CA2384637A1 - Nucleotide sequences derived from genes coding for trimethylamine n-oxide reductase, uses thereof in particular for detecting bacteria - Google Patents

Abstract

The invention concerns the use of nucleotide sequences selected among those comprising a sequence coding for a protein of the trimethylamine N-oxide reductase (TMAO reductase) system in bacteria, or a fragment or a sequence derived from said sequence for implementing a method for detecting the presence of all bacteria involved in the process of decaying of aquatic animal flesh, in a host suspected of carrying such bacteria. The invention also concerns nucleotide sequences such as defined above, said detecting methods as well as kits for implementing said methods.

Description

NUCLEOTIDE SEQUENCES FROM GENES ENCODING THE
TRIMETHYLAMINE N-OXIDE REDUCTASE, AND USES THEREOF, IN PARTICULAR FOR THE DETECTION OF BACTERIA.
The subject of the present invention is nucleotide sequences derived from coding genes for trimethylamine N-oxide reductase (TMAO reductase) and usable, especially as primers, for the implementation of bacteria detection methods involved in i the alteration of the flesh of aquatic animals, and more particularly of marine animals.
Trimethylamine N oxide (TMAO) is one of the main constituents of small weight molecular of marine animals, where it can represent up to 1% of their dry weight. In anaerobic, some bacteria use TMAO as an exogenous acceptor electrons for their breathing. The TMAO is then reduced to trimethylamine (TMA), a compound very volatile, largely responsible for the foul odor of endangered fish decomposition. The reduction of TMAO to TMA has been demonstrated in marine bacteria, like those of the genus Shewanella, Photobacterium or Yibrio (1, 2), in bacteria photosynthetic isolated in brackish waters, like those of the genus Rhodobacter (3), but also at several enterobacteria, such as Escherichia coli, Salmonella typhimuriur ~ c or Proteus vulgaris (4).
The enzyme responsible for the reduction of TMAO to TMA, corresponds to the trimethylamine N-oxide reductase (TMAO reductase), the properties of which have been studied at different organizations. For example, there are two enzymes in E. coli separate responsible for the reduction of TMAO: TMAO reductase and 1â DMSO reductase.
The TMAO
reductase, which is responsible for 90% of the reduction in TMAO, is a molybdoenzyme 90 kDa periplasmic, which can be found as a monomer or dimer (5). The TMAO reductases of the different bacteria studied are all 80 molybdoenzymes 100 kDa which can be found in the form of monomer, dimer or tetramer. These proteins are all inducible anaerobically by TMAO, but also by DMSO
(dimethyl sulfoxide). Finally, apart from P. vulgaris, TMAO reductases are all located in the periplasm of the bacteria.
The study of the TMAO reductase system at the genetic and molecular level has been carried out in E. coli enterobacteria and those of the genus Rhodobacter. Both TMAO / DMSO
reductases from bacteria of the genus Rhodobacter and inducible TMAO reductase of. coli show homology at the level of their primary sequence (6, 7, 8). The overall structure of TMAO reductase from E. coli could approach those described for Rhodobacter sphaeroides or Rhodobacter capsulatus. In E. coli, the genes that code for the different components of the TMAO reductase system are organized into operons: the operon torCAD, which encodes for the three proteins TorC, TorD and TorA (6). In Rhodobacter, the genes who code for the various components of the TMAO / DMSO reductase system are also organized in operon: foperon dorCDA, which codes for three proteins DorC, DorD (also called Dora) and DorA, having homologies with the proteins TorC, TorD respectively and TorA
of E. coli (9, 10).
A phylogenetic study by sequencing of 16S rDNA was carried out on a bacterium isolated from a fish caught in the bay of Marseille, and kept at ambient temperature in filtered sea water. The results obtained show that this bacteria, which has a significant TMAO reductase activity, corresponds to a new species of the genus Shewanella, close to Shewanella putrefaciens. She was called Shewanella massilia.
Except protein TorE, the gene products of the torECAD operon of S. massilia have strong homologies with the proteins involved in the TMAO reductase systems of E.
coli and bacteria of the genus Rhodobacter (19). Likewise, the overall structure of the TMAO
reductase S. massilia presents homologies with the structures already described of TMAO / DMSO
reductases of bacteria of the genus Rhodobacter (20).
Alteration of fish flesh, like most foods, is essentially due to the metabolic activity of certain bacteria, which may represent up to 10g cells per gram of product. The first post-mortem changes that occur at level of fish flesh is not necessarily due to bacteria; but at some reactions autolysis. However, microbial development very quickly constitutes the major element of spoilage, the muscle tissue of the fish constituting a substrate for growth bacterial. Several factors intrinsic to fish flesh can explain their very high alterability compared to other food products: a significant hydration, a high post-mortem pH (> 6), high content of non-nitrogenous substances protein, including TMAO forms the essential part and, low support protein content (11, 12). These different characteristics contribute to the development of a flora spoilage microbial specific to fish flesh. Interestingly, the diversity of fish species do does not appear to affect the nature or number of bacteria initially present on

2 fish: In contrast, the bacterial microflora of fish seem to directly related to the natural environment where it evolves. So the contamination of the water that depends basically temperature, salinity or oxygen concentration dissolved, seems play a decisive role. The origin of bacteria found in fish meat does not only come from the microflora of live fish:
contaminating bacteria can in particular come from the cold chain (ice, containers containing the fish, processing steps ...). Among all the flora microbial found only a part of the fish flesh is made responsible of the phenomenon of putrefaction. While bacterial contamination does not make necessarily the flesh of unfit fish, some bacteria cause quickly unpleasant changes in texture, flavor or odor (13).
Seafood therefore represents' a highly perishable commodity, posing from many conservation problems for fishing industry. The mastery quality freshness of these products is therefore a growing concern important for the entire fishing sector (producers, processors or distributors), leading for a few years manufacturers have been looking for new techniques allowing to report the state of freshness of the fish in a very short time.
Currently, the determination of (state of freshness of seafood East essentially based on a sensory appreciation (appearance, smell, flavor) which allows the better to conclude with a fresh product, moderately fresh or unfit for consumption. This organoleptic method is only applicable to whole products, and analyzes more reliable in the laboratory are often essential.
Methods based on bacteriological analysis (total bacterial flora) applicable to processed products are increasingly used to appreciate the state of fish freshness. Direct estimation of the number of cultivable bacteria on a sample fish remains the most commonly used method: after growth on different media (for example iron citrate medium), each cell will form a colony visible on petri dishes, allowing a count. Although this technique be simple of use and quite sensitive, it however requires time incubation for growth important (about 3 to 5 days). In addition, the composition of the middle of growth, or temperatures used for these tests (20-30 ° C), do not allow always detect all of the spoilage bacteria. So the current regulations which advocates a

