 84,7atpD; F1F0-ATPase subunit beta (EC
(480 aa) 3.6.1.34) LJ 0954 10492121050366 70,8nifS; pyridoxal-phosphate dependent (384 aa) aminotransferase (EC 4.4.1.- ).
(458 aa) LJ 0996 10939851095841 70 elongation factor Tu family protein (618 aa) LJ 1007 (89 11039991104268 71,9rps0; 30S ribosomal protein S15 aa) LJ_1010 (39611072391108429 '74EF-Tu; Elongation factor Tu aa) LJ 1033 (37211292761130394 85,8Sequence from patent aa) LJ_1079 (31911813841182343 76,1K6PF; 6-phosphofructokinase (EC
2.7.1.11) aa) LJ_1080 (58911823781184147 83 pyk; Pyruvate kinase (EC 2.7.1.40) aa) LJ 1092 (91 11937101193985 77,5hu; DNA-binding protein II
aa) LJ 1111 (17412153821215906 83,9hslU;heatshockinduced protein HtpI
aa) LJ 1112 (46412159171217311. 76,1HSLU; ATP-dependent hsl protease ATP-binding aa) subunit hslU.
LJ 1138 264 12555821256376 70 ABC transporter ATP-binding protein aa).
LJ_1170 (66112878171289802 71 topoisomerase IV B subunit (EC 5.99.1.*) aa) -LJ_1200 (43213242751325573complement83,5asnAl; Asparaginyl-tRNA synthetase aa) (EC 6.1.1.22) LJ 1207 (35713380651339138 70,1pmk; phosphomevalonate kinase (EC
2.7.1.36) aa) LJ 1298 (75 14252441425471 71,8tpnA; transposase, fragment only aa) LJ 1303 (41.514285751429822complement85,4pepT, PEPTIDASE T (EC 3.4.11: ) (aminotripeptidase) aa) (tripeptidase) LJ 1304 (26514298341430631complement81,1Hypothetical protein aa) LJ 1317 (37214410161442134complement81,4rpoD; RNA polymerase sigma factor rpoD
aa) (Sigma-42) LJ_1320 (30514460501446967complement70,4glyQ; Glycyl-tRNA synthetase alpha aa) chain (EC 6.1.1.14) LJ_1389 (14214571281457556complement71,7Peptide methionine sulfoxide reductase aa) (EC 1.8.4.6) LJ 1356 (32614849031485883complement99 conjugated bile salt hydrolase bile aa) (EC 3.5.1.24) LJ_1384 (47015103221511734complement100orf338; putative portal protein (prophage aa) protein) LJ 1385 (42215117461513014complement100orf42; terminase large subunit (prophage aa) protein) LJ 1386 (15115130071513462complement100orf155; terminase sriiall subunit (prophage aa) protein) LJ 1387 (21815135191514175complement100Orf221 (prophage protein) aa) LJ 1388 (17415143571514881complement90,5Orf174 (prophage protein) aa) LJ_1389 (14615159251516365complement100Orf154 (prophage protein) aa) LJ 1390 (73 15164541516675complement100OrfbS (prophage protein) ' aa) LJ 1391 (18415166951517249complement93,4Orf197 (prophage protein) aa) LJ 1392 (13215172511517649complement82,6Orf79 (prophage protein) aa) LJ 1393 (71 15176501517865complement96,5Orf78a (prophage protein) aa) LJ_1394 (29615180251518915complement98,1Ort212 (prophage protein) aa) . , LJ 1395 (26115189281519713complement.93,9Orf223 (prophage protein) aa) LJ_1396 (29715197151520608complement100Orf309 (prophage protein) aa) LJ 1397 (71 15212851521500complement100Orf73 (prophage protein) aa) LJ 1415 (31815340641535020complement71,4thyA; thymidylate synthase (EC
2.1.1.45) aa) LJ_1423 (62415454101547284complement85,3dnaK; heat shock protein DnaK
aa) LJ_1431 (88015537961556438complement70,3IF2; Translation initiation factor aa) IF-2.
LJ 1442 (24115682881569013complement70,5pyres; UMP-kinase (EC 2.7.4.-) aa) LJ_1444 (26115701861570971complement75,6RS2; 30S ribosomal protein S2.
aa) LJ 1446 (12515810601581437complement76,1Khl9; 505 ribosomal protein L19.
aa) LJ 1447 16057461606000 80 RL28; 50S ribosomal protein L28.
