EP1379549A2 - Sequence du genome de photorhabdus luminescens souche tto1 et utilisations - Google Patents

Sequence du genome de photorhabdus luminescens souche tto1 et utilisations

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Publication number
EP1379549A2
EP1379549A2 EP20020751498 EP02751498A EP1379549A2 EP 1379549 A2 EP1379549 A2 EP 1379549A2 EP 20020751498 EP20020751498 EP 20020751498 EP 02751498 A EP02751498 A EP 02751498A EP 1379549 A2 EP1379549 A2 EP 1379549A2
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EP
European Patent Office
Prior art keywords
seq
escherichia coli
polypeptide
protein
sequence
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.)
Withdrawn
Application number
EP20020751498
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German (de)
English (en)
French (fr)
Inventor
Eric Duchaud
Sead Taourit
Philippe Glaser
Lionel Frangeul
Frederik Kunst
Antoine Danchin
Carmen Buchrieser
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Centre National de la Recherche Scientifique CNRS
Institut Pasteur de Lille
Original Assignee
Centre National de la Recherche Scientifique CNRS
Institut Pasteur de Lille
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Application filed by Centre National de la Recherche Scientifique CNRS, Institut Pasteur de Lille filed Critical Centre National de la Recherche Scientifique CNRS
Publication of EP1379549A2 publication Critical patent/EP1379549A2/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • 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
    • C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia

Definitions

  • the subject of the invention is the genomic sequence and nucleotide sequences coding for photorhabdus luminescens polypeptides, such as polypeptides involved in operons for biosynthesis of antibiotics, or toxins, or polypeptides with activity of toxin or antibiotic type which can be used as a pesticide, bactericide or fungicide as well as vectors including said sequences and cells or animals transformed by these vectors.
  • Photorhabdus luminescens is an entomopathogenic bacteria, intestinal commensal of a nematode and insect parasite.
  • This bacterium is both a model for the study of host-parasite interactions and a bacterium whose industrial applications are numerous due to its ability to synthesize many toxins (insecticides, bactericides and fungicides) and to secrete many enzymes.
  • the photorhabdus luminescens genome has been sequenced.
  • the present invention thus relates to the nucleotide and polypeptide sequences of Photorhabdus luminescens strain TT01.
  • the Photorhabdus luminescens TT01 strain is also identified in the present application by Photorhabdus luminescens, interchangeably.
  • the invention also relates to new tools for typing strains of Photorhabdus. These tools could be of the DNA "chip” type or of another type. The new features of these typing tools will be as follows:
  • the present invention therefore relates to an isolated nucleotide sequence derived from the genome of Photorhabdus luminescens, characterized in that it comprises a sequence chosen from the sequences SEQ ID No. 1 to SEQ ID No. 41 and the sequences SEQ ID No. 5826 to SEQ ID No. 5834.
  • sequences SEQ ID No. 1 to SEQ ID No. 41 represent the sequences of 41 contigs, the whole of which covers the genomic sequence of Photorhabdus luminescens TTOl.
  • sequences SEQ 1) N ° 1 to SEQ ID N ° 41 could be reassembled and reread in 9 new contigs, all of which also covers the genomic sequence of Photorhabdus luminescens TTOl.
  • sequences SEQ ID N ° 5826 to SEQ ID N ° 5834 represent the sequences of these 9 contigs
  • the nucleotide sequences SEQ ID No.1 to SEQ ID No.41 and SEQ ID No.5826 to SEQ ID No.5834 were obtained by sequencing the genome of Photorhabdus luminescens TTOl by the so-called shot-gun technique (cf. Examples) .
  • shot-gun technique cf. Examples
  • SEQ ⁇ ) N ° 1 to SEQ ⁇ ) N ° 41 or SEQ ⁇ ) N ° 5826 to SEQ ⁇ ) N ° 5834 it is possible that these do not represent perfectly 100 % after assembly the nucleotide sequence of the genome of Photorhabdus luminescens TTOl and that some rare sequencing errors or indeterminacies remain in these sequences.
  • the corrected nucleotide sequence obtained would still comprise at least 97%, preferably at least 98%, 98.5%, 99% or 99.9% of identity with the genomic sequence obtained after assembly of these nucleotide sequences SEQ ⁇ ) N ° 1 to SEQ ⁇ ) N ° 41 or SEQ ⁇ ) N ° 5826 to SEQ ⁇ ) N ° 5834.
  • the present invention also relates to an isolated nucleotide sequence derived from the Photorhabdus luminescens genome, characterized in that it is chosen from: a) a nucleotide sequence comprising at least 75%, 80%, 85%, 90%, 95%, 98% or 99% identity with a sequence chosen from the sequences SEQ ⁇ )
  • the present invention also relates to a nucleotide sequence included in one of the sequences SEQ ⁇ ) N ° 1 to SEQ ⁇ ) N ° 41 or one of the sequences SEQ ⁇ ) N ° 5826 to SEQ ⁇ ) N ° 5834 , and in that it codes for a polypeptide chosen from the polypeptides of sequence SEQ ⁇ ) N ° 42 to SEQ ⁇ ) N ° 3855 or from the polypeptides coded by a sequence SEQ ⁇ ) N ° 5835 to SEQ ⁇ ) N ° 10784.
  • SEQ ⁇ ) N ° 10784 are the polypeptides whose sequence of at least 5 amino acids is obtained by taking as reading frame the first nucleotide of the sequences SEQ ⁇ ) N ° 5835 to SEQ ⁇ ) N ° 10784.
  • the invention also relates to a nucleotide sequence, characterized in that it codes for a polypeptide whose function annotated in Table I below, last column or in Table ⁇ below, penultimate column, corresponds to a toxin and / or antibiotic type activity, or to an operon involved in the synthesis of toxin and / or antibiotic, polypeptide preferably chosen from: a) polypeptides of sequences SEQ 1), No.
  • polypeptides of sequence as defined in paragraph a) above the function of which is associated with a toxin or antibiotic type activity, or their homologous polypeptide from Table ⁇ as defined in paragraph b) above, have could be identified, for example, by the presence of a consensus motif linked to these functions or by the presence of sequences adjoining them on the genome and involved in this type of activity.
  • the present invention also relates more generally to the nucleotide sequences originating from SEQ ⁇ ) N ° 1 to SEQ ⁇ ) N ° 41 or SEQ ⁇ ) N ° 5826 to SEQ ⁇ )
  • nucleotide sequences characterized in that they comprise a nucleotide sequence chosen from: a) a nucleotide sequence coding for a polypeptide chosen from the sequences SEQ ⁇ ) N ° 42 to SEQ ⁇ ) N ° 3855 or from the encoded polypeptides by the sequences SEQ 1) No. 5835 to SEQ 1) No.
  • 10784 preferably from the 82 sequences of polypeptides above selected for their function associated with an activity of toxin or antibiotic type or their homologous peptide as defined in the table ⁇ in the last column; b) a nucleotide sequence comprising at least 75% identity with a nucleotide sequence as defined in a), preferably at least 80%, 85%,
  • c) a complementary nucleotide or RNA sequence corresponding to a sequence as defined in a) or b); d) a nucleotide sequence of a fragment representative of a sequence as defined in a) or c); and e) a sequence as defined in a), or c) modified, are also objects of the invention.
  • nucleic acid nucleic or nucleic acid sequence, polynucleotide, ohgonucleotide, polynucleotide sequence, nucleotide sequence, terms which will be used interchangeably in the present description, is intended to denote a precise sequence of nucleotides, modified or not, making it possible to define a fragment or region of a nucleic acid, which may or may not contain unnatural nucleotides, and which may correspond both to double-stranded DNA, single-stranded DNA and to transcripts of said DNAs.
  • the nucleic acid sequences according to the invention also include PNA (Peptid Nucleic Acid).
  • nucleotide sequences in their natural chromosomal environment that is to say in the natural state. These are sequences which have been isolated and / or purified, that is to say that they have have been taken directly or indirectly, for example by copy, their environment having been at least partially modified. This also means the nucleic acids obtained by chemical synthesis.
  • percentage of identity between two nucleic acid or amino acid sequences within the meaning of the present invention is meant a percentage of identical nucleotides or amino acid residues between the two sequences to be compared, obtained after the best alignment, this percentage being purely statistical and the differences between the two sequences being distributed randomly and over their entire length.