3 temperature of 30 ° C to carry out these tests (1979 decree), does not allow not able to detect certain spoilage bacteria such as Photobacterium phosphoreum which not grow to this temperature. So even if the nonspecific measure of the load total microbial constitutes a good indicator of the state of hygiene of fish, it does not allow however not assess the sensory quality of this type of product. Microscopic examination food is a quick technique to estimate the level of bacterial contamination. The colored cells with orange facridine are visualized by a microscopy technique of fluorescence; the main drawback of this method, however, is its low sensitivity (between 104 and 105 cells).
Degradation of cellular components of fish leads to production of many substances that can serve as an indirect indicator of a contamination bacterial. An alternative to the methods listed above therefore consisted to be followed by chemical techniques, different degradation products of fish (14). The determination of total volatile basic nitrogen (ABVT), including ammonia (NH3) and the trimethylamine (TMA) form the essential part, is a technique widely widespread at the current time to estimate the degree of decomposition of the flesh of Pisces. The dosage of ABVT has the advantage of being simple, fast and very low cost.
The determination of the rate of TMA in the ABVT, makes it possible to bring a qualitative element additional to ABVT criterion. This criterion seems less dependent on variations which affect generally the level of TMA or ABVT (the species of fish, its content in fats etc ...). The same methodology as for ABVT can be used to measure TMA.
Others colorimetric, enzymatic or chromatographic techniques have also been described for quantify the TMA (15, 16). During the reaction of reduction of TMAO to TMA
by the bacteria, the rotting fish flesh undergo different changes: decrease in redox potential, increased pH, electrical conductivity.
Several studies have so also used these different indicators to try to track indirectly the release of TMA. However, all of these studies have never allowed establish a definitive correlation between the level of TMA released and the freshness quality samples of Pisces. Many other degradation products have also been tested afm of power determine by chemical dosages the level of spoilage of the fish: the sulfide hydrogen, hypoxanthine, indol, histamine and amino acids.
However, these