(84 aa) LJ 1429 16567251659109 71,5pepX; Xaa-Pro dipeptidyl-peptidase (794 aa) (EC 3.4.14.11) LJ~,1537 16661621667067complement71,2galU; UDP-glucose pyrophosphorylase, (301 aa) (EC 2.7.7.9) LJ 1558 16868631688200complement72,8Glutamine synthetase (EC 6.3.1.2) (445 aa) (Glutamate--ammonia ligase) LJ 1584 17133301713686complement70,7RL20; 50S ribosomal protein L20 (118 aa) LJ_1681 18269011827287 72,6tagD; Glycerol-3-phosphate cytidylyltransferase (128 aa) (EC 2.7.7.39) LJ_1741 19029451903592complement72,5Pyrrolidone-carboxylate peptidase (215 aa) (EC 3.4.19.3) I
LJ 1767 19306101931257 100deoxyadenosine ltinase (EC 2.7.1.76) (215 aa) j LJ 1768 19312791931953 99,1deoxyguanosine kinase (EC 2.7.1.113) (224 aa) * complement = on the reverse strand * as = amino acids The ORFs corresponding to various poplypeptides are. shown in table 1, supra, and are represented by their position in the genomic sequence as identified by SEQ.
ID. NO. 1.
The open reading frames.have been identified via homology analyses as well as via analyses of potential ORF start sites. It is to be understood that each identified ORF
comprises a nucleotide sequence that spans the contiguous nucleotide sequence from the codon immediately 3' to the stop codon of the preceding ORF and through the 5' codon to the next stop codon of SEQ. ID. NO. 1 in frame to the ORF nucleotide sequence.
Table 1 also depicts the results of homology searches that compared the sequences of the polypeptides encoded by each of the ORFs to sequences present in databases.
The sequence information disclosed in the present application may be utilized for selecting a r polynucleotide of interest, i.e. a nucleic acid containing an open reading frame encoding a known or an unknown, putative polypeptide and transforming micro-organisms with the selected polynucleotide. As transformation vehicles the well known plasmids, phage vectors (trans-fection) or F-vectors (conjugation) may be utilized. The nucleic acid introduced into the micro-organism selected may be expressed and its biological function may be either utilized as such, if known, or elucidated, in case a so far unknown polypeptide is expressed. The micro-organism selected may be a Lactobacillus itself or other well known micro-organisms, such as bacteria, e.g.
E.coli, Streptococci or yeast, insect cells or even animal and plant cells.
It will be understood that the polypeptides may be expressed as such or as a fusion polypeptide.
The skilled person is well aquatinted with techniques performing such a ligation and expressing the corresponding fusion-polypeptide in an appropriate cell.
In view of the present invention also new recombinant vectors for the cloning and/or the expression of a nucleotide sequence according to the present invention may be devised. The vectors comprise elements necessary to enable expression and/or secretion of the nucleotide sequences 'in a given host cell, such as a promoter, signals for initiation and for termination of translation, as well as appropriate regions for regulation of transcription.
For example, expression of a protein or peptide may be controlled by any promoter/enhancer element known. in the art.
Exemplary promotors are the CMV promoter, the SV40 early promoter region, the promoter contained in the 3' long terminal repeat of the roux sarcoma virus, the herpes thymidine kinase promoter, the regulatory sequences of the metallothionein gene, or, for prokaryotic expression systems, the 13-lactamase promoter, the tac promoter or the T7 promoter.
The vector should be capable of being stably maintained in the host cell and may optionally possess particular signals specifying the secretion of the translated protein.
These different elements are chosen according to the host cell utilized. To this effect the nucleotide sequences according to the invention may be inserted into autonomously-replicating vectors within the chosen host, or integrative vectors in the chosen host, such as e.g. yeast artificial chromosomes, plasmids or viral vectors.
Any of the standard methods known to those skilled in the art for inserting DNA fragments into a vector may be used to construct expression vectors containing a chimeric gene consisting of appropriate transcriptional/trans'lational control signals and the protein coding sequences. These methods may include in vitro recombinant DNA and synthetic techniques and in vivo recombinants (genetic recombination).
The vector may be used for transcription and/or translation of a nucleic acid comprised in SEQ.
ID. NO. l, to produce RNA or antisense RNA, respectively. Such a vector can remain episomal or become chromosomally integrated, as long as it can be transcribed to produce the desired transcript.
The antisense nucleic acids of the invention comprise a sequence complementary to at least a portion of a RNA transcript of a polynucleotide sequence in SEQ. >D. NO. 1, designating a sequence having sufficient complementarity to be able to hybridize with the RNA, forming a stable~duplex. In the case of double-stranded antisense nucleic acid sequence, a single strand of the duplex DNA may thus be tested, or triplex formation may be assayed.