  • the term “best alignment” or “optimal alignment” is intended to denote the alignment for which the percentage of identity determined as below is the highest. Sequence comparisons between two nucleic acid or amino acid sequences are traditionally carried out by comparing these sequences after having optimally aligned them, said comparison being carried out by segment or by "comparison window" to identify and compare the regions. sequence similarity locale.
  • the optimal alignment of the sequences for comparison can be achieved, besides manually, by means of the local homology algorithm of Smith and Waterman (1981, Ad. App. Math., 2: 482), by means of the local homology algorithm of Neddleman and Wunsch (1970, J. Mol. Biol., 48: 443), using the similarity search method of Pearson and Lipman (1988, Proc. Natl. Acad. Sci. USA, 85: 2444), using computer software using these algorithms (GAP, BESTFIT, BLAST P, BLAST N, FASTA and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI).
  • the BLAST program is preferably used with the BLOSUM 62 matrix.
  • the PAM or PAM250 matrices can also be used.
  • the percentage of identity between two nucleic acid or amino acid sequences is determined by comparing these two optimally aligned sequences, the nucleic acid or amino acid sequence to be compared can include additions or deletions by compared to the reference sequence for optimal alignment between these two sequences.
  • the percentage identity is calculated by determining the number of identical positions for which the nucleotide or the amino acid residue is identical in the two sequences, by dividing this number of identical positions by the total number of positions compared and by multiplying the result obtained by 100 to obtain the percentage of identity between these two sequences.
  • nucleic acid sequences having a percentage identity of at least 75%, preferably 80%, 85%, 90%, 95%, 98% or 99%, after optimal alignment with a reference sequence is meant the sequences nucleic acids presenting, with respect to the reference nucleic acid sequence, certain modifications such as in particular a deletion, a truncation, an elongation, a chimeric fusion and / or a substitution, in particular punctual, and whose nucleic sequence presents at least 75%, preferably 80%, 85%, 90%, 95%, 98% or 99% of identity after optimal alignment with the reference nucleic sequence.
  • They are preferably sequences whose complementary sequences are capable of hybridizing specifically with the reference sequences.
  • the specific hybridization conditions or high stringency will be such that they ensure at least 75%, preferably 80%, 85%, 90%, 95%, 98% or 99% of identity after optimal alignment between one of the two sequences and its complementary sequence.
  • Hybridization under conditions of high stringency means that the conditions of temperature and ionic strength are chosen in such a way that they allow hybridization to be maintained between two complementary DNA fragments.
  • high stringency conditions of the hybridization step for the purpose of defining the polynucleotide fragments described above are advantageously as follows.
  • DNA-DNA or DNA-RNA hybridization is carried out in two stages: (1) prehybridization at 42 ° C for 3 hours in phosphate buffer (20 mM, pH 7.5) containing 5 x SSC (1 x SSC corresponds to a 0.15 M NaCl + 0.015 M sodium citrate solution), 50% formamide, 7% sodium dodecyl sulfate (SDS), 10 x Denhardt's, 5% dextran sulfate and 1% salmon sperm DNA; (2) actual hybridization for 20 hours at a temperature depending on the size of the probe (ie: 42 ° C, for a probe of size> 100 nucleotides) followed by 2 washes of 20 minutes at 20 ° C in 2 x SSC + 2% SDS, 1 wash for 20 minutes at 20 ° C in 0.1 x SSC + 0.1% SDS.
  • the last washing is carried out in 0.1 ⁇ SSC + 0.1% SDS for 30 minutes at 60 ° C. for a probe of size> 100 nucleotides.
  • the high stringency hybridization conditions described above for a polynucleotide of defined size can be adapted by the skilled person for oligonucleotides of larger or smaller size, according to the teaching of Sambrook et al. (1989, Molecular cloning: a laboratory manual. 2 nd Ed. Cold Spring Harbor).
  • fragment representative of sequences according to the invention is intended to denote any nucleotide fragment having at least 15 nucleotides, preferably at least 20, 25, 30, 50, 75, 100, 150, 200, 300 and 450 consecutive nucleotides.
  • representative fragment in particular a nucleic sequence coding for a biologically active fragment of a polypeptide, as defined below.
  • representative fragment is also meant the intergenic sequences, and in particular the nucleotide sequences carrying the regulatory signals (promoters, terminators, or even enhancers, etc.).
  • the numbering of the ORFs nucleotide sequences which will be used subsequently in the present description corresponds to the numbering of the amino acid sequences of the proteins encoded by said ORFs for the sequences SEQ 1) No. 42 to SEQ 1) No. 3855.
  • the numbering of the nucleotide sequences ORFs SEQ ⁇ ) N ° 5835 to SEQ ⁇ ) N ° 10784 will be used subsequently in the present description for the numbering of the amino acid sequences of the proteins coded by said ORFs SEQ ⁇ ) N ° 5835 to SEQ II) No. 10784.
  • the representative fragments according to the invention can be obtained for example by specific amplification such as PCR or after digestion with appropriate restriction enzymes of nucleotide sequences according to the invention, this method being described in particular in the book by Sambrook et al. .. Said representative fragments can also be obtained by chemical synthesis when their size is not too large, according to methods well known to those skilled in the art.
  • sequences containing sequences of the invention or representative fragments, one also understands the sequences which are naturally framed by sequences which present at least 75%, 80%, 85%, 90%, 95%, 98% or 99% identity with the sequences according to the invention.
  • modified nucleotide sequence any nucleotide sequence obtained by mutagenesis according to techniques well known to those skilled in the art, and comprising modifications with respect to the normal sequences, preferably at most 10% of nucleotides modified with respect to these normal sequences, for example mutations in the regulatory and / or promoter sequences for the expression of the polypeptide, in particular leading to a modification of the expression or activity level of said polypeptide.
  • modified nucleotide sequence is also meant any nucleotide sequence coding for a modified polypeptide as defined below.
  • the representative fragments according to the invention can also be probes or primers, which can be used in methods of detection, identification, assay or amplification of nucleic sequences.
  • a probe or primer is defined, within the meaning of the invention, as being a fragment of single-stranded nucleic acids or a denatured double-stranded fragment comprising for example from 12 bases to a few kb, in particular from 15 to a few hundred bases, preferably from 15 to 50 or 100 bases, and having a specificity of hybridization under determined conditions to form a hybridization complex with a target nucleic acid.
  • the probes and primers according to the invention can be labeled directly or indirectly with a radioactive or non-radioactive compound by methods well known to those skilled in the art, in order to obtain a detectable and / or quantifiable signal (patent FR 78 10975 and bDNA of Chiron EP 225 807 and EP 510 085).
  • the unlabeled polynucleotide sequences according to the invention can be used directly as a probe or primer.
  • sequences are generally marked to obtain sequences which can be used for numerous applications.
  • the labeling of the primers or probes according to the invention is carried out with radioactive elements or with non-radioactive molecules.
  • Non-radioactive entities are selected from ligands such as biotin, avidin, streptavidin, dioxygenin, haptens , dyes, luminescent agents such as radioluminescent, chemoluminescent, bioluminescent, fluorescent, phosphorescent agents.
  • the polynucleotides according to the invention can thus be used as a primer and / or probe in methods using in particular the PCR technique (polymerase chain reaction) (Rolfs et al., 1991, Berlin: Springer-Verlag).
  • This technique requires the choice of pairs of oligonucleotide primers framing the fragment which must be amplified.
  • the amplified fragments can be identified, for example after agarose or polyacrylamide gel electrophoresis, or after a chromatographic technique such as gel filtration or ion exchange chromatography, and then sequenced.
  • the specificity of the amplification can be controlled by using the nucleotide sequences of polynucleotides of the invention as template, plasmids containing these sequences or even the amplification products derived therefrom.
  • the amplified nucleotide fragments can be used as reagents in hybridization reactions in order to demonstrate the presence, in a biological sample, of a target nucleic acid of sequence complementary to that of said amplified nucleotide fragments.
  • the invention also relates to the nucleic acids capable of being obtained by amplification using primers according to the invention.
  • Other techniques for amplifying the target nucleic acid can advantageously be used as an alternative to PCR (PCR-like) using pairs of primers of nucleotide sequences according to the invention.