4 different dosages are only significant in certain species of fish, or at a level very advanced alteration.
The polymerase chain reaction (PCR) is a molecular technique who amplifies large amounts of a target DNA sequence in order to of the view. In recent years, the technique of PCR is more and more used for the detection and identification of microorganisms especially in the field of the clinical bacteriology or food safety (17, 18).
Currently, PCR tests applied to food products mainly allow the detection of bacteria well-defined pathogens (Shigella, Salmonella, Listeria ...).
The present invention follows from the discovery by the inventors of the makes them DNA sequences encoding a protein of the TMAO reductase system, have a homology sufficient within various bacteria, especially within bacteria responsible for the deterioration of the tissues of aquatic organisms, and more particularly breast bacteria marine, to allow the implementation of detection methods of all bacteria responsible for (tissue damage by their TMAO reductase activity, said processes being applicable on all aquatic animals, and in particular on all marine animals.
One of the aims of the present invention is to provide a quality test freshness aquatic products, and more particularly seafood, to the times fast, little expensive, and offering high reliability in terms of specificity and sensitivity.
One of the other aims of the present invention is to provide a test for detection not of a bacterial type, but of all the bacteria responsible for tissue alteration of aquatic organisms, including the putrefaction of the flesh of fish, namely also well marine bacteria, as enterobacteria or isolated bacteria at water level brackish.
Another object of the present invention is to provide a test for detecting bacteria responsible for the deterioration of tissues of marine organisms, applicable to different species of fish from different geographic regions.
Another object of the present invention is to provide a test for detecting bacteria responsible for tissue damage to marine organisms, sufficiently sensitive for detect early stages of alteration.
The object of the present invention is and the use of nucleotide sequences chosen from those comprising a sequence coding for a protein of the system trimethylamine N-oxide s reductase (TMAO reductase) in bacteria, or a fragment of this sequence such as probe or a primer of about 15 to about 25 nucleotides, or a sequence derived by addition, deletion, and / or substitution of one or more nucleotides of this coding sequence for a protein from the TMAO reductase system or a fragment thereof last said fragment and said derived sequence being capable of hybridizing, in particular in the conditions of hybridization defined below, with said sequence coding for a protein of the system TMAO reductase, for the implementation of a method for detecting presence of all bacteria involved in the degradation process of animal flesh aquatic, at a host likely to carry such bacteria.
Advantageously, the nucleotide sequences coding for a protein of the system TMAO reductase mentioned above, are chosen from those corresponding to the torCAD operon or the dorCDA operon mentioned above, of the TMAO reductase system in bacteria.
Preferably, the sequence coding for a protein of the TMAO reductase system mentioned above, is chosen from the sequences coding for TMAO reductase at the bacteria, also known as TorA or DorA protein, or coding for a cytochrome type c in bacteria, also known as TorC or DorC protein.
According to a particularly advantageous embodiment of the invention, the sequences nucleotides coding for a protein of the TMAO reductase system are chosen from those bacteria following - marine bacteria, such as those of the genus Shewanella, Photobacterium or Vibrio, said sequences being chosen in particular from the following * the sequence SEQ> D NO: 1 coding for the protein TorA of Shewanella massilia shown in figure 1, * the sequence SEQ) D NO: 2 coding for the protein TorA of Shewanella putref'aciens shown in figure 1, * the sequence SEQ ID NO: 3 coding for the protein TorA of Shewanella c shown in Figure 2, * the partial sequence SEQ m NO: 4 coding for the protein TorA of Photobacterium phosphoreum represented in FIG. 3, * the sequence coding for the protein TorC SEQ m NO: 5 from Shewanella massilia shown in figure 14, - bacteria from brackish water, such as those of the genus Rhodobacter, or Roseobacter, said sequences being chosen in particular from the following * the sequence SEQ> D NO: 6 coding for the DorA protein of Rhodobacter sphaeroides shown in Figure 4, * the sequence SEQ ID NO: 7 coding for the DorA protein of Rhodobacter capsulatus shown in Figure 4, * the sequence coding for the DorC protein SEQ ID NO: 8 from Rhodobacter sphaeroides shown in Figure 14, - enterobacteria, such as those of the genus Escherichia, or Salmonella, said sequences being chosen in particular from the following * the sequence SEQ) D NO: 9 coding for the protein TorA of Escherichia coli shown in Figure 4, * the partial sequence SEQ) D NO: 10 coding for the protein TorA of Salmonella typhimurium represented in FIG. 5, * the sequence coding for the protein TorC SEQ> D NO: 11 from Escherichia 1 S coli shown in Figure 14.
The invention more particularly relates to the aforementioned use of sequences nucleotides chosen from the fragments of the sequences defined above, or among sequences derived from these fragments, said fragments or sequences derived including about 15 to 25 nucleotides, and are used in pairs to form priming couples allowing the amplification of fragments of genes coding for a protein of the TMAO system-reductase of bacteria involved in the degradation of animal flesh according to the PCR technique.
Advantageously, the abovementioned nucleotide sequences are used form pairs of primers chosen from any one of the three groups of primers following (1) the group of primers “DDN” amplifying DNA fragments of the torA gene, this group including ~ the following “DDN +” nucleotide sequence compositions - DDN1 + (SEQ ID NO: 12): S 'CGG vGA yTA CTC bAC hGG TGC 3': mixture of 54 nucleotide sequences, - DDNS + (SEQ) D NO: 13): S 'ATy GAT GCG ATy CTC GAA CC 3': mixture of 4 nucleotide sequences, 1 the following “DDN-” nucleotide sequence compositions - DDN2- (SEQ ID NO: 14): 5'CGT Amw sGT CGA kAT CGT TrC GCT C 3 ' mixture of 32 nucleotide sequences, - DDN3- (SEQ ID NO: 15): 5 'GAC TCA CAy Awy TGy GAG TG 3': mixture of 16 nucleotide sequences, - DDN4- (SEQ ID NO: 16): S 'TGr CCd CGr kCG TT ~ AAG AC 3': mixture of 24 nucleotide sequences, - DDNS- (SEQ ID NO: 17): S 'CCv GGT TCG AGr ATC GCA TC 3': mixture of 6 nucleotide sequences, (2) the group of primers "BN" amplifying DNA fragments of the torA gene, this group including ~ the following “BN +” nucleotide sequence compositions - BN1 + (SEQ ID NO: 18): 5 'C bGA yAT CsT rCT GCC 3': mixture of 16 sequences nucleotides, - BN3 + (SEQ> D NO: 19): S 'GGm GAy TAy TCb ACm GGy GC 3': mixture of 96 nucleotide sequences, - BN6 + (SEQ ID NO: 20): 5 'Twy GAr CGy AAC GAy mTC GA 3': mixture of 64 nucleotide sequences, ~ the following “BN-” nucleotide sequence compositions - BN2- (SEQ m NO: 21): 5 'GG vyC rTA CCA bsC vCC TTC 3': mixture of 216 nucleotide sequences, - BN4- (SEQ> D NO: 22): 5 'ATC Arr CCn swv GGC GTG CC 3': mixture of 192 nucleotide sequences, - BNS- (SEQ) D NO: 23): S'GbC ACr TCd GTy TGy GG 3 ',: mixture of 72 sequences nucleotides, (3) the group of primers "BC" amplifying DNA fragments of the torC gene, this group including 1 the following “BC +” nucleotide sequence compositions - BC1 + (SEQ ID NO: 24): 5 'ACn CCn GAr AAr TTy GAr GC 3': mixture of 256 nucleotide sequences, - BC2 + (SEQ ID NO: 25): 5 'TGy ATh GAy TGy CAy AAr GG 3': mixture of 96 nucleotide sequences, 1 the following “BC-” nucleotide sequence compositions - BC2- (SEQ) D NO: 26): S 'CCy TTr TGr CAr TCd ATr CA 3': mixture of 96 nucleotide sequences, - BC3- (SEQ m NO: 27): S 'TTn GCr TCr AAr TGn GC 3': mixture of 128 sequences nucleotides, where n = (A, C, G, T), y = (C, T), r = (A, G), h = (A, C, T), d = (G, A, T), m = (A, C), w = (A, T), b = (G, T, C), s = (G, C), v = (G, A, C), and k = (G, T), the pairs of primers being chosen such that one of the primers of a pair corresponds to one of the compositions of nucleotide sequence DDN +, BN + or BC +
above, while the other primer corresponds respectively to one compositions of the above-mentioned DDN-, BN- or BC- nucleotide sequences, said pairs primers being chosen in particular from any one of the following four couples (a) the DDN1 + / DDNS- pair, leading to the amplification of gene fragments coding for the protein TorA in bacteria, about 820 pairs in size bases (bp), and in particular to the amplification of a fragment of 821 bp of the gene coding for the TorA protein in bacteria of the genus Shewanella, such as the fragment of 821 bp delimited by the nucleotides located at positions 620 to 1450 of the torA gene of S. massilia shown in the figure 4, (b) the BN6 + / BN2- pair, leading to the amplification of gene fragments coding for the protein TorA in bacteria, of a size of approximately 710 bp, and especially at amplification of a 727 bp fragment of the gene coding for the protein TorA
in bacteria of the genus Shewanella, such as the 727 bp fragment delimited by nucleotides located at positions 1657 to 2403 of the torA gene of S. massilia represented in FIG. 4, (c) the BN6 + / BN4- pair, leading to the amplification of gene fragments coding for TorA protein in bacteria, about 360 bp in size, and especially at amplification of a 355 bp fragment of the gene coding for the protein TorA
in bacteria of the genus Shewanella, such as the fragment of 355 bp delimited by nucleotides located at positions 1657 to 2023 of the torA gene of S. massilia represented in FIG. 4, (d) the BC1 + / BC2- pair, leading to the amplification of gene fragments coding for TorC protein in bacteria, about 170 bp in size, and especially at amplification of a 197 bp fragment of the gene coding for the protein TorC
in bacteria of the genus Shewanella, such as the 197 bp fragment encoding the fragment polypeptide delimited by the amino acids located at positions 114 to 179 of the protein TorC of S. massilia shown in Figure 14.
Hosts likely to be carriers of bacteria involved in process of degradation of the flesh of aquatic animals, as described above, are organizations aquatic, including marine organisms such as fish and shellfish, and more particularly Atlantic fish such as sole, cod, or the fish of the Mediterranean Sea such as red mullet, sea bream, as well as some water animals sweet or brackish.
The invention more particularly relates to the aforementioned use of sequences i nucleotides described above, for the implementation of a method of detection of presence of all bacteria involved in the degradation of the flesh aquatic animals, as part of an animal freshness assessment process aquatic on which sample is taken when the latter are extracted of their environment natural.
The invention also relates to any nucleotide sequence corresponding to one of following sequences - DDN1 +: 5 'CGG vGA yTA CTC bAC hGG TGC 3', - DDNS +: 5 'ATy GAT GCG ATy CTC GAA CC 3', - DDN2-: S'CGT Amw sGT CGA kAT CGT TrC GCT C 3 ', - DDN3-: 5 'GAC TCA CAy Awy TGy GAG TG 3', - DDN4-: 5 'TGr CCd CGr kCG TTA AAG AC 3', - DDNS-: S 'CCv GGT TCG AGr ATC GCA TC 3', - BN1 +: 5 'C bGA yAT CsT rCT GCC 3', - BN3 +: S 'GGm GAy TAy TCb ACm GGy GC 3', - BN6 +: S 'Twy GAr CGy AAC GAy mTC GA 3', - BN2-: 5 'GG vyC rTA CCA bsC vCC TTC 3', - BN4-: 5 'ATC Arr CCn swv GGC GTG CC 3', - BNS-: 5'GbC ACr TCd GTy TGy GG 3 ', - BC 1 +: 5 'ACn CCn GAr AAr TTy GAr GC 3', - BC2 +: 5 'TGy ATh GAy TGy CAy AAr GG 3', - BC2-: 5 'CCy TTr TGr CAr TCd ATr CA 3', - BC3-: S 'TTn GCr TCr AAr TGn GC 3', where n = (A, C, G, T), y = (C, T), r = (A, G), h = (A, C, T), d = (G, A, T), m = (A, C), 1o w = (A, T), b = (G, T, C), s = (G, C), v = (G, A, C), and k = (G, T).
The subject of the invention is also and any composition of sequences nucleotides in mixture, corresponding to one of the following compositions ~ the following “DDN +” nucleotide sequence compositions - DDN1 +: 5 'CGG vGA yTA CTC bAC hGG TGC 3': mixture of 54 sequences nucleotides, - DDNS +: S 'ATy GAT GCG ATy CTC GAA CC 3'. mixture of 4 sequences nucleotides, ~ the following “DDN-” nucleotide sequence compositions - DDN2-: S'CGT Amw sGT CGA kAT CGT TrC GCT C 3 ': mixture of 32 sequences nucleotides, - DDN3-: 5 'GAC TCA CAy Awy TGy GAG TG 3': mixture of 16 sequences nucleotides, - DDN4-: 5 'TGr CCd CGr kCG TTA AAG AC 3': mixture of 24 sequences nucleotides, - DDNS-: S 'CCv GGT TCG AGr ATC GCA TC 3': mixture of 6 sequences nucleotides, 1 the following “BN +” nucleotide sequence compositions - BN1 +: 5 'C bGA yAT CsT rCT GCC 3': mixture of 16 nucleotide sequences, - BN3 +: 5 'GGm GAy TAy TCb ACm GGy GC 3': mixture of 96 sequences nucleotides, - BN6 +: 5 'Twy GAr CGy AAC GAy mTC GA 3': mixture of 64 sequences nucleotides, ~ the following “BN-” nucleotide sequence compositions - BN2-: 5 'GG vyC rTA CCA bsC vCC TTC 3': mixture of 216 sequences nucleotides, - BN4-: 5 'ATC Arr CCn swv GGC GTG CC 3': mix of 192 sequences nucleotides, - BNS-: 5'GbC ACr TCd GTy TGy GG 3 ': mixture of 72 nucleotide sequences, 1 the following “BC +” nucleotide sequence compositions - BC1 +: S 'ACn CCn GAr AAr TTy GAr GC 3': mixture of 256 sequences nucleotides, - BC2 +: 5 'TGy ATh GAy TGy CAy AAr GG 3': mixture of 96 sequences nucleotides, ~ the following “BC-” nucleotide sequence compositions - BC2-: 5 'CCy TTr TGr CAr TCd ATr CA 3': mixture of 96 sequences nucleotides, - BC3-: S 'TTn GCr TCr AAr TGn GC 3': mixture of 128 sequences nucleotides, where n = (A, C, G, T), y = (C, T), r = (A, G), h = (A, C, T), d = (G, A, T), m = (A, C), w = (A, T), b = (G, T, C), s = (G, C), v = (G, A, C), and k = (G, T).