In knowledge of the present invention also host cells may be obtained transformed with a nucleic acid or a vector according described herein. These cells may be attained by introducing into an appropriate cell a nucleotide sequence or a vector as defined above, and then culturing the said cell under conditions allowing the replication andlor the expression of the transformed/trans-fected nucleotide sequence.
The host cell may be chosen from eukaryotic or prokaryotic system, such as for example bacterial cells, yeast cells, animal cells as well as plant cells. In the context of this invention a cell shall be understood to comprise higher biological systems. Such as animals, whole plants or parts thereof.
Furthermore, a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired.
A preferred host cell for the expression of the proteins of the invention consists of prokaryotic cells, such as gram negative or gram positive bacteria. A further preferred host cell .according to the invention is a bacterium belonging to the Lactobacillus family, more preferably belonging to the species Lactobacillus johnsonii or chosen from a microorganism associated with the species Lactobacillus.
The transformed/transfected cells according to the invention may advantageously serve as a model and may be used in methods for studying, identifying and/or selecting compounds capable of being responsible for any of the beneficial effects brought about by the present Lactobacillus strain.
The invention further enables the synthesis of polypeptides encoded by the Lactobacillus johnsonii ORFs, in particular those listed in table 1. In the present description, the terms polypeptide, peptide and protein are used interchangeably. Furthermore the present invention also enables to carry out method for preparing such polypeptides by recombinant means comprising the steps of , (a) culturing a host cell according to the present invention under conditions suitable to produce the polypeptide encoded by the polynucleotide;
and (b) recovering the polypeptide from the culture.
It will be appreciated that the above polypeptides may also be obtained using combinatory chemistry, wherein the polypeptide is modified at some locations before testing them in model systems, so as to select the compounds which are the most active or which exhibit the desired properties.
In this context, chemical synthesis has the advantage of being able to use non-natural amino acids or non-peptide bonds. Accordingly, in order to e.g. extend the life of the polypeptides according to the invention, it may be advantageous to use such non-natural amino acids, for example in the D form, or alternatively amino acid analogues, preferably sulphur-containing forms.
Finally, the structure of the polypeptides according to the invention, its homologous or modified forms, as well as the corresponding fragments may be integrated into chemical structures of the polypeptide type and the like. Accordingly, in order to preserve the polypeptide in an in vivo environment it will be preferred to provide at the N- and C-terminal ends compounds which convey a resistance to degradation to proteases.
It will also be appreciated that the different polypeptides according to the present invention and produced by the above method may represent antigens to the immune system of a host animal, so that antibodies may be produced directed against said polypeptides. These antibodies may be used for the detection of a polypeptide of interest in a mixture or generically of a strain of Lactobacillus in a sample. In addition they may be used as research tools by e.g. producing antibodies against cellular surface epitopes and determining the effect of blocking certain polypeptides on the bacterial cell wall.
According to another aspect the present invention also provides a method for the detection and/or identification of Lactobacilli, preferably Lactobacillus johnsonii in a biological sample. This method may comprise several techniques known in the art, such as PCR or simply hybridization with a suitable probe. Alternatively, an antibody raised against a cell wall epitope of Lactobacillus, preferably Lactobacillus johnsonii may be used for said purpose. It will be appreciated that the above method may also be reversed and the presence of antibodies against Lactobacillus may be determined by contacting the sample to be tested with a polypeptide of Lactobacillus under conditions to allow formation of immune complexes.
The polypeptides and antibodies obtainable in knowledge of the present invention and the nucleotide sequences described herein may be used in in vitro and/or in vivo methods for the detection and/or the identification of bacteria belonging to the species Lactobacillus in a biological sample (biological tissue or fluid) which is likely to contain them. These methods, depending on the specificity of the polypeptides, of the antibodies and of the nucleotide sequences described herein, which will be used, may detect and/br identify the bacterial variants belonging to the species Lactobacillus as well as associated microorganisms capable of being detected by the polypeptides, the antibodies and the nucleotide sequences according to the invention which will be chosen. It may, for example, be advantageous to choose a polypeptide, an antibody or a nucleotide sequence according to the invention, which is capable of detecting any bacterium of the Lactobacillus family by choosing a polypeptide, an antibody and/or a nucleotide sequence according to the invention which is specific to the family.
The sequences referred to herein SEQ ID. NO. 1 is listed in the attached sequence listings which is to be considered as part of the specification.
The invention also comprises, the nucleotide sequences or polypeptides according to the invention covalently or non-covalently immobilized on a solid support. In the first case such a support may serve to capture, through specific hybridization, the target nucleic acid obtained from a biological sample to be tested. If necessary, the solid support is separated from the sample and the hybridization complex formed between the capture probe and the target nucleic acid is then detected by means of a second probe, called detection probe, labeled with an easily detectable element.