  • PCR-like is meant to denote all the methods implementing direct or indirect reproductions of the nucleic acid sequences, or in which the labeling systems have been amplified, these techniques are of course known. In general, it is the amplification of DNA by a polymerase; when the original sample is an RNA, a reverse transcription should be carried out beforehand.
  • the target polynucleotide to be detected is an mRNA
  • an enzyme of reverse transcriptase type in order to obtain a cDNA from the mRNA contained in the biological sample.
  • the cDNA obtained will then serve as a target for the primers or probes used in the amplification or detection method according to the invention.
  • the probe hybridization technique can be performed in various ways (Matthews et al., 1988, Anal. Biochem., 169, 1-25).
  • the most general method consists in immobilizing the nucleic acid extracted from cells of different tissues or cells in culture on a support (such as nitrocellulose, nylon, polystyrene) and incubating, under well defined conditions, the target nucleic acid immobilized with the probe. After hybridization, the excess probe is eliminated and the hybrid molecules formed are detected by the appropriate method (measurement of radioactivity, fluorescence or enzymatic activity linked to the probe).
  • the latter can be used as capture probes.
  • a probe called a “capture probe”
  • a probe is immobilized on a support and is used to capture by specific hybridization the target nucleic acid obtained from the biological sample to be tested and the target nucleic acid is then detected.
  • a second probe called a “detection probe”, marked by an easily detectable element.
  • the antisense oligonucleotides that is to say those whose structure ensures, by hybridization with the target sequence, an inhibition of the expression of the corresponding product. Mention should also be made of sense oligonucleotides which, by interaction with proteins involved in the regulation of the expression of the corresponding product, will induce either an inhibition or an activation of this expression.
  • the probes or primers according to the invention are immobilized on a support, covalently or non-covalently.
  • the support can be a DNA chip or a high or medium density filter, also subject of the present invention (patents WO 97/29212, WO 98/27317, WO 97/10365 and WO 92/10588).
  • DNA chip or high density filter is intended to denote a support on which DNA sequences are fixed, each of which can be identified by its geographic location. These chips or filters differ mainly in their size, the material of the support, and possibly the number of DNA sequences attached to them.
  • the probes or primers according to the first invention can be fixed on solid supports, in particular DNA chips, by various manufacturing methods.
  • a synthesis can be carried out in situ by photochemical addressing or by ink jet.
  • Other techniques consist in carrying out an ex situ synthesis and in fixing the probes on the support of the DNA chip by mechanical, electronic or inkjet addressing. These different methods are well known to those skilled in the art.
  • a nucleotide sequence (probe or primer) according to the invention therefore allows the detection and / or amplification of specific nucleic sequences.
  • the detection of these said sequences is facilitated when the probe is fixed to a DNA chip, or to a high density filter.
  • DNA chips or high density filters makes it possible to determine the expression of genes in an organism having a genomic sequence close to P. luminescens and the typing of the strain in question.
  • the genomic sequence of P. luminescens supplemented by the identification of the genes of these organisms, as presented in the present invention, serve as the basis for the construction of these DNA chips or filter.
  • the preparation of these filters or chips consists in synthesizing oligonucleotides, corresponding to the 5 ′ and 3 ′ ends of the genes or to more internal fragments to amplify fragments of a suitable size, for example between approximately 300 and 800 bases.
  • oligonucleotides are chosen using the genomic sequence and its annotations disclosed by the present invention.
  • the pairing temperature of these oligonucleotides at the corresponding places on the DNA must be approximately the same for each oligonucleotide. This makes it possible to prepare DNA fragments corresponding to each gene by the use of appropriate PCR conditions in a highly automated environment.
  • the amplified fragments are then immobilized on filters or supports in glass, silicon or synthetic polymers and these media are used for hybridization.
  • the differences between the genomic sequences of the different strains or species can greatly affect the intensity of the hybridization and therefore disturb the interpretation of the results. It may therefore be necessary to have the precise sequence of genes of the strain that one wishes to study.
  • the method of detecting genes described later in detail, involving determining the sequence of random fragments of a genome, and organizing them according to the genome sequence of P. luminescens, in particular of P. luminescens TTOl, disclosed in the present invention can be very useful.
  • the nucleotide sequences according to the invention can be used in DNA chips to carry out the analysis of mutations. This analysis is based on the constitution of chips capable of analyzing each base of a nucleotide sequence according to the invention. In particular, it will be possible to implement microsequencing techniques on a DNA chip.
  • the mutations are detected by extension of immobilized primers hybridizing to the matrix of the sequences analyzed, just in position adjacent to that of the mutated nucleotide sought.
  • a single-stranded matrix, RNA or DNA, of the sequences to be analyzed will advantageously be prepared according to conventional methods, from products amplified according to PCR type techniques.
  • the single-stranded DNA or RNA matrices thus obtained are then deposited on the DNA chip, under conditions allowing their specific hybridization to the immobilized primers.
  • a thermostable polymerase for example Tth or Taq DNA polymerase, specifically extends the 3 'end of the immobilized primer with a labeled nucleotide analog complementary to the nucleotide at the variable site position; for example, thermal cycling is carried out in the presence of fluorescent dideoxyribonucleotides.
  • the experimental conditions will be adapted in particular to the chips used, to the immobilized primers, to the polymerases used, and to the chosen labeling system.
  • DNA chips according to the invention are also preferred, which also contain at least one nucleotide sequence of a microorganism other than Photorhabdus luminescens, immobilized on the support of said chip.
  • the microorganism chosen is from bacteria of the genus Photorhabdus (hereinafter designated as bacteria associated with P. luminescens), or variants of Photorhabdus luminescens TTOl.
  • a DNA chip or a filter according to the invention is a very useful element in certain kits or necessary for the detection and / or identification of microorganisms, in particular bacteria belonging to the species Photorhabdus luminescens, also subject of the invention.
  • DNA chips or filters according to the invention containing probes or primers specific to Photorhabdus luminescens, are very advantageous elements of kits or necessary for the detection and / or quantification of the expression of photorhabdus genes luminescens.
  • the control of gene expression is a critical point for optimizing the growth and yield of a strain, either by allowing the expression of one or more new genes, or by modifying the expression of genes already present in the cell.
  • the present invention provides all the naturally active sequences in P. luminescens allowing gene expression. It thus allows the determination of all the sequences expressed in P. luminescens. It also provides a tool for identifying genes whose expression follows a given pattern. To achieve this, the DNA of all or part of the P. luminescens genes can be amplified using primers according to the invention, then fixed to a support such as for example glass or nylon or a DNA chip, in order to build a tool to monitor the expression profile of these genes.
  • This tool consisting of this support containing the coding sequences, serves as a hybridization matrix for a mixture of labeled molecules reflecting the messenger RNAs expressed in the cell (in particular the labeled probes according to the invention). By repeating this experience at different times and by combining all of these data by appropriate processing, the expression profiles of all of these genes are then obtained.
  • Knowledge of the sequences which follow a given regulatory scheme can also be used to search in a directed manner, for example by homology, for other sequences following globally, but in a slightly different manner the same regulatory scheme.
  • each control sequence present upstream of the segments serving as probes and to monitor their activity using an appropriate means such as a reporter gene (luciferase, ⁇ -galactosidase, GFP).
  • a reporter gene luciferase, ⁇ -galactosidase, GFP
  • the invention also relates to the polypeptides encoded by a nucleotide sequence according to the invention, preferably, by a fragment representative of the preceding sequences and corresponding to an ORF sequence.
  • the photorhabdus luminescens TTO1 polypeptides of SEQ ⁇ ) N ° 42 to SEQ ⁇ ) N ° 3855 or coded by SEQ ⁇ ) N ° 5835 to SEQ ⁇ ) N ° 10784 are subject of the invention.
  • the invention also includes the polypeptides characterized in that they comprise a polypeptide chosen from: a) a polypeptide of sequence SEQ ⁇ ) N ° 42 to SEQ ⁇ ) N ° 3855 or coded by a sequence SEQ ⁇ ) N ° 5835 to SEQ 11) No. 10784; b) a polypeptide having at least 80%, preferably 85%, 90%, 95% and 98% identity with a polypeptide according to the invention; c) a fragment of at least 5 amino acids of a polypeptide as defined in a); d) a biologically active fragment of a polypeptide as defined in a); and e) a polypeptide as defined in a), b), c) or d) modified.
  • the nucleotide sequences coding for the polypeptides described above are also subject of the invention.