The invention also relates to the pairs of primers chosen within one groups following primers (1) the group of primers “DDN” comprising 1 the following “DDN +” nucleotide sequence compositions - DDN1 +: 5 'CGG vGA yTA CTC bAC hGG TGC 3': mixture of 54 sequences nucleotides, - DDNS +: 5 'ATy GAT GCG ATy CTC GAA CC 3': mixture of 4 sequences nucleotides, ~ the following “DDN-” nucleotide sequence compositions - DDN2-: 5'CGT Amw sGT CGA kAT CGT TrC GCT C 3 ': mixture of 32 sequences nucleotides, - DDN3-: 5 'GAC TCA CAy Awy TGy GAG TG 3': mixture of 16 sequences nucleotides, - DDN4-: S 'TGr CCd CGr kCG TTA AAG AC 3': mixture of 24 sequences nucleotides, - DDNS-: 5 'CCv GGT TCG AGr ATC GCA TC 3': mixture of 6 sequences nucleotides, (2) the group of primers "BN" comprising ~ the following “BN +” nucleotide sequence compositions - BN1 +: S 'C bGA yAT CsT rCT GCC 3': mixture of 16 nucleotide sequences, - BN3 +: 5 'GGm GAy TAy TCb ACm GGy GC 3': mixture of 96 sequences nucleotides, - BN6 +: 5 'Twy GAr CGy AAC GAy mTC GA 3': mixture of 64 sequences nucleotides, 1 the following “BN-” nucleotide sequence compositions - BN2-: 5 'GG vyC rTA CCA bsC vCC TTC 3'. mix of 216 sequences nucleotides, - BN4-: 5 'ATC Arr CCn swv GGC GTG CC 3'. mix of 192 sequences nucleotides, - BNS-: 5'GbC ACr TCd GTy TGy GG 3 ': mixture of 72 nucleotide sequences, (3) the group of primers "BC" comprising ~ the following “BC +” nucleotide sequence compositions - BC1 +: S 'ACn CCn GAr AAr TTy GAr GC 3': mixture of 256 sequences nucleotides, - BC2 +: 5 'TGy ATh GAy TGy CAy AAr GG 3': mixture of 96 sequences nucleotides, 1 the following “BC-” nucleotide sequence compositions - BC2-: 5 'CCy TTr TGr CAr TCd ATr CA 3': mixture of 96 sequences nucleotides, - BC3-: 5 'TTn GCr TCr AAr TGn GC 3': mixture of 128 sequences nucleotides, where n = (A, C, G, T), y = (C, T), r = (A, G), h = (A, C, T), d = (G, A, T), m = (A, C), w = (A, T), b = (G, T, C), s = (G, C), v = (G, A, C), and k = (G, T), the pairs of primers being chosen such that one of the primers of a pair corresponds to one of the compositions of nucleotide sequence DDN +, BN + or BC +
above, while the other primer corresponds respectively to one compositions of the above-mentioned DDN-, BN- or BC- nucleotide sequences, said pairs primers being chosen in particular from any one of the following four couples (a) the DDN1 + / DDNS- pair, leading to the amplification of gene fragments coding for the protein TorA in bacteria, about 820 pairs in size bases (bp), and in particular to the amplification of a fragment of 821 bp of the gene coding for the TorA protein in bacteria of the genus Shéwanella, such as the fragment of 821 bp delimited by the nucleotides located at positions 620 to 1450 of the torA gene of S. massilia shown in the figure 4, (b) the BN6 + / BN2- pair, leading to the amplification of gene fragments coding for the protein TorA in bacteria, of a size of approximately 710 bp, and especially at amplification of a 727 bp fragment of the gene coding for the protein TorA
in bacteria of the genus Shewanella, such as the 727 bp fragment delimited by nucleotides located at positions 1657 to 2403 of the torA gene of S. massilia represented in FIG. 4, (c) the BN6 + BN4- pair, leading to the amplification of gene fragments coding for TorA protein in bacteria, about 360 bp in size, and especially at amplification of a 355 bp fragment of the gene coding for the protein TorA
in bacteria of the genus Shewanella, such as the fragment of 355 bp delimited by nucleotides located at positions 1657 to 2023 of the torA gene of S. massilia represented in FIG. 4, (d) the couple BC1 + BC2-, leading to the amplification of fragments of the gene coding for TorC protein in bacteria, about 170 bp in size, and especially at amplification of a 197 bp fragment of the gene coding for the protein TorC
in bacteria of the genus Shewanella, such as the 197 bp fragment encoding the fragment polypeptide delimited by the amino acids located at positions 114 to 179 of the protein TorC of S. massilia shown in Figure 14.
The subject of the invention is also a. method of detecting all bacteria involved in the degradation of aquatic animal flesh in a host likely to be carrying such bacteria, said method being carried out from a biological sample taken from this host, this biological sample corresponding in particular to a fragment under-cutaneous of flesh of the aquatic animal in question, and being characterized in that she understands a hybridization step of at least one nucleotide sequence such as defined above, with fragments of genes coding for a protein of the TMAO-reductase system of bacteria involved in the degradation of the flesh of susceptible aquatic animals to be present in the biological sample taken from said host, followed by a step of revelation, especially by electrophoresis, of the possible presence in said sample of genes coding for a protein of the TMAO-reductase system, or fragments of these genes, the number of copies has been amplified if necessary.
The invention more particularly relates to a detection method such that defined above, characterized in that it comprises the following stages - processing a biological sample taken from this afm host extract total DNA
of this host and to make the genome of these bacteria accessible to sequences nucleotides or primers defined above, this treatment being carried out in particular using of a technique rapid DNA extraction based on the attachment of nucleic acids to silica beads, - the amplification of the number of copies of genes coding for the proteins of the system TMAO-reductase of bacteria involved in the degradation of the flesh aquatic animals, or fragments of these genes, which may be present in this sample, using nucleotide sequences or primers mentioned above, - the detection of the possible presence of an amplified number of copies of coding genes for a protein of the TMAO-reductase system of the above-mentioned bacteria, or fragments of these genes, and therefore of the presence of such bacteria in the sample biological studied.
Advantageously, the detection methods according to the invention are characterized in that that the amplification of the number of genes coding for proteins in the system TMAO-reductase includes the following steps - the predenaturation of the total double stranded DNA of the host into single-stranded DNA, preferably in a buffer consisting of IOmM Tris-HCl pH 8.3, SOmM KCI, l, SmM
MgCl2, 0.01% gelatin, of the 4 deoxynucleotides that make up DNA (dCTP, dATP, dGTP, dTTP) at a concentration of 100 p, M each, and pairs of primers, such as defined above, by heating between about 90 ° C and about 100 ° C, preferably at 94 ° C, for approximately 1.5 minutes, - the actual amplification by addition to the medium obtained in step previous DNA polymerase, for example Taq polymerase, 1 heating at approximately 94 ° C for approximately 30 seconds, which correspond to the actual denaturation step, 1 then heating between about 35 ° C to about 60 ° C, and especially at around 45 ° C
or 55 ° C, for about 30 seconds, which corresponds to the step priming hybridization with the genes coding for the proteins of the TMAO-reductase system of bacteria, or fragments of these genes, likely to be present in the sample biological studied, ~ and finally, heating at 72 ° C, for about 45 seconds, which correspond to the stage of elongation of the primers, hybridized in the preceding stage, one towards the other, producing thus complementary nucleotide sequences of gene fragments coding for proteins of the TMAO-reductase system of bacteria, these latter sequences being demarcated by nucleotides hybridizing with the aforementioned primers, - repetition of the previous amplification step between around 15 and about 35 times, preferably about 30 times.
The invention also relates to the kits or kits for the implementation of a aforementioned detection method, characterized in that they include - one or more nucleotide sequences or primers as defined above above, - a DNA polymerase, a reaction medium advantageously consisting of IOmM Tris-HCl pH 8.3, SOmM
KCI, l, SmM MgClz, 0.01% gelatin, of the 4 deoxynucleotides constituting the DNA (dCTP, dATP, dGTP, dTTP) at a concentration of 100 pM each.
The invention also relates to any nucleotide sequence comprising - the sequence represented in FIG. 2 of the torA gene coding for the protein TorA of the marine bacteria Shewanella C, - or any sequence derived from the above-mentioned sequence by degeneration of the coded genetic, and encoding the protein TorA of Shewanella C, whose sequence peptide is represented in FIG. 6, - or any sequence derived from the above-mentioned nucleotide sequence, especially by substitution, deletion or addition of one or more nucleotides, said derived sequence preferably having a homology of about 35% to 100% with the sequence nucleotide mentioned above shown in FIG. 2, - or any fragment of the above-mentioned nucleotide sequence, or of a sequence derived from the latter as defined above, said fragment being of corporate preference at least about 15 nucleotides.
A subject of the invention is also all peptide sequence coded by the sequence above-mentioned nucleotide shown in FIG. 2, and comprising - the amino acid sequence shown in Figure 6 of the TorA protein of Shewanella c, - or a sequence derived from the above-mentioned peptide sequence, in particular through substitution, deletion or addition of one or more amino acids, said derived sequence preferably having a homology of about 35% to 100% with the sequence peptide mentioned above shown in FIG. 6, - or a fragment of the above-mentioned peptide sequence, or of a sequence derived from the latter as defined above, said fragment preferably being made up of at minus about S amino acids.
The invention also relates to any nucleotide sequence comprising - the partial sequence shown in FIG. 3 of the gene coding for the TorA protein from the marine bacteria Photobacterium phosphoreum, - or any sequence derived from the above-mentioned sequence by degeneration of the coded genetic, and coding for the fragment of the protein TorA of Photobacterium phosphoreum whose peptide sequence is represented in FIG. 7, - or any sequence derived from the above-mentioned nucleotide sequence, especially by substitution, deletion or addition of one or more nucleotides, said derived sequence preferably having a homology of about 35% to 100% with the sequence nucleotide mentioned above shown in FIG. 3, - or any fragment of the above-mentioned nucleotide sequence, or of a sequence derived from the latter as defined above, said fragment being of corporate preference at least about 15 nucleotides.
The invention also relates to any peptide sequence encoded by the sequence above-mentioned nucleotide shown in FIG. 3, and comprising - the partial amino acid sequence shown in FIG. 7 of the TorA protein from Photobacterium phosphoreum, - or a sequence derived from the above-mentioned peptide sequence, in particular through substitution, deletion or addition of one or more amino acids, said derived sequence preferably having a homology of about 35% to 100% with the sequence peptide mentioned above represented in FIG. 7, - or a fragment of the above-mentioned peptide sequence, or of a sequence derived from the latter as defined above, said fragment preferably being made up of at minus about 5 amino acids.
The subject of the invention is also any nucleotide sequence comprising the partial sequence represented in FIG. 5 of the gene coding for the TorA protein from the marine bacteria Salmonella typhimurium, - or - any sequence derived from the above-mentioned sequence by degeneration code genetic, and coding for the TorA protein of Salmonella typhimurium whose sequence peptide is shown in Figure 8, - or any sequence derived from the above-mentioned nucleotide sequence, especially by substitution, deletion or addition of one or more nucleotides, said derived sequence preferably having a homology of about 35% to 100% with the sequence nucleotide mentioned above shown in FIG. 5, - or any fragment of the above-mentioned nucleotide sequence, or of a sequence derived from the latter as defined above, said fragment being of corporate preference at least about 15 nucleotides.