Such support may take the form of so-called DNA array or DNA chips, a multitude of molecular probes precisely organized or arrayed on a solid support, which will allow sequencing genes, studies of mutations contained therein and the expression of genes, and which are currently of interest given their very small size and their high capacity in terms of number of analyses.
The function of these arrays/chips is based on molecular probes, mainly oligonucleotides which are attached to a carrier having a size of generally a few square centimetres or more as desired. For an analysis the carrier (DNA array/chip) is coated with probes that are arranged at a predetermined location of the carrier. A sample containing fragments of a target nucleic acid to be analyzed, for example DNA or RNA or cDNA, that has been labeled beforehand, is subsequently contacted with the DNA array/chip leading to the formation;
through hybridization, of a duplex. After a washing step, analysis of the surface of the chip allows the effective hybridization to be located by means of the signals emitted by the labels tagging the target. A hybridization fingerprint results from this analysis which, by appropriate computer processing, allows to retrieve information such as the expression of genes, the presence of specific fragments in the sample, the determination of sequences and the presence of mutations.
The hybridization between the probes of the invention, deposited or synthesized in situ on the DNA chips, and the sample to be analyzed, may, e.g. be determined by means of fluorescence, radioactivity or by electronic detection.
The nucleotide sequences according to the invention may be used in DNA
arrays/chips to carry out analyses of the expression of the Lactobacillus genes. This analysis is based on DNA arrays/chips on which probes, chosen for their specificity to characterize a given gene, are present. The target sequences to be analyzed are labeled before being hybridized onto the chip. After. washing the labeled compounds are detected and quantified, with the hybridization being carried out at least in duplicate. Comparative analyses of the signal intensities obtained with respect to the same probe for different samples and/or for different probes with the same sample, determine a differential transcription of RNA derived from the sample.
The DNA arrays/chips according to the present invention may also contain nucleotide probes specific for other microorganisms, which will enable a serial testing allowing rapid identification of the presence of a microorganism in a sample.
The principle of the DNA chip, as detailed above may also be used to produce-protein chips on which the support has been coated with a polypeptide or an antibody according to the invention, or arrays thereof, in place of the DNA. These protein chips make it possible to analyze the biomolecular interactions (BIA) induced by the affinity capture of target analytes onto a support coated e.g. with proteins, by surface plasma resonance (SPR).
The polypeptides or antibodies according to the invention, capable of specifically binding anti-bodies or polypeptides derived from the sample to be analyzed, may thus be used in protein chips for the detection and/or the identification of proteins in samples.
The present invention also relates to a computer readable medium having recorded thereon one or more nucleotide and/or a polypeptide sequences according to the invention. This medium may also comprise additional information extracted from the present invention, such as e.g. analogies with already known sequences and/or information relating to the nucleotide and/or polypeptide sequences of other microorganisms so as to facilitate the comparative analysis and the exploitation of the results obtained. Preferred media are e.g. magnetic, optical, electrical and hybrid media such as, for example, floppy disks, CD-ROMs or recording cassettes.
The invention also relates to kits or sets for the detection and/or the identifi-ration of bacteria belonging to the species Lactobacillus johnsonii or to associated microorganisms, which comprises, a polypeptide according to the invention, where appropriate, the reagents for constituting the medium appropriate for the immunological or specific reaction, the reagents allowing the detection of the antigen-antibody complexes produced by the immunological reaction between the polypeptide (s) of the invention and the antibodies which may be present in the biological sample, it being possible for these reagents also to carry a label, or to be capable of being recognized in turn by a labeled reagent, more particularly in the case where the polypeptide according to the invention is not labeled, a reference biological sample (negative control) free of antibodies recognized by a polypeptide according to the invention, a reference biological sample (positive control) containing a predetermined quantity of antibodies recognized by a polypeptide according to the invention.
The invention also relates to a kit or set for the detection and/or the identification of bacteria belonging to the species Lactobacillus johnsonii or to an associated microorganism; or for the detection and/or the identification of a microorganism, wherein the kit comprises a protein chip according to the invention.
Claims (19)
1. Use of a DNA sequence as identified by SEQ ID. No.1 or fragments of a DNA
sequence as identified by SEQ ID. No.1 or sequences having a percentual identity with the sequence of SEQ ID. No.1 of at least 90 % for elucidating interactions between a host and bacteria.
sequence as identified by SEQ ID. No.1 or sequences having a percentual identity with the sequence of SEQ ID. No.1 of at least 90 % for elucidating interactions between a host and bacteria.