  • polypeptides in the present description, the terms polypeptides, polypeptide sequences, peptides and proteins are interchangeable.
  • polypeptide includes any amino acid sequence used to generate an antibody response. It should be understood that the invention does not relate to polypeptides in natural form, that is to say that they are not taken in their natural environment. On the other hand, it concerns those which could have been isolated or obtained by purification from natural sources, or else obtained by genetic recombination, or by synthesis chemical, and that they can then contain unnatural amino acids as will be described later.
  • polypeptide having a certain percentage of identity with another which will also be designated by homologous polypeptide, is intended to denote the polypeptides having, with respect to the natural polypeptides, certain modifications, in particular a deletion, addition or substitution of at least an amino acid, truncation, elongation, chimeric solution and / or mutation, or polypeptides with post-translational modifications.
  • homologous polypeptides those whose amino acid sequence has at least 80%, preferably 85%, 90%, 95%, 98% or 99% of identity after optimal alignment with the amino acid sequences are preferred. polypeptides according to the invention.
  • one or more consecutive or non-consecutive amino acid (s) may be replaced by “equivalent” amino acids.
  • equivalent amino acids is intended here to denote any amino acid capable of being substituted for one of the amino acids of the basic structure without, however, essentially modifying the biological activities of the corresponding peptides as defined by after.
  • Leucine can thus be replaced by valine or isoleucine, aspartic acid by glutamine acid, glutamine by asparagine, Targinine by lysine, etc., the reverse substitutions being naturally possible under the same conditions.
  • the homologous polypeptides also correspond to the polypeptides encoded by the nucleotide sequences having a certain percentage of identity with the nucleotide sequences of the invention or identical, as defined above and thus include in the present definition polypeptides which are mutated or correspond to inter variations or intra-species, which may exist in Photorhabdus, and which correspond in particular to truncations, substitutions, deletions and / or additions, of at least one amino acid residue. It is understood that the percentage of identity between two polypeptides is calculated in the same way as between two nucleic acid sequences. Thus, the percentage of identity between two polypeptides is calculated after optimal alignment of these two sequences, over a window of maximum homology. To define said maximum homology window, the same algorithms can be used as for the nucleic acid sequences.
  • biologically active fragment of a polypeptide according to the invention is intended to denote in particular a fragment of polypeptide, as defined below, having at least one of the biological characteristics of the polypeptides according to the invention, in particular in that it is able to exercise in general even a partial activity, such as for example:
  • an enzymatic (metabolic) activity an activity which may be involved in the biosynthesis or biodegradation of organic or inorganic compounds, or preferably a toxic or antibiotic activity, in particular for insects or micro-organisms (bacteria or fungi), or else an activity involved in the biosynthesis of these toxins or antibiotics; such proteins with enzymatic activity may in particular be used in methods of screening and / or selection of compounds capable of modifying this activity, in particular of inhibiting it.
  • proteins corresponding in particular to extramembrane proteins could in particular be used as immunogen for the production of antibodies, mono- or polyclonal directed specifically against these extramembrane proteins;
  • polypeptide fragment according to the invention is intended to denote a polypeptide comprising at least 5 amino acids, preferably 10, 15, 25, 50, 100 and 150 amino acids.
  • Polypeptide fragments can also be prepared by chemical synthesis, from hosts transformed by an expression vector according to the invention which contain a nucleic acid allowing the expression of said fragment, and placed under the control of regulatory elements and / or appropriate expression.
  • modified polypeptides mention should be made of the polypeptides in which up to five amino acids can be modified, truncated at the N or C-terminus, or else deleted, or added.
  • Chemical synthesis also has the advantage of being able to use unnatural amino acids or non-peptide bonds. Thus, it may be advantageous to use unnatural amino acids, for example in D form, or analogs of amino acids, in particular sulfur forms.
  • the invention further relates to the nucleic or peptide sequences according to the present invention with the exception of the nucleic or peptide sequences described in WO 99/54472, WO 99/42589, WO 99/03328, WO 98/08932 and EP 0 823 215.
  • the present invention provides the nucleotide sequence of the genome of Photorhabdus luminescens TTOl in the form of 41 contigs or in the form of 9 contigs, as well as certain polypeptide sequences.
  • nucleic or peptide sequences characterized by their function can also be identified by their nucleotide and amino acid sequence by referring to Table I.
  • the invention relates to a nucleotide sequence according to the invention, characterized in that it codes for a polypeptide of Photorhabdus luminescens TTOl with activity of toxin and / or antibiotic type, or involved in the synthesis of these toxins and / or antibiotics.
  • the invention relates to a nucleotide sequence according to the invention, characterized in that it codes for a polypeptide of Photorhabdus luminescens TTOl or one of its fragments involved in the biosynthesis of amino acids.
  • the invention relates to a nucleotide sequence according to the invention, characterized in that it codes for a photorhabdus luminescens TTOl polypeptide or one of its fragments involved in the biosynthesis of co factors, prosthetic groups and transporters.
  • the invention relates to a nucleotide sequence according to the invention, characterized in that it codes for a cell envelope polypeptide or present on the surface of Photorhabdus luminescens TTOl or for one of its fragments.
  • the invention relates to a nucleotide sequence according to the invention, characterized in that it codes for a polypeptide of Photorhabdus luminescens TTOl or one of its fragments involved in the cellular machinery.
  • the invention relates to a nucleotide sequence according to the invention, characterized in that it codes for a polypeptide of Photorhabdus luminescens TTOl or one of its fragments involved in central intermediate metabolism.
  • the invention relates to a nucleotide sequence according to the invention, characterized in that it codes for a polypeptide of Photorhabdus luminescens TTOl or one of its fragments involved in energy metabolism.
  • the invention relates to a nucleotide sequence according to the invention, characterized in that it codes for a polypeptide of Photorhabdus luminescens TTOl or one of its fragments involved in the metabolism of fatty acids and phospholipids.
  • the invention relates to a nucleotide sequence according to the invention, characterized in that it codes for a polypeptide of Photorhabdus luminescens TTOl or one of its fragments involved in the metabolism of nucleotides, purines, pyrimidines or nucleosides.
  • the invention relates to a nucleotide sequence according to the invention, characterized in that it codes for a polypeptide of Photorhabdus luminescens TTOl or one of its fragments involved in regulatory functions.
  • the invention relates to a nucleotide sequence according to the invention, characterized in that it codes for a polypeptide of Photorhabdus luminescens TTOl or one of its fragments involved in the replication process.
  • the invention relates to a nucleotide sequence according to the invention, characterized in that it codes for a polypeptide of Photorhabdus luminescens TTOl or one of its fragments involved in the transcription process.
  • the invention relates to a nucleotide sequence according to the invention, characterized in that it codes for a polypeptide of Photorhabdus luminescens TTOl or one of its fragments involved in the translation process.
  • the invention relates to a nucleotide sequence according to the invention, characterized in that it codes for a photorhabdus luminescens TTOl polypeptide or one of its fragments involved in the protein transport and binding process.
  • the invention relates to a nucleotide sequence according to the invention, characterized in that it codes for a polypeptide of Photorhabdus luminescens TTOl or one of its fragments involved in the adaptation to atypical conditions.
  • the invention relates to a nucleotide sequence according to the invention, characterized in that it codes for a polypeptide of Photorhabdus luminescens TTOl or one of its fragments in the sensitivity to drugs and the like.
  • the invention relates to a nucleotide sequence according to the invention, characterized in that it codes for a photorhabdus polypeptide luminescens TTOl or one of its fragments involved in the functions relating to transposons.
  • the invention relates to a nucleotide sequence according to the invention, characterized in that it codes for a polypeptide specific for Photorhabdus luminescens TTOl or one of its fragments.
  • the invention relates to a polypeptide according to the invention, characterized in that it is a polypeptide of Photorhabdus luminescens TTOl or one of its activity fragments of toxin type and / or antibiotic, or involved in the synthesis of these toxins and / or antibiotics.
  • the invention relates to a polypeptide according to the invention, characterized in that it is a polypeptide of Photorhabdus luminescens TTOl or one of its fragments involved in the biosynthesis of acids amines.
  • the invention relates to a polypeptide according to the invention, characterized in that it is a polypeptide of Photorhabdus luminescens TTOl or one of its fragments involved in the biosynthesis of cofactors , prosthetic groups and carriers.