The invention also relates to any peptide sequence encoded by the sequence above-mentioned nucleotide shown in FIG. 5, and comprising - the amino acid sequence shown in FIG. 8 of the protein TorA
of Salmonella typhimurium, - or a sequence derived from the above-mentioned peptide sequence, in particular through substitution, deletion or addition of one or more amino acids, said derived sequence preferably having a homology of about 35% to 100% with the sequence peptide mentioned above represented in FIG. 8, - or a fragment of the above-mentioned peptide sequence, or of a sequence derived from the latter as defined above, said fragment preferably being made up of at minus about S amino acids.
The invention will be further illustrated by means of the detailed description which follows from obtaining primers of the invention, and their use for setting process work detection according to (invention.
1 S Description of figures - Figure 1 shows the alignment of the nucleotide sequences of the genes torA of Shewanella massilia (torA / Sm), Shewanella c (torA / Sc), and Shewanella putrefaciens (torA / Sp).
- Figure 2 shows the complete nucleotide sequence of the torA gene coding for the TMAO reductase from Shewanella c.
- Figure 3 shows the partial nucleotide sequence of the torA gene coding for the TMAO reductase from Photobacterium phosphoreum.
FIG. 4 represents the alignment of the nucleotide sequences of the genes torA of Shewanella massilia (torA / Sm), from E. coli (torA / E. C.), From Rhodobacter sphaeroids (TorA / Rs), and Rhodobacter capsulatus (TorA / Rc). The arrows indicate the position of different primers on the torA gene of the bacterium Shewanella massilia, developed from conserved regions. More specifically, the positions of the different “DDN” primers and "BN" on the torA gene of S. massilia are as follows DDNl +: 620 BNl +: 1628 3 0 DDNS +: 1428 BN3 +: 621 DDN2-: 1684 BN6 +: 1657 DDN3-: 2201 BN2-: 2403 DDN4-: 2325 BN4-: 2023 DDNS-: 1450 BNS-: 946 - Figure 5 shows the partial nucleotide sequence of the torA gene coding for the TMAO reductase from Salmonella typhimurium.
- Figure 6 shows the complete peptide sequence of TMAO reductase of Shewanella c.
FIG. 7 represents the alignment of the peptide sequences of the fragment of protein TorA of Photobacterium phosphoreum (TorA / Pp), derived from the DNA fragment amplified in P. phosphoreum using the molecular primers DDN1 + / DDNS-, with the regions protein corresponding to the TorA proteins of Shewanella massilia (TorA / Sm), of E.
coli (TorA / Ec), and Rhodobacter sphaeroides (DorA / Rs).
FIG. 8 represents the alignment of the peptide sequences of the fragment of protein TorA of Salmonella typhimurium (TorA / St), deduced from the amplified DNA fragment at S.
typhimurium using the molecular primers DDN1 + / DDNS-, with the regions protein corresponding to the TorA protein of E. coli (TorA / E. c.), of the protein DorA's Rhodobacter sphaeroides (DorA / Rs), and TorA protein from Shewanella massilia (TorA / Sm).
- Figure 9 shows the alignment of nucleic sequences of a region very preserved of the gene which codes for the TMAO reductase of bacteria of the genus Shewanella and from E. coli, used for the preparation of one of the molecular primers "DDN" according to the invention.
(a) Approximate position and orientation of the different DDN primers on the torA gene coding for TMAO reductase of different bacteria of the genus Shewanella and from E. coli, (b) Example of a strategy for developing one of the primers .DDN (DDNl +); The area boxed corresponds to a very conserved nucleic region of the gene which codes for TMAO
reductase of bacteria of the genus Shewanella [Shewanella massilia (torAlS.
massilia), Shewanella putrefaciens (torAlS. Putrefaciens), Shewanella c (torAlS. C)] and from E. coli.
- Figure 10 shows the electrophoresis on agarose gel of the products of PCR when amplification is carried out using chromosomal DNA from Shewanella massilia (tracks 1, 2, 3 and 4), of Shewanella c (tracks 5, 6, 7 and 8) and of Shewanella putrefaciens MR-1 (tracks 9, 10, 11 and 12) using the molecular primers DDN1 + / DDNS- (lanes 1, 5 and 9; cut expected: 820 bp), DDNS + / DDN2- (tracks 2, 6 and 10; expected size: 234 bp), DDNS + / DDN3-(tracks 3, 7 and 11; expected size: 740 bp), DDNS + / DDN4- (tracks 4, 8 and 12 ; expected size 866 bp). Tracks M: Size markers.
- Figure 11 shows the agarose gel electrophoresis of the products of PCR when the amplification is carried out from the chromosomal DNA of Photobacterium phosphoreum using the pairs of primers DDN1 + / DDNS- (lane 1; expected size:
820 bp), DDNS + / DDN2- (lane 2; expected size: 234 bp), DDNS + / DDN3- (lane 3;
expected size 740 bp) and DDNS + / DDN4- (lane 4: expected size: 866 bp). M tracks:
size markers.
- Figure 12 shows the agarose gel electrophoresis of the products of PCR when (amplification is carried out from chromosomal DNA from S. massilia (tracks l, 4, 7 and 10), E. coli (tracks 2, 5, 8 and 11) and S. typhimurium (tracks 3, 6 and 9), using the pairs of molecular primers DDN1 + lDDNS- (tracks 1 to 3; expected size:
820 bp), DDNS + / DDN2- (tracks 4 to 6; expected size: 234 bp), DDNS + / DDN3- (tracks 7 to 9; cut expected: 740 bp), DDNS + / DDN4- (tracks 10 and 11; expected size: 866 bp).
Tracks M
Size markers.
- Figure 13 shows the alignment of nucleic sequences of a region very from the gene that codes for TMAO reductase in bacteria of the genus Shewanella, Rhodobacter and E. coli, used for the preparation of one of the primers "BN" molecules according to the invention.
(a) Approximate position and orientation of the different BN primers on the torA gene coding for the TMAO reductase of different bacteria of the genus Shewanella, Rhodobacter and E. coli, (b) Example of a strategy for developing one of the BN primers (BN3 +); The area framed corresponds to a very conserved nucleic region of the gene which codes for TMAO
reductase bacteria Shewanella massilia (torAlS. massilia), E. coli, Rhodocbacter sphaeroides (dorAlR. sphaeroides), and Rhodocbacter capsulatus (dorAlR. capsulatus).
- Figure 14 shows the alignment of cytochrome protein sequences TorC by S.
massilia (TorC / Sm), E. coli (TorC / Ec) and cytochrome DorC from R.
sphaeroids (DorC / Rs). The arrows indicate the position of the different primers on the torC gene bacteria Shewanella massilia, made from preserved regions. AT
As an example, the couple BC1 + BC2- leads to the amplification of the fragment with a size of 197 bp of the coding gene for the protein TorC in S. massilia, this fragment coding for the polypeptide 66 acids amino acids delimited by the amino acids located in positions 114 and 179 of the peptide sequence of TorC of S. massilia. The couple BC1 + / BC3- leads to the amplification of the fragment of a size of 719 bp of the gene coding for the protein TorC in S. massilia, this fragment coding for the 240 amino acid polypeptide delimited by the amino acids located at positions 114 and 353 of the TorC peptide sequence of S. massilia. The BC2 + / BC3- pair leads to amplification of the 542 bp size fragment of the gene encoding for TorC protein in S.
massilia, this fragment coding for the polypeptide of 181 amino acids delimited by acids amines located at positions 173 and 353 of the peptide sequence of TorC of S.
massilia.
- Figure 15 represents the electrophoresis on agarose gel (2%) of the products of PCR
when the amplification is carried out from the chromosomal DNA of E.
coli (lane 1), from S.
typhimurium (lane 2), P. phosphoreum (lane 3), S. massilia (lane 4), by R. sphaeroides (lane 5), or Erwinia chrysanthemi (lane 6) using the primers BC1 + molecules and BC2-. The expected DNA fragment size (177 base pairs) is indicated by an arrow.
Tracks M: Size markers.
- Figure 16 shows the influence of chromosomal fish DNA on the specificity of the molecular primers according to the invention. PCR amplification is performed using molecular primers DDNl + and DDNS- in the presence of 2.5 p, g (Tracks 1 and 4), 5 pg (Tracks 2 and S), or 10 pg (Tracks 3 and 6) of purified herring chromosomal DNA. The tracks 4, 5 and 6 correspond to the tests carried out by adding 2.5 p1 of cell suspension from Shewanella massilia to the reaction mixture. Tracks M: size markers.
- Figure 17 shows the agarose gel electrophoresis (2%) of the products of PCR
when the amplification is carried out using total DNA extracted from fish meats in decomposition (cod: track 1 to 3; sole: track 4 to 6; sea bream: track 7 at 9 and red mullet rock: track 10 to 12) using primers BN6 + / BN4- (tracks 1, 4, 7 and 10, expected size: 364 bp), BN6 + / BN2- primers (tracks 2, 5, 8 and 11, expected size: 711 bp), primers DDN1 + / DDNS- (tracks 3, 6, 9 and 12, expected size: 820 bp). M tracks:
standard size (1 Kb BRL scale).
- Figure 18 shows the electrophoresis on agarose gel (2%) of the products of PCR
when the amplification is carried out using molecular primers BC1 + / BC2- on two different preparations of total DNA from a red mullet in process of putrefaction (track 1 and 2).
The arrow indicates the amplified DNA fragment of 177 base pairs.
FIG. 19 represents the alignment of the nucleotide sequences of the genes torA of Photobacterium phosphoreum (torA / Pp), from Shewanella putrefaciens (torA / Sp), from Shewanella massilia (torA / Sm), Shewanella c (torA / Sc), and Salmonella typhimurium (torA / St).
A) METHODS OF STUDY
Development of specific primers from the gene coding for the enzyme TMAO
reductase: molecular probes "DDN", "BN" and "BC".
The first stage of the study below concerns the development of primers (or probes) molecules usable for a PCR reaction, and on the study of their specificity for different bacteria involved in the alteration of fish flesh. The second step relates to the application of the PCR technique on fish flesh.
> 7 Development and validation of molecular primers used for the PCR reaction.
The choice of nucleotide primers is a critical step for success of a PCR test.
These primers should be developed from highly conserved regions of the gene that codes for TMAO reductase in all bacteria responsible for degradation of fish flesh. Several strategies were used to develop these primers.
(1) Molecular primers produced from conserved regions of the torA gene in bacteria of the genus Shewanella and in E. coli: primers "DDN".
The development of very little degenerate primers from protein regions kept is very difficult due to the degeneracy of the genetic code.
Molecular primers, called "DDN", were defined from alignment of nucleic acid sequences of the torA gene of different bacteria of the genus Shewanella and E. coli (Figure 9): Indeed, several regions of DNA of the torA gene in these bacteria present a high degree of sequence identity with very few mismatches.
The molecular primers DDN are the molecular primers DDN1 +, DDN2-, DDN3-, DDN4-, DDNS- and DDNS + as defined above.
For all PCR reactions, the pairing temperature is brought to 55 ° C so to make the amplification more specific and therefore to limit the appearance of DNA bands contaminating. This is made possible by the slight degeneration of newly primers synthesized. PCR amplification involves 30 reaction cycles successive. Each cycle includes a denaturation step at 94 ° C for 30 s, a step hybridization at 45 ° C
for 30 s and an elongation step at 72 ° C for 45 s.