2. ~The use according to claim 1, wherein the interaction is based on probiotic properties of bacterial strains, preferably on stimulating the immune system, on anti-pathogenic properties and/or anti-viral properties.
3. ~The use according to any of the claims 1 or 2, wherein the sequence is a fragment of SEQ.
ID. No. 1 selected from the group consisting-of from nucleotide 1-54596, from 56070-77430, from 81302-308537, from309588-342757, from 378458-389217, from 389779-404510, from 405561-501116, from503873-558194, from 563262-696518, from 697569-721736, from 722787-756845, from 761682-860446, from 860723-865550, from 867260-867490, from 868541-1448288, from1463851-1526077, from 1527278-1552024, from1563147-1809115, from1810166-1858190, from 1863258-1872871, from 1877939-1930430, from1932063-1983043, based on the numbering of SEQ ID. No.1.
ID. No. 1 selected from the group consisting-of from nucleotide 1-54596, from 56070-77430, from 81302-308537, from309588-342757, from 378458-389217, from 389779-404510, from 405561-501116, from503873-558194, from 563262-696518, from 697569-721736, from 722787-756845, from 761682-860446, from 860723-865550, from 867260-867490, from 868541-1448288, from1463851-1526077, from 1527278-1552024, from1563147-1809115, from1810166-1858190, from 1863258-1872871, from 1877939-1930430, from1932063-1983043, based on the numbering of SEQ ID. No.1.
4. ~A method for the detection, identification and/or selection of a Lactobacillus strain, preferably Lactobacillus johnsonii in a biological sample, comprising:
(a) contacting the sample with a nucleotide sequence derived from a polynucleotide sequence as identified by SEQ ID. No. 1 in the presence of a polymerase enzyme and nucleotides under conditions which permit extension of the nucleotide; and (b) detecting the presence of extension products in the sample in which the detection of primer extension products indicates the presence of a Lactobacillus strain in the sample.
(a) contacting the sample with a nucleotide sequence derived from a polynucleotide sequence as identified by SEQ ID. No. 1 in the presence of a polymerase enzyme and nucleotides under conditions which permit extension of the nucleotide; and (b) detecting the presence of extension products in the sample in which the detection of primer extension products indicates the presence of a Lactobacillus strain in the sample.
5. A method for the detection, identification and/or selection of a Lactobacillus strain, preferably Lactobacillus johnsonii in a biological sample, comprising:
(a) contacting the sample with a nucleotide sequence derived from a polynucleotide sequence as identified by SEQ ID. NO. 1 under conditions which permit hybridization of complementary base pairs; and (b) detecting the presence of hybridization complexes in the sample in which the detection of hybridization complexes indicates the presence of a Lactobacillus strain in the sample.
(a) contacting the sample with a nucleotide sequence derived from a polynucleotide sequence as identified by SEQ ID. NO. 1 under conditions which permit hybridization of complementary base pairs; and (b) detecting the presence of hybridization complexes in the sample in which the detection of hybridization complexes indicates the presence of a Lactobacillus strain in the sample.
6. ~A method for the detection, identification and/or selection of a Lactobacillus strain, preferably Lactobacillus johnsonii in a biological sample, comprising:
(a) contacting the sample with an antibody raised against a polypeptide derived from SEQID.
No. 1 under conditions suitable for the formation of immune complexes ; and (b) detecting the presence of immune complexes in the sample, in which the detection of immune complexes indicates the presence of a Lactobacillus strain in the sample.
(a) contacting the sample with an antibody raised against a polypeptide derived from SEQID.
No. 1 under conditions suitable for the formation of immune complexes ; and (b) detecting the presence of immune complexes in the sample, in which the detection of immune complexes indicates the presence of a Lactobacillus strain in the sample.
7. A method for the detection, identification and/or selection of antibodies directed to Lactobacillus johnsonii polypeptides in a biological sample, comprising:
(a) contacting the sample with a polypeptide produced according to claim 4 or 5 under conditions suitable for the formation of immune complexes; and (b) detecting the presence of immune complexes in the sample, in which the detection of immune complexes indicates the presence of Lactobacillus polypeptides in the sample.
(a) contacting the sample with a polypeptide produced according to claim 4 or 5 under conditions suitable for the formation of immune complexes; and (b) detecting the presence of immune complexes in the sample, in which the detection of immune complexes indicates the presence of Lactobacillus polypeptides in the sample.
8. A DNA array/chip containing an array of polynucleotides comprising at least a polynucleotide derived from SEQ ID. No. 1.
9. A protein array/chip containing an array of polypeptides comprising at least one of the polypeptides obtainable by expressing a polypeptide as identified by an open reading frame derived from SEQ. ID. No. 1.