  • the invention relates to a polypeptide according to the invention, characterized in that it is a cell envelope or surface polypeptide of Photorhabdus luminescens TTOl or one of its fragments.
  • the invention relates to a polypeptide according to the invention, characterized in that it is a polypeptide of Photorhabdus luminescens TTOl or one of its fragments involved in cellular machinery.
  • the invention relates to a polypeptide according to the invention, characterized in that it is a polypeptide of Photorhabdus luminescens TTOl or one of its fragments involved in central intermediate metabolism .
  • the invention relates to a polypeptide according to the invention, characterized in that it is a polypeptide of Photorhabdus luminescens TTOl or one of its fragments involved in energy metabolism.
  • the invention relates to a polypeptide according to the invention, characterized in that it is a polypeptide of Photorhabdus luminescens TTOl or one of its fragments involved in the metabolism of acids fatty and phospholipids.
  • the invention relates to a polypeptide according to the invention, characterized in that it is a polypeptide of Photorhabdus luminescens TTOl or one of its fragments involved in the metabolism of nucleotides , purines, pyrimidines or nucleosides.
  • the invention relates to a polypeptide according to the invention, characterized in that it is a polypeptide of Photorhabdus luminescens TTOl or one of its fragments involved in the regulatory functions .
  • the invention relates to a polypeptide according to the invention, characterized in that it is a polypeptide of Photorhabdus luminescens TTOl or one of its fragments involved in the replication process .
  • the invention relates to a polypeptide according to the invention, characterized in that it is a polypeptide of Photorhabdus luminescens TTOl or one of its fragments involved in the transcription process .
  • the invention relates to a polypeptide according to the invention, characterized in that it is a polypeptide of Photorhabdus luminescens TTOl or one of its fragments involved in the translation process .
  • the invention relates to a polypeptide according to the invention, characterized in that it is a polypeptide of Photorhabdus luminescens TTOl or one of its fragments involved in the transport process and protein binding.
  • the invention relates to a polypeptide according to the invention, characterized in that it is a polypeptide of Photorhabdus luminescens TTOl or one of its fragments involved in the adaptation to atypical conditions.
  • the invention relates to a polypeptide according to the invention, characterized in that it is a polypeptide of Photorhabdus luminescens TTOl or a fragment thereof in sensitivity to drugs and the like.
  • the invention relates to a polypeptide according to the invention, characterized in that it is a polypeptide of Photorhabdus luminescens TTOl or one of its fragments involved in the functions relating to transposons.
  • the invention relates to a polypeptide according to the invention, characterized in that it is a polypeptide specific for Photorhabdus luminescens TTOl or one of its fragments.
  • the invention also relates to the operons involved in the synthesis of antibiotics and / or toxins.
  • Table I provides the list of certain polypeptides according to the invention, as well as their location in the contigs represented by SEQ 1) No. 1 to SEQ 1) No. 41, and the analogies observed after comparison in the databases.
  • Table ⁇ provides the list of certain polypeptides according to the invention, as well as their location in the contigs represented by SEQ ⁇ ) N ° 5826 to SEQ ⁇ ) N ° 5834, and the analogies observed after comparison in the databases.
  • contig 1 to 9 are identified by the sequences SEQ ⁇ ) N ° 5826 to SEQ ⁇ ) N ° 5834.
  • the subject of the invention is also the polypeptides whose functions annotated with table I, last column, or whose functions annotated in table ⁇ , penultimate column, correspond to toxin and / or antibiotic type activities, or to polypeptides involved in the synthesis of these toxins and / or antibiotics, preferably polypeptides chosen from: a) the polypeptides of sequence SEQ ⁇ ) N ° 61, SEQ ⁇ ) N ° 62, SEQ ⁇ ) N ° 67, SEQ ⁇ ) N ° 171, SEQ ⁇ ) N ° 221, SEQ ⁇ ) N ° 268 , SEQ ⁇ ) N ° 288, SEQ ⁇ ) N ° 380, SEQ ⁇ ) N ° 426, SEQ ⁇ ) N ° 438, SEQ ⁇ ) N ° 448, SEQ ⁇ ) N ° 453, SEQ ⁇ ) N ° 455, SEQ ⁇ ) N ° 456, SEQ ⁇ ) N °
  • a subject of the present invention is also the nucleotide and / or polypeptide sequences according to the invention, characterized in that said sequences are recorded on a recording medium the shape and nature of which facilitate reading, analysis and / or the exploitation of said sequence (s).
  • These supports can also contain other information extracted from the present invention, in particular analogies with already known sequences, and / or information concerning the nucleotide and / or polypeptide sequences of a plant cell, of an animal or a micro-organism other than P.
  • luminescens in particular a cell or micro-organism sensitive to a toxin or an antibiotic produced by P. luminescens, a bacterium of the genus Photorhabdus, or a variant of P.
  • luminescens this in order to facilitate comparative analysis and exploitation of the results obtained.
  • recording media particular preference is given to media readable by a computer, such as magnetic, optical, electrical or hybrid media, in particular computer floppy disks, CD-ROMs, computer servers.
  • Such recording media are also subject of the invention.
  • the recording media according to the invention are very useful for the choice of primers or nucleotide probes for the determination of genes in Photorhabdus luminescens TTOl or strains close to this organism.
  • the use of these supports for the study of the genetic polymorphism of strains close to Photorhabdus luminescens TTOl, in particular by the determination of the regions of collinearity is very useful insofar as these supports provide not only the nucleotide sequence of the genome of Photorhabdus luminescens TTOl, but also the genomic organization in said sequence.
  • sequence comparison software such as software
  • the invention also relates to the cloning and / or expression vectors, which contain a nucleotide sequence according to the invention.
  • the vectors according to the invention preferably comprise elements which allow the expression and / or the secretion of the nucleotide sequences in a determined host cell.
  • the vector must then include a promoter, translation initiation and termination signals, as well as suitable regions for transcription regulation. It must be able to be maintained stably in the host cell and may possibly have specific signals which specify the secretion of the translated protein. These various elements are chosen and optimized by a person skilled in the art according to the cell host used. To this end, the nucleotide sequences according to the invention can be inserted into vectors with autonomous replication within the chosen host, or can be vectors integrating with the chosen host.
  • vectors are prepared by methods commonly used by those skilled in the art, and the resulting clones can be introduced into an appropriate host by standard methods, such as lipofection, electroporation, heat shock, or chemical methods .
  • the vectors according to the invention are for example vectors of plasmid or viral origin. They are useful for transforming host cells in order to clone or express the nucleotide sequences according to the invention.
  • the cloning and / or expression vectors according to the invention are also preferred, characterized in that they contain a nucleotide sequence chosen from the sequences SEQ ⁇ ) N ° 3856 to SEQ ⁇ ) N ° 5825 , SEQ ⁇ ) N ° 5835 to SEQ ⁇ ) N ° 10784 or their fragment derived from the genome of P. luminescens, in particular the sequences coding for the polypeptides with toxin activity, antibiotics or involved in these activities, in particular those mentioned above whose functions annotated in table I below correspond to these activities.
  • the invention also includes host cells transformed with a vector according to the invention.
  • the cell host can be chosen from prokaryotic or eukaryotic systems, for example bacterial cells but also yeast cells or animal cells, in particular mammalian cells. You can also use insect cells or plant cells.
  • the preferred host cells according to the invention are in particular prokaryotic cells, preferably bacteria belonging to the genus Photorhabdus or to the species Photorhabdus luminescens, more particularly Photorhabdus luminescens TTOl.
  • the invention also relates to plants and animals, except humans, which comprise a transformed cell according to the invention.
  • the cells transformed according to the invention can be used in processes for the preparation of recombinant polypeptides according to the invention.
  • the methods for preparing a polypeptide according to the invention in recombinant form characterized in that they use a vector and / or a cell transformed with a vector according to the invention are themselves included in the present invention.
  • a cell transformed with a vector according to the invention is cultivated under conditions which allow the expression of said polypeptide and said recombinant peptide is recovered.
  • the cell host can be chosen from prokaryotic or eukaryotic systems.
  • a vector according to the invention carrying such a sequence can therefore be advantageously used for the production of recombinant proteins, intended to be secreted. Indeed, the purification of these recombinant proteins of interest will be facilitated by the fact that they are present in the supernatant of the cell culture rather than inside the host cells.