a) Use of DDN molecular primers for the detection of the TMAO system reductase from bacteria of the genus Shewanella.
The first series of experiments, using the pairs of primers DDN1 + / DDNS-, DDNS + / DDN2-, DDNS + / DDN3-, DDNS + / DDN4-, was performed on chromosomal DNA
of the strain Shewanella massilia (S. massilia). The priming couples DDN1 + / DDN2-, DDNl + / DDN3- and DDN1 + / DDN4- were purposely discarded due to the remoteness of their positions on the torA gene.
The results, grouped in Figure 10 (tracks 1 to 4), reveal for the majority of pairs of molecular primers used, amplification of a single fragment DNA
specific to the expected size. Although for the pair of primers DDNS + / DDN2-(track 2), a non-specific DNA fragment, of approximately 300 base pairs, is amplified, DDN molecular primers appear to be specific to the TMAO system S. reductase massilia.
The pairs of DDN primers were then tested on other bacteria belonging to like Shewanella. The same series of experiments was performed using as matrix the chromosomal DNA of the strain Shewanella c and S. putrefaciens MR-1.
All of the primer pairs amplify a size-specific DNA fragment expected for these two bacteria (Figure 10).
The different pairs of DDN primers therefore make it possible to detect the set of Shewanella bacteria tested.
b) Use of the DDN molecular primers for the detection of the coding gene for TMAO reductase in the marine bacterium Photobacterium phosphoreum, and for setting in the sequencing of said gene.
Photobacterium phosphoreum (P. phosphoreum) is a bacteria which, like S.
putrefaciens is in a number of cases made responsible for the alteration of the flesh of marine fish (12, 21). It belongs to the family of Vibrionaceae, and has a optimal growth temperature of 15 ° C. It is a very little bacteria studied, whose Putative TMAO reductase system is absolutely unknown to date.
In order to verify the specificity of the molecular primers according to the invention on bacteria distinct from the Shewanella group, and likely to contaminate the flesh fish, one collection P. phosphoreum strain was obtained by ATCC (American Type Culture Collection No. 11040). The first growth tests carried out on this bacteria on medium marine (Difco) at 15 ° C have shown that the addition of TMAO in the middle of culture improves significantly its growth rate. The optical density (D06 ~) obtained after 24 hours of growth is 0.28 for the strain cultivated in the absence of TMAO, and 0.47 for the strain grown in the presence of TMAO. This result is in agreement with the presence of a TMAO
respiratory reductase in P. phosphoreum. Genetic amplification was made from the chromosomal DNA of this bacterium (prepared from 10 ml of culture in marine environment) using the set of pairs of molecular primers DDN.
The results, presented in FIG. 11, show that the DDN primers allow in all cases the amplification of a specific DNA fragment. The different sizes of DNA fragments are in agreement with those obtained for amplification performed on the chromosome of bacteria of the genus Shewanella.
To confirm that the DNA fragment amplified in P. phosphoreum matches well to a part of the gene coding for the TMAO reductase of P. phosphoreum, the sequencing of said fragment has been produced. So the sequencing of around 600 nucleotides of the fragment amplified from the pair of molecular primers DDN1 + / DDNS- was carried out.
The product PCR directly purified by Geneclean (Bio101) was sequenced by the technique termination chains (22) using the molecular primer DDN1 +. The sequence protein deduced from this nucleic sequence has been aligned with those of various TMAO reductases (figure 'n.
This protein sequence has approximately 60% identity with a region of TMAO
S. massilia reductase. The homologies observed are sufficiently high to conclude that the sequenced DNA fragment corresponds to a part of the gene that codes for TMAO
P. phosphoreum reductase.
These results highlight the presence of a TMAO reductase system in the P. phosphoreum bacteria, and validate the detection method according to the invention by the PCR test, since the latter makes it possible to detect the TMAO-reductase system in bacteria relatively distant alteration from a phylogenetic point of view.
c) Use of the DDN molecular primers for the detection of genes encoding for TMAO reductase of enterobacteria, and for the implementation of sequencing said Genoa.
To verify that the PCR test is valid for detection enterobacteria such as E.
coli or certain pathogenic bacteria like Salmonella typhimurium, the same experience PCR as that described above was carried out, with as DNA template of DNA