10. An antibody chip containing an array of antibodies comprising at least one antibody directed to a polypeptide obtainable by expressing an open reading frame in SEQ ID. No. 1.
11. A screening assay, comprising:
(a) contacting a test compound with a polynucleotide as identified by SEQ ID.
No. 1 or with a fragment of a DNA sequence as identified by SEQ ID. No.1; and (b) detecting whether binding occurs.
(a) contacting a test compound with a polynucleotide as identified by SEQ ID.
No. 1 or with a fragment of a DNA sequence as identified by SEQ ID. No.1; and (b) detecting whether binding occurs.
12. A screening assay, comprising:
(a) contacting a test compound with a polypeptide obtainable by expressing an open reading frame derived from SEQ ID. No. 1; and (b) detecting whether binding occurs.
(a) contacting a test compound with a polypeptide obtainable by expressing an open reading frame derived from SEQ ID. No. 1; and (b) detecting whether binding occurs.
13. A screening assay, comprising:
(a) contacting a test compound with an antibody raised against a polypeptide obtainable by expressing an open reading frame derived from SEQ ID. No.1 ; and (b) detecting whether binding occurs.
(a) contacting a test compound with an antibody raised against a polypeptide obtainable by expressing an open reading frame derived from SEQ ID. No.1 ; and (b) detecting whether binding occurs.
14. A kit comprising a polynucleotide as identified by SEQ. ID. No. 1 fragments of a DNA
sequence as identified by SEQ ID. No.1.
sequence as identified by SEQ ID. No.1.
15. The kit according to claim 13, wherein the polynucleotide is a primer or a probe and wherein the kit optionally contains a polymerase and deoxynucleotide triphosphates.
16. A kit comprising containing an antibody raised against a polypeptide obtainable by expressing an open reading frame in SEQID. No. 1.
17. A computer readable medium having recorded thereon a nucleic acid sequence as identified by SEQID. No. 1 or fragments of a DNA sequence as identified by SEQ
ID. No.1 or a polypeptide sequence derived from the nucleotide sequence as identified by SQEID. No.1.
ID. No.1 or a polypeptide sequence derived from the nucleotide sequence as identified by SQEID. No.1.
18. The computer readable medium according to claim 17, wherein said medium is selected from the group consisting of:
(a) a floppy disc;
(b) a hard disc;
(c) random access memory (RAM) ;
(d) read only memory(ROM) ; and (e) CD-ROM.
(a) a floppy disc;
(b) a hard disc;
(c) random access memory (RAM) ;
(d) read only memory(ROM) ; and (e) CD-ROM.
19. A computer-based system for identifying fragments of the Lactobacillus johnsonii La1 genome comprising the following elements:
(a) a data storage means comprising a nucleic acid sequence as identified by SEQ ID. No.1;
(b) search means for comparing a target sequence to the nucleotide sequence of the data storage means of step (a) to identify homologous sequence (s); and (c) retrieval means for obtaining said homologous sequence (s) of step (b).
(a) a data storage means comprising a nucleic acid sequence as identified by SEQ ID. No.1;
(b) search means for comparing a target sequence to the nucleotide sequence of the data storage means of step (a) to identify homologous sequence (s); and (c) retrieval means for obtaining said homologous sequence (s) of step (b).
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| EP02007932 | 2002-04-09 | ||
| EP02007932.3 | 2002-04-09 | ||
| PCT/EP2003/002882 WO2003084989A2 (en) | 2002-04-09 | 2003-03-19 | La1 - the genome of a lactobacillus strain |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CA2482138A1 true CA2482138A1 (en) | 2003-10-16 |
| CA2482138C CA2482138C (en) | 2014-05-27 |
Family
ID=28685848
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA2482138A Expired - Fee Related CA2482138C (en) | 2002-04-09 | 2003-03-19 | La1 - the genome of a lactobacillus strain |
Country Status (11)
| Country | Link |
|---|---|
| US (2) | US20080044820A1 (en) |
| EP (2) | EP1816196A1 (en) |
| JP (1) | JP2006506042A (en) |
| CN (1) | CN1659280A (en) |
| AT (1) | ATE403718T1 (en) |
| AU (1) | AU2003223971A1 (en) |
| CA (1) | CA2482138C (en) |
| DE (1) | DE60322689D1 (en) |