  • the polypeptides according to the invention can also be prepared by chemical synthesis. Such a preparation process is also an object of the invention.
  • a person skilled in the art knows the chemical synthesis processes, for example the techniques implementing solid phases (see in particular Steward et al., 1984, Solid phase peptides synthesis, Pierce Chem. Company, Rockford, 111, 2nd ed., (1984)) or techniques using partial solid phases, by condensation of fragments or by synthesis in conventional solution.
  • the polypeptides obtained by chemical synthesis and possibly comprising corresponding non-natural amino acids are also included in the invention.
  • hybrid polypeptides according to the invention are very useful for obtaining monoclonal or polyclonal antibodies capable of specifically recognizing the polypeptides according to the invention.
  • polyclonal or monoclonal antibodies can be obtained by standard methods well known to those skilled in the art, after immunization of a mammal using these polypeptides (or their corresponding nucleic acid) or for example according to the conventional method of hybridoma culture described by Kohler and Milstein (1975, Nature, 256, 495) for monoclonal antibodies.
  • Such monoclonal or polyclonal antibodies, their fragments, or chimeric antibodies, recognizing the polypeptides according to the invention, are also objects of the invention.
  • the antibodies according to the invention are, for example, chimeric antibodies, humanized antibodies, Fab fragments, or F (ab ') 2 . They can also be in the form of immunoconjugates or labeled antibodies in order to obtain a detectable and / or quantifiable signal.
  • the antibodies according to the invention can be used in a method for the detection and / or identification of bacteria belonging to the genus Photorhabdus and / or to the species Photorhabdus luminescen in a biological sample, characterized in that it comprises the following steps: a) bringing the biological sample into contact with an antibody according to the invention; b) highlighting of the antigen-antibody complex possibly formed.
  • the antibodies according to the present invention are also usable in order to detect an expression of a gene of Photorhabdus luminescens TTOl. Indeed, the presence of the expression product of a gene recognized by an antibody specific for said expression product can be detected by the presence of an antigen-antibody complex formed after contacting the strain of Photorhabdus luminescens TTOl with an antibody according to the invention.
  • the bacterial strain used may have been "prepared", that is to say centrifuged, lysed, placed in a reagent suitable for constituting the medium suitable for the immunological reaction.
  • a method of detecting expression in the gene, corresponding to a Western blot which can be carried out after an electrophoresis on polyacrylamide gel of a lysate of the bacterial strain, is preferred, in the presence or in the absence of reducing conditions (SDS- PAGE).
  • SDS- PAGE reducing conditions
  • the present invention also comprises the kits or kits necessary for the implementation of a method as described (for detecting the expression of a gene of Photorhabdus luminescens TTOl, or for the detection and / or identification of bacteria belonging to the species Photorhabdus luminescens), comprising the following elements: a) a polyclonal or monoclonal antibody according to the invention; b) optionally, the reagents for constituting the medium suitable for the immunological reaction; c) optionally, the reagents allowing the detection of the antigen-antibody complexes produced by the immunological reaction.
  • polypeptides and antibodies according to the invention can advantageously be immobilized on a support, in particular a protein chip.
  • a protein chip is an object of the invention, and may also contain at least one polypeptide from a microorganism other than Photorhabdus luminescens or an antibody directed against a compound of a microorganism other than Photorhabdus luminescens.
  • the protein chips or high density filters containing proteins according to the invention can be constructed in the same way as the DNA chips according to the invention.
  • the latter method is preferable, when it is desired to attach proteins of large size to the support, these proteins being advantageously prepared by genetic engineering.
  • the protein chips according to the invention can advantageously be used in kits or necessary for the detection and / or identification of bacteria associated with the species Photorhabdus luminescens or with a microorganism, or more generally in kits or necessary for the detection and / or identification of microorganisms.
  • the polypeptides according to the invention are fixed on the DNA chips, one searches for the presence of antibodies in the samples tested, the fixing of a antibody according to the invention on the support of the protein chip allowing the identification of the protein of which said antibody is specific.
  • an antibody according to the invention is fixed on the support of the protein chip, and the presence of the corresponding antigen, specific for Photorhabdus luminescens, is detected.
  • a protein chip described above can be used for the detection of gene products, to establish an expression profile of said genes, in addition to a DNA chip according to the invention.
  • the protein chips according to the invention are also extremely useful for proteomics experiments, which studies the interactions between the different proteins of a plant cell, an animal, such as an insect, or a micro- organism other than P. luminescens.
  • the invention also comprises a protein chip according to the invention, characterized in that it also contains at least one polypeptide from a plant cell, from an animal or from a microorganism other than P luminescens, immobilized on the support of said chip, preferably said cell or other microorganism is chosen from a cell or microorganism sensitive to a toxin or an antibiotic produced by P. luminescens.
  • peptides representative of the various proteins of an organism are fixed on a support. Then, said support is brought into contact with labeled proteins, and after an optional rinsing step, interactions between said labeled proteins and the peptides fixed on the protein chip are detected.
  • protein chips comprising a polypeptide sequence according to the invention or an antibody according to the invention are subject of the invention, as well as the kits or kits containing them.
  • the present invention also covers a method for detecting and / or identifying bacteria belonging to the species Photorhabdus luminescens in a biological sample, which implements a nucleotide sequence according to the invention.
  • biological sample relates in the present invention to samples taken from a living organism (in particular blood, tissues, organs or the like taken from a mammal) or a sample containing biological material, that is, DNA or RNA.
  • a biological sample also includes food compositions containing bacteria (for example cheeses, dairy products), but also food compositions containing yeasts (beers, breads) or others.
  • the term biological sample also relates to bacteria isolated from these samples or food compositions.
  • the detection and / or identification process using the nucleotide sequences according to the invention can be of various nature.
  • a method is preferred comprising the following steps: a) optionally, isolation of the DNA from the biological sample to be analyzed, or obtaining a cDNA from the RNA of the biological sample; b) specific amplification of the DNA of bacteria belonging to the species Photorhabdus luminescens using at least one primer according to the invention; c) highlighting of the amplification products.
  • This process is based on specific amplification of DNA, in particular by an amplification chain reaction.
  • a method is also preferred comprising the following steps: a) bringing a nucleotide probe according to the invention into contact with a biological sample, the nucleic acid contained in the biological sample having, if necessary, previously been made accessible to hybridization, under conditions allowing hybridization of the probe to the nucleic acid of a bacterium belonging to the species Photorhabdus luminescens; b) demonstration of the hybrid possibly formed between the nucleotide probe and the DNA of the biological sample.
  • Such a method should not be limited to the detection of the presence of the DNA contained in the biological sample to be tested, it can also be implemented to detect the RNA contained in said sample.
  • This process includes in particular the Southern and Northern blot.
  • Another preferred method according to the invention comprises the following steps: a) bringing a nucleotide probe immobilized on a support according to the invention into contact with a biological sample, the nucleic acid of the sample, having, where appropriate , been previously made accessible to hybridization, under conditions allowing hybridization of the probe to the nucleic acid of a bacterium belonging to the species Photorhabdus luminescens; b) bringing the hybrid formed into contact between the nucleotide probe immobilized on a support and the nucleic acid contained in the biological sample, where appropriate after elimination of the DNA from the biological sample which has not hybridized with the probe, with a labeled nucleotide probe according to the invention; c) highlighting of the new hybrid formed in step b).
  • This method is advantageously used with a DNA chip according to the invention, the desired nucleic acid hybridizing with a probe present on the surface of said chip, and being detected by the use of a labeled probe.
  • This method is advantageously implemented by combining a prior step of amplification of the DNA or of the complementary DNA optionally obtained by reverse transcription, using primers according to the invention.
  • kits or kits for the detection and / or identification of bacteria belonging to the species Photorhabdus luminescens characterized in that it comprises the following elements: a) a nucleotide probe according to the invention ; b) optionally, the reagents necessary for carrying out a hybridization reaction; c) optionally, at least one primer according to the invention as well as the reagents necessary for a DNA amplification reaction.
  • kits or kits for the detection and / or identification of bacteria belonging to the species Photorhabdus luminescens TTOl characterized in that it comprises the following elements: a) a nucleotide probe, called capture probe according to the invention; b) an oligonucleotide probe, called the revelation probe, according to the invention; c) optionally, at least one primer according to the invention as well as the reagents necessary for a DNA amplification reaction.