chromosome of E. coli and S. typhimurium.
In S. typhimurium, the promoter region of the TMAO reductase system could be setting demonstrated by a PCR approach (23).
The results obtained are grouped in Figure 12. The four couples primers molecular DDN1 + / DDNS-, DDNS + / DDN2-, DDNS + / DDN3- and DDNS + / DDN4- allow amplification of a specific DNA fragment in E. coli (lane 2, 6, 10 and 16). In the case of S. typhimurium, the pair of primers DDNl + / DDNS- also allows amplification of a DNA fragment, 820 base pairs, compatible with a specific fragment of the system TMAO reductase (lane 3).
To confirm that the DNA fragment amplified in S. typhimurium fits well to a part of the gene coding for the TMAO reductase of S. typhimurium, the sequencing of said fragment has been made. The protein sequence, deduced from a part of this fragment DNA (477 nucleotides), has important homologies with the sequence protein from various TMAO reductases (Figure 8). It also has more than 88%
identity with the TMAO reductase sequence of E. coli and 45% with that of the enzyme in S.
massilia. The DNA fragment amplified using the primers DDN1 + / DDNS- therefore corresponds presumably a part of the gene that codes for TMAO reductase in S.
typhimurium.
d) Conclusion As previously described, the pair of primers DDN1 + / DDNS-allows detect the torA gene of marine bacteria (Shewanella and P. phosphoreum), but also enterobacteria (E. coli and S. typhimurium).
The same PCR tests were carried out on bacteria isolated at the level of waters brackish- (Rhodobacter) which can possibly be found at the level flesh of Pisces. In order to perform these tests, samples of chromosomal DNA from the bacteria R.
sphaeroides were used as template DNA.
The bacterium R. sphaeroides has a DMSO / TMAO reductase system close to the one described in E. coli (9, 10). Although the terminal enzyme of this bacteria may reduce indifferently TMAO and DMSO, it has many homologies of sequences protein with TMAO reductase from E. coli or S. massilia (respectively 47.5% and 45%
identity).
However, PCR tests performed with DDN molecular probes did not permit to amplify a DNA fragment corresponding to the TMAO reductase gene from this bacteria.
2s (2) Molecular primers made from conserved regions of the torA gene in all the bacteria studied: “BN” primers.
In order to further broaden the detection spectrum of the molecular primers of the test PCR, the nucleic acid sequence of the TMAO / DMSO reductase gene of bacteria of the genus Rhodobacter (R. sphaeroides and R. capsulatus) was integrated into the alignment carried out at from the sequences of torA gene from bacteria of the genus Shewanella and E. coli.
A new series of primers, called "BN", was thus developed (Figure 13).
For all of the experiments, the PCR amplification is carried out with a temperature SS ° C hybridization. At this temperature, only a small part primers present in the mixture will be able to hybridize specifically to the template DNA.
For this reason, a larger amount of molecular primers is added to the reaction mixture (approximately 100 pmoles in SO ~ l final).
The primers BN obtained are the primers BNl +, BN2-, BN3 +, BN4-, BNS-, BN6 +
as defined above.
Table 1 below represents the results obtained with regard to the application primers BN molecules on the DNA of different bacteria.
The amplification is carried out using 5 ng of bacterial chromosomal DNA.
The PCR reaction involves 30 successive reaction cycles.
+: A DNA fragment specific to the expected size is amplified : No amplification Table 1 Bactries Primers Primers Primers Primers Primers BN1 + BN2- BN1 + BN4-BN3 + BNS- BN6 + BN4- BN6 + BN2-S. massilia + + + + +