| DK (1) | DK1497422T3 (en) |
| ES (1) | ES2311700T3 (en) |
| WO (1) | WO2003084989A2 (en) |
Families Citing this family (15)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP4850698B2 (en) | 2003-04-25 | 2012-01-11 | ノース・キャロライナ・ステイト・ユニヴァーシティ | Lactobacillus acidophilus nucleic acid sequences encoding cell surface protein homologues and uses thereof |
| EP1651750B1 (en) | 2003-06-23 | 2011-04-06 | North Carolina State University | Lactobacillus acidophilus nucleic acids encoding fructo-oligosaccharide utilization compounds and uses thereof |
| US7455992B2 (en) | 2004-02-23 | 2008-11-25 | North Carolina State University | Lactobacillus acidophilus nucleic acid sequences encoding protease homologues and uses therefore |
| US7608700B2 (en) | 2004-03-08 | 2009-10-27 | North Carolina State University | Lactobacillus acidophilus nucleic acid sequences encoding stress-related proteins and uses therefor |
| US7459289B2 (en) | 2004-03-08 | 2008-12-02 | North Carolina State University | Lactobacillus acidophilus nucleic acid sequences encoding carbohydrate utilization-related proteins and uses therefor |
| US7919277B2 (en) * | 2004-04-28 | 2011-04-05 | Danisco A/S | Detection and typing of bacterial strains |
| US7550576B2 (en) | 2004-08-09 | 2009-06-23 | North Carolina State University | Nucleic acid sequences encoding two-component sensing and regulatory proteins, antimicrobial proteins and uses therefor |
| US7468182B2 (en) | 2004-10-27 | 2008-12-23 | North Carolina State University | Lactobacillus acidophilus nucleic acids and uses thereof |
| US7495092B2 (en) | 2005-01-14 | 2009-02-24 | North Carolina State University | Compositions comprising promoter sequences and methods of use |
| JP5138197B2 (en) * | 2006-10-04 | 2013-02-06 | 茂 藤村 | LG21 strain primer and detection method using the primer |
| JP4457364B2 (en) | 2007-04-27 | 2010-04-28 | 勇志 相場 | New lactic acid bacteria and various products processed using the lactic acid bacteria |
| WO2009150856A1 (en) * | 2008-06-13 | 2009-12-17 | 株式会社ヤクルト本社 | Gene that imparts oxygen resistance and application thereof |
| GB0914878D0 (en) * | 2009-08-26 | 2009-09-30 | Plant Bioscience Ltd | Competetive exclusion compositions and methods |
| EP3011966A1 (en) * | 2014-08-08 | 2016-04-27 | Institut National De La Recherche Agronomique | Compositions for the inhibition of Giardia Lamblia |
| CN108315402B (en) * | 2018-04-28 | 2021-02-12 | 内蒙古农业大学 | Amplification primer pair for sequencing bifidobacterium in intestinal tract and sequencing method |
Family Cites Families (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DK0577904T3 (en) * | 1992-07-06 | 1997-10-27 | Nestle Sa | The lactic acid bacteria. |
| JPH08185676A (en) * | 1994-12-28 | 1996-07-16 | Sony Corp | Magnetic disk and disk device |
| AU5552396A (en) | 1995-04-21 | 1996-11-07 | Human Genome Sciences, Inc. | Nucleotide sequence of the haemophilus influenzae rd genome, fragments thereof, and uses thereof |
| KR0159441B1 (en) * | 1995-08-28 | 1999-01-15 | 김광호 | Disc recording medium and its servo controlling method for high quantity hard disc driver |
| US5652838A (en) * | 1996-05-20 | 1997-07-29 | Lovett; Donna M. | Smart disc cd-rom |
| DK0875579T3 (en) * | 1997-05-03 | 2007-10-22 | Nestle Sa | Preparation of L (+) lactate |
| JP3028214B2 (en) * | 1997-06-03 | 2000-04-04 | カルピス株式会社 | How to administer live birds |
| EP1110555A1 (en) * | 1999-12-22 | 2001-06-27 | Societe Des Produits Nestle S.A. | Antiadhesive agent for the pathogen flora of the skin |
| EP1311684A4 (en) * | 2000-08-08 | 2005-10-19 | Genesis Res & Dev Corp Ltd | Lactobacillus rhamnosus polynucleotides, polypeptides and methods for using them |
| GB0027761D0 (en) * | 2000-11-14 | 2000-12-27 | Nestle Sa | Nutritional composition for an immune condition |
-
2003
- 2003-03-19 ES ES03720340T patent/ES2311700T3/en not_active Expired - Lifetime
- 2003-03-19 EP EP07104120A patent/EP1816196A1/en not_active Withdrawn
- 2003-03-19 AU AU2003223971A patent/AU2003223971A1/en not_active Abandoned
- 2003-03-19 WO PCT/EP2003/002882 patent/WO2003084989A2/en active IP Right Grant
- 2003-03-19 DK DK03720340T patent/DK1497422T3/en active
- 2003-03-19 DE DE60322689T patent/DE60322689D1/en not_active Expired - Lifetime