  • kits or kits for the detection and / or identification of bacteria belonging to the Photorhabdus luminescens species characterized in that they comprise the following elements: a) at least one primer or probe according to the invention; b) optionally, the reagents necessary to carry out a DNA amplification reaction; c) optionally, a component making it possible to verify the sequence of the amplified fragment, more particularly an oligonucleotide probe according to the invention, are also objects of the present invention.
  • said primers and / or probes and / or polypeptides and / or antibodies according to the present invention used in the methods and / or kits or necessary according to the present invention are chosen from primers and / or probes and / or polypeptides and / or specific antibodies for the species Photorhabdus luminescens.
  • these elements are chosen from the nucleotide sequences coding for a secreted protein, from secreted polypeptides, or from antibodies directed against secreted polypeptides of Photorhabdus luminescens.
  • the subject of the present invention is also the strains of Photorhabdus luminescens TTOl containing one or more mutation (s) in a nucleotide sequence according to the invention, in particular an ORF sequence, or their regulatory elements (in particular promoters).
  • the strains of Photorhabdus luminescens TTOl having one or more mutation (s) in the nucleotide sequences coding for polypeptides preferably with activity of toxin or antibiotic type, or involved in their biosynthesis, or alternatively under a another aspect involved in cellular machinery, in particular secretion, central intermediate metabolism, energy metabolism, the processes of amino acid synthesis, transcription and translation, synthesis of polypeptides.
  • Said mutations can lead to inactivation of the gene, or in particular when they are located in the regulatory elements of said gene, to overexpression of the latter.
  • the strains of Photorhabdus luminescens TTOl having one or more mutation (s) can be used to validate the wild gene function of Photorhabdus luminescens.
  • the invention further relates to the use of a nucleotide sequence according to the invention, a polypeptide according to the invention, an antibody according to the invention, and / or a cell according to the invention, for the selection of an organic or inorganic compound capable of modulating, regulating, inducing or inhibiting the expression of genes of a plant or animal cell, or of a microorganism other than P. luminescens, of which, by For example, we want to modify the resistance or the sensitivity to at least one toxin or antibiotic produced by P. luminescens.
  • the invention also includes a method of selecting compounds capable of binding to a polypeptide or a fragment thereof according to the invention, capable of binding to a nucleotide sequence according to the invention, or capable of recognizing an antibody according to invention, and / or capable of modulating, regulating, inducing or inhibiting gene expression, and / or modifying the growth or cellular replication of eukaryotic or prokaryotic cells, or capable of inducing, inhibit or increase in an animal or plant organism resistance or sensitivity to at least one toxin or antibiotic produced by P.
  • luminescens said method comprising the following steps: a) bringing said compound into contact with said polypeptide, said nucleotide sequence and / or with a cell transformed according to the invention; b) determining the capacity of said compound to bind with said polypeptide or said nucleotide sequence, or to modulate, regulate, induce or inhibit the expression of genes, or to modulate cell growth or replication, or to induce, inhibit or increase in an animal or plant organism resistance or sensitivity to at least one toxin or antibiotic produced by P. luminescens.
  • the cells transformed according to the invention can advantageously serve as a model and be used in methods for studying, identifying and / or selecting compounds capable of being responsible for the resistance or the sensitivity to at least one toxin or antibiotic produced by P luminescens.
  • the compounds which can be selected can be organic compounds such as polypeptides or carbohydrates or any other organic or inorganic compounds already known, or new organic compounds produced from molecular modeling techniques and obtained by chemical or biochemical synthesis. , these techniques being known to those skilled in the art.
  • the invention relates to compounds capable of being selected by a selection method according to the invention.
  • the invention also relates to a composition, in particular pesticide or pharmaceutical, comprising a compound chosen from the following compounds: a) a nucleotide sequence according to the invention; b) a polypeptide according to the invention; c) a vector according to the invention; d) an antibody according to the invention; and e) a compound capable of being selected by a selection method according to the invention, optionally in combination with a pharmaceutically acceptable vehicle.
  • the invention also relates to a pharmaceutical composition according to the invention for the prevention or treatment of an infection by a microorganism, such as a bacteria or a fungus, sensitive to at least one toxin or antibiotic produced by P. luminescens.
  • a microorganism such as a bacteria or a fungus
  • the invention also relates to a pesticidal composition, in particular against insects, bacteria and / or fungi, according to the invention, for the prevention or treatment of plants infected by animals, such as insects, or by a microorganism, such as '' a bacterium or a fungus, sensitive to at least one toxin or antibiotic produced by P. luminescens.
  • the invention also includes the use of a transformed cell according to the invention, for the preparation of a toxin or an antibiotic produced by P. luminescens.
  • pharmaceutically acceptable vehicle is intended to denote a compound or a combination of compounds entering into a pharmaceutical composition which does not cause side reactions and which allows for example the facilitation of the administration of the active compound, the increase in its lifespan and / or of its effectiveness in the organism, the increase of its solubility in solution or the improvement of its conservation.
  • pharmaceutically acceptable vehicles are well known and will be adapted by those skilled in the art depending on the nature and the mode of administration of the active compound chosen.
  • these pharmaceutical compounds will be administered by the systemic route, in particular by the intravenous route, by the intramuscular, intradermal or subcutaneous route, or by the oral route.
  • dosages and dosage forms can be determined according to the criteria generally taken into account in establishing a treatment adapted to a patient such as for example the patient's age or body weight, the severity of his general condition, tolerance to treatment and side effects observed.
  • the invention comprises the use of a composition according to the invention, for the preparation of a medicament intended for the prevention or the treatment of an infection by a microorganism, such as a bacterium or a fungus , sensitive to at least one toxin or antibiotic produced by P. luminescens.
  • the present invention also relates to a genomic DNA library of a bacterium of the genus Photorhabdus, preferably, Photorhabdus luminescens, preferably the strain TTOl.
  • the genomic DNA libraries described in the present invention in particular the BAC library deposited at the CNCM on May 12, 2000 under the number 1-2478 and which covers the genome of Photorhabdus luminescens TTOl.
  • the invention also relates to a method of identifying at least one nucleotide sequence of P. luminescens not present in the genome of another species of bacteria, in particular pathogenic, and / or of identifying at least one nucleotide sequence of a genome of a bacterium, in particular pathogenic, of a species other than P. luminescens and not present in the genome of P. luminescens, characterized in that it comprises the following stages: a) the nucleotide sequences are aligned of P.
  • luminescens according to the invention or contained in a genomic library according to the invention, with the genomic sequence of the other species of bacteria, or one of its fragments; and b) the data obtained by this alignment are processed to isolate and identify the said sequence or sequences only present in one or the other genome.
  • a method also called “subtractive genomics” can be used here to identify a sequence responsible for the pathogenicity of a bacterium, such as a gram negative bacterium, of which the non-pathogenic P. luminescens bacterium can serve as a model for comparison.
  • the present invention thus relates to methods for the isolation of a polynucleotide of interest present in a strain of Photorhabdus and absent in another strain or species, which uses at least one DNA library based for example on a plasmid pcDNA2. 1 containing the Photorhabdus genome.
  • the method according to the invention for the isolation of a polynucleotide of interest can comprise the following steps: a) isolating at least one polynucleotide contained in a clone of the original DNA library of Photorhabdus; b) isolate:
  • At least one genomic polynucleotide or cDNA of a bacterium said bacterium belonging to a strain or species different from the Photorhabdus strain used for the construction of the DNA library of step a) or, alternatively,
  • step b at least one polynucleotide contained in a clone of a DNA bank prepared from the genome of a bacterium belonging to a strain or species different from the Photorhabdus strain used for the construction of the DNA bank of step a) and hybridize the polynucleotide of step a) to the polynucleotide of step b); c) selecting the polynucleotides of step a) which have not formed a hybridization complex with the polynucleotides of step b); d) characterize the selected polynucleotide.
  • the polynucleotide of step a) can be prepared by digestion of at least one recombinant clone with an appropriate restriction enzyme, and optionally, the amplification of the resulting polynucleotide insert.
  • the method of the invention allows a person skilled in the art to carry out comparative genomic studies between a bacterium of the genus Photorhabdus, and for example between a strain or pathogenic species.