- 2003-03-19 JP JP2003582184A patent/JP2006506042A/en active Pending
- 2003-03-19 CA CA2482138A patent/CA2482138C/en not_active Expired - Fee Related
- 2003-03-19 CN CN038128209A patent/CN1659280A/en active Pending
- 2003-03-19 EP EP03720340A patent/EP1497422B1/en not_active Expired - Lifetime
- 2003-03-19 AT AT03720340T patent/ATE403718T1/en active
-
2004
- 2004-10-08 US US10/961,644 patent/US20080044820A1/en not_active Abandoned
-
2008
- 2008-10-02 US US12/244,581 patent/US7875421B2/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| ES2311700T3 (en) | 2009-02-16 |
| CA2482138C (en) | 2014-05-27 |
| EP1497422B1 (en) | 2008-08-06 |
| JP2006506042A (en) | 2006-02-23 |
| AU2003223971A1 (en) | 2003-10-20 |
| DE60322689D1 (en) | 2008-09-18 |
| EP1816196A1 (en) | 2007-08-08 |
| US7875421B2 (en) | 2011-01-25 |
| AU2003223971A8 (en) | 2003-10-20 |
| WO2003084989A3 (en) | 2004-02-05 |
| ATE403718T1 (en) | 2008-08-15 |
| DK1497422T3 (en) | 2008-12-15 |
| CN1659280A (en) | 2005-08-24 |
| EP1497422A2 (en) | 2005-01-19 |
| US20090098053A1 (en) | 2009-04-16 |
| WO2003084989A2 (en) | 2003-10-16 |
| US20080044820A1 (en) | 2008-02-21 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US7875421B2 (en) | La1—the genome of a lactobacillus strain | |
| Axelsson | Lactic acid bacteria: classification and physiology | |
| US5849295A (en) | Nucleotide sequences coding for a protein with urease activity | |
| Blum et al. | Excision of large DNA regions termed pathogenicity islands from tRNA-specific loci in the chromosome of an Escherichia coli wild-type pathogen | |
| CA2435664A1 (en) | Ncc2705-the genome of a bifidobacterium | |
| JP2008220376A (en) | Nucleic acid sequence derived from salmonella typhi genome and use thereof particularly for in vitro diagnosis of existence of genus salmonella bacteria in foodstuff | |
| US20070020625A1 (en) | Sequence of the photorhabdus luminescens strain tt01 genome and uses | |
| Christmann et al. | Epitope mapping and affinity purification of monospecific antibodies by Escherichia coli cell surface display of gene-derived random peptide libraries | |
| JP2006500951A (en) | Polynucleotides and polypeptides, materials containing them, and methods of using the same | |
| CA2252372A1 (en) | Pathogenic escherichia coli associated protein | |
| JP3570737B2 (en) | Methods and reagents for Listeria detection | |
| Scherl et al. | Correlation of proteomic and transcriptomic profiles of Staphylococcus aureus during the post-exponential phase of growth | |
| US7026463B2 (en) | Polynucleotides and polypeptides, materials incorporating them and methods for using them | |
| Nelson et al. | Detection of Bordetella pertussis in clinical specimens by PCR and a microtiter plate-based DNA hybridization assay | |
| Mujahid et al. | Improved solubilization of surface proteins from Listeria monocytogenes for 2‐DE | |
| Klijn et al. | Identification of Clostridium tyrobutyricum and related species using sugar fermentation, organic acid formation and DNA probes based on specific 16S rRNA sequences | |
| JP2001522605A5 (en) | ||
| WO1996013608A3 (en) | Polynucleic acid sequences for use in the detection and differentiation of prokaryotic organisms | |
| JP4336814B2 (en) | Target nucleic acid detection method using zinc finger protein | |
| JP3578233B2 (en) | Method for detecting and measuring anti-Chlamydia pneumoniae antibody and reagent thereof, and diagnostic agent for Chlamydia pneumoniae infection | |
| US6905865B1 (en) | Detection of a gene, vatE, encoding an acetyltransferase inactivating streptogramin | |
| US20060099623A1 (en) | Polynucleotides and polypeptides isolated from lactobacillus and methods for their use | |
| US20080076672A1 (en) | Methods for Identifying Genes Essential to the Growth of an Organism | |
| Douglas et al. | Genomics and Proteomics of Foodborne Microorganisms | |
| KR100493824B1 (en) | Antibiotics screening composition comprising Streptococcus pneumoniae spr0049 gene or its protein product, and antibiotics screening method using said composition |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| EEER | Examination request | ||
| MKLA | Lapsed |
Effective date: 20170320 |