  • the present invention also relates to the use of the nucleic sequences or polypeptides according to the present invention:
  • bio-pesticides in particular entomotoxins, antibiotics, antifungals or cytotoxins
  • the present invention also relates to the use of the polypeptides according to the present invention for the screening of compounds capable of modulating the activity of these polypeptides, in particular the polypeptides with enzymatic activity.
  • Other characteristics and advantages of the invention appear in the following examples:
  • Example 1 Materials and methods The genome sequencing strategy for Photorhabdus luminescens strain TTOl is based on randomized shot-gun sequencing. The first step in this work consisted in cloning the genomic DNA of the bacterium Photorhabdus luminescens into different vectors (plasmids and BAC). Genomic DNA libraries of Photorhabdus luminescens used.
  • a dry pellet of genomic DNA from the TTOl strain prepared on 10/30/98 was taken up in 200 ⁇ l of TE 10: 1 and then dissolved for 30 min at 65 ° C. Its concentration has been estimated at 0.15 ⁇ g / ⁇ l.
  • DNA precipitation This DNA was then precipitated with sodium acetate (2 ml of DNA + 0.2 ml of 3M Na acetate pH 5.2 + 5 ml of absolute ethanol; 2 h at -20 ° C; centrifuged at 30 min at 14,000 rpm, 4 ° C then redissolved in 100 ⁇ l of water.
  • DNA fragments of the expected size 500 bpd to 3 kb were visualized.
  • This DNA was then precipitated overnight at -20 ° C with sodium acetate (1/10 of the volume of Na acetate and 2.5 volume of absolute ethanol), centrifuged and the DNA pellet obtained was air-dried and redissolved in 30 ⁇ l of water.
  • the region of interest (between 1 and 3 Kb) was cut into four fragments.
  • the genomic DNA bank obtained in step 10 was integrated into the ultra-competent XL2 Blue cells (Stratagene) according to the conditions recommended by the manufacturer.
  • the partial filling technique (PARTIAL FTLL-IN), developed in the laboratory, allowed us to build a genomic DNA bank of the TTOl strain of the bacterium Photorhabdus luminescens cloned in the plasmid pSYX34 (size of the inserts approximately 10 Kb) .
  • the PARTIAL F ⁇ X-IN was carried out in the presence of the deoxynucleotides dCTP and dTTP.
  • Plasmid pSYX34 was prepared, by carrying out in parallel two midipreps, KIT QIAGEN according to the conditions recommended by the manufacturer.
  • Chloroform extraction makes it possible to stop enzymatic digestion, and to eliminate all protein traces.
  • nucleotide C 100 mM
  • TRIS 10 mM
  • the nucleotides are thus diluted 1/10, for a concentration of 10 mM.
  • a second 1/10 dilution in 10 mM TRIS buffer will be made to have a concentration of 1 M.
  • the nucleotides are diluted to 1/20 (2.5 ⁇ l in 50 ⁇ l), a final concentration thus obtained of 50 ⁇ M in nucleotides.
  • a dry pellet of genomic DNA of the TTOl strain was taken up in 200 ⁇ l of TE 10: 1 and then dissolved for 30 min at 65 ° C. Its concentration has been estimated at 0.15 ⁇ g / ⁇ l. 2) Partial digestion of genomic DNA by the restriction enzyme Sau3A
  • the chloroform extraction allows us to stop the enzymatic digestion, and to eliminate all protein traces.
  • Vortex centrifuge 1 min at 1000 rpm. Recover the aqueous phase, upper phase, comprising chromosomal DNA.
  • nucleotides G 100 mM
  • TRIS 10 mM pH - 7.5.
  • the nucleotides are thus diluted to 1/10, there is a concentration of 10 mM.
  • a second 1/10 dilution in HO will be carried out, so there is a concentration of 1 mM.
  • the chloroform extraction makes it possible to stop the enzymatic digestion, and to eliminate all protein traces.
  • Chloroform extraction eliminates all protein traces.
  • Vortex centrifuge 1 min at 1000 rpm.
  • the genomic DNA bank obtained in the previous step was integrated into the XL10 Gold Kanr ultra-competent cells (Stratagene) according to the conditions recommended by the manufacturer.
  • the agarose pieces are balanced twice in 300 ⁇ l of Hind III digestion buffer (1x) (Boehringer or Biolabs) for 30 minutes at room temperature;
  • molten agarose / DNA solution is balanced for 15 minutes at 45 ° C; - gelase (Epicenter Technologies) is added, at a rate of 1 U per 100 ⁇ l of gel strip (do not add digestion buffer which causes certain problems at the time of ligation);
  • the ligation is carried out in 50 ⁇ l of a solution containing pBeloBAC ⁇ (2 ⁇ l), of T4 ligase (1 ⁇ l at 1:10), a T4 lOx buffer (5 ⁇ l), and the DNA / agarose solution
  • the ligation medium is heated for 15 minutes at 65 ° C;
  • the ligation medium is dyalized against a Tris-EDTA buffer using Millipore membranes with a pore size VS 0.025 mM; - 1 or 2 ⁇ l of the ligation solution are introduced by electroporation into the E. coli DH10B cells (Gibco BRL) using electroporation cuvettes with a width of 2 mm with the following settings 2.5 kV, 25 ⁇ F and 200 ⁇ ; - After electroporation, the cells are resuspended in 600 ⁇ l of SOC or NYZ medium and then incubated for 45 minutes at 37 ° C with shaking;
  • Plasmid DNA extraction was carried out by the alkaline lysis technique in 24-well plates.
  • the sequencing of the clone inserts was carried out by the KIT PE Big Dye according to the conditions recommended by the manufacturer using the specific oligonucleotides of each vector.
  • the reactions are then introduced into the thermal cycler in order to undergo 35 cycles composed of the following three stages:
  • Precipitation is then carried out with 76% ethanol for 20 minutes at room temperature.
  • the plates are then centrifuged 35 minutes at 2200 g and then drained on absorbent paper and then centrifuged inverted on absorbent paper for 1 minute at 500 g so as to leave no trace of ethanol.
  • the DNA pellets are then: - either taken up in a solution of formamide-EDTA-blue dextran, then denatured for 2 minutes at 96 ° C.
  • the plates are then immediately put in ice, an aliquot then being deposited on acrylamide gel of an automatic sequencer PE-377,
  • genomic DNA sequences were assembled contiguously by the PHRED-PHRAP software and visualized by the CONSED software. Analysis and annotation The contigs were first analyzed and annotated automatically by the GMPTB software.
  • Example 2 Genes of Interest The various characteristics linked to the lifestyle of Photorhabdus luminescens and reported in the literature (Forst et al., 1997; Hu et al., 1998, etc.), led to research in the genome of this bacterium the presence of genes involved in the biosynthesis of antibiotics and toxins. Antibiotic biosynthesis operons The search for peptide-synthesized hases was undertaken in silico. To do this, we first used the different motifs classically described in the literature (Stachelhaus & Marahiel, 1995, FEMS Microbiology Letters 125: 3- 14) as a probe in order to identify by sequence homology in the genome of Photorhabdus luminescens, genes likely to be involved in the biosynthesis of antibiotics.
  • His- HHILxDGW Racemization peptide module (optional) His- HfflLxDGW A - AYxTExND ⁇ XTAxG
  • Photorhabdus luminescens W-14 insecticidal activity consists of at least two similar but distinct protems. Purification and characterization of toxin A and toxin B. J Biol Chem., 274: 9836-42.
  • Photorhabdus luminescens strain TTOl clones in the vector pBelo BAC ⁇ (Kim et al, Genomics, 34, 213, 1996) at the Hind III site.
  • the average size of the inserts is 60 kb.
  • the protein bank used is constituted by the non-redundant fusion of these banks, such as the Genpept bank (automatic translation of GenBank and NCBI).
  • the BLAST software package (public domain, Altschul et al., 1990) was used to search for homologies between a sequence and protein or nucleic databases.
  • the significance thresholds used depend on the length and complexity of the region tested as well as the size of the reference bank. They were adjusted and adapted to each analysis.
  • the results of search for homologies between a sequence according to the invention and protein or nucleic databases are presented and summarized in Table I and III below.
  • nucleic acid sequences of sequence SEQ ⁇ ) N ° 5835 to SEQ ⁇ ) N ° 10784 can be easily identified by their start (column “CONTIG”, “from”) and end position (column "CONTIG””to”) on each of the legend contig sequences in table ⁇ :

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