Shewanella + + + + +
vs S. putrefaciens + + + + +

P. phosphoreum + + + + +

E. coli + - + + +

S. typhimurium- - + + +

R. sphaeroides- - - + +

Two pairs of primers: BN6 + / BN2- and BN6 + / BN4-, allow to amplify a specific DNA fragment in all of the bacteria tested, and in particular at R.
sphaeroides.
BN primers therefore prove to be perfectly suitable for the detection of bacteria who have a TMAO reductase system, and who are likely to contaminate the flesh of Pisces.
On the other hand, the use during PCR of a hybridization temperature high (55 ° C), and a larger amount of degenerate molecular primers, allowed to improve significantly the specificity of the amplification.
(3) Molecular primers based on the gene coding for cytochrome TMAO reductase TorC: “BC” primers.
The study of the TMAO reductase system in different bacteria made it possible to to show that the electron transfer chain to the terminal enzyme TorA does intervene in all case a type c pentahemic cytochrome, cytochrome TorC (or DorC) (6, 9, 10, 19).
To highlight the TMAO reductase system in bacteria alteration, nucleic acid primers directed against the gene encoding this cytochrome have been developed.
The mufti-alignment of protein sequences, presented in FIG. 14, makes appear many protein regions conserved between the cytochromes of the system TMAO
reductase of different bacteria: S. massilia, E. coli, R. sphaeroides.
Some of these regions preserved correspond to the c-type heme binding sites (motif CxxCH) and are therefore found in a large number of class tetrahemic cytochromes NapC / NirT
involved in other respiratory systems (24). Cytochrome TorC
nevertheless has the peculiarity of containing an additional carboxy-terminal domain which fixes a fifth heme (6, 19).
The molecular primers BC are the primers BC1 +, BC2 +, BC2- and BC3- such than defined above. To develop said primers, the protein regions preserved have been sought from all of the NapC / NirT class cytochromes (BC2 + probes and BC2-), but also only among TorC cytochromes (BC1 + and BC3- probes). The position and the orientation of the four degenerate molecular primers BC are indicated in figure 14.
The BC molecular primers are capable of hybridizing to the gene coding for the TMAO reductase cytochrome TorC.

The PCR reaction is carried out under the same conditions as those used with the BN molecular primers. It involves 30 successive reaction cycles.
Each cycle is consisting of a denaturation step at 94 ° C for 30 s, a step hybridization at 55 ° C
for 30 s and an elongation step for 45 s.
About 100 pmol of each molecular primer is added to the mixture reaction (final volume 50 p.1). Among the three pairs of primers molecular tested (BC1 + BC2-, BC1 + BC3- and BC2 + BC3-), the couple BC1 + BC2- allowed to amplify a DNA fragment of the expected size (177 base pairs) for all of the bacteria previously tested (Figure 15; tracks 1 to 5). This result allows us to consider the use of the pair of molecular primers BC1 + BC2- for the PCR test.
In parallel, the same PCR experiment was carried out with the pair of primers BC1 + BC2- from the chromosome of the bacterium Erwinia chrysanthemi, enterobacterium phytopathogenic which would be deprived of a system TMAO reductase (4). In this case, none DNA fragment of a size compatible with that expected for a amplification from torC gene is not observed (Figure 15, lane 6).
The pair of primers BC1 + BC2- therefore seems to specifically recognize the cytochrome TorC of the TMAO reductase system.
(4) Application of the PCR technique to whole cells For carrying out a PCR test intended to be used directly from samples of fish, it is important to get rid of the preparation stage of chromosomal DNA
bacteria. For this reason, PCR experiments using couples DDN primers, BN and BC- were reproduced from whole bacteria of S. massilia.
Colonies bacteria isolated on a dish are resuspended in 200 p1 of water sterile distilled (D06oo between 0.1 and 0.5). The PCR reaction is then carried out in the conditions previously described with 2.5 ~ 1 matrix of cell suspension (in 50 ~ 1 of final volume). For all the PCR reactions carried out (with the primers DDN, BN and BC), the result obtained is equivalent to that observed with DNA
purified chromosome.
Bacteria are therefore well lysed during the first stage of PCR
that corresponds to incubation at 94 ° C of the samples for 1.5 min. This result positive lets consider use of the PCR test directly from fish flesh.

II) Application of the PCR-TMAO test to fish flesh After verifying the specificity of the molecular primers according to the invention on the bacteria responsible for spoilage of seafood, the stage next therefore consists in direct application of the PCR test to fish flesh.
The four pairs of primers DDN1 + / DDNS-, BN6 + BN4-, BN6 + BN2- and BCl + BC2-were chosen to perform these tests because they allow amplification of a DNA fragment specific for the vast majority of bacteria previously tested.
(1) Influence of fish chromosomal DNA during PCR amplification of TMAO reductase gene in bacteria.
For the use of the PCR test on fish flesh samples, it must first verify that the chromosomal DNA of the fish does not compete with DNA
bacterial, during the hybridization of molecular primers.
In order to test the influence of chromosomal DNA from fish on the specificity of 1 S molecular primers according to the invention, variable concentrations of DNA
chromosome purified herring sperm were added to the PCR reaction mixture containing or no 2.5 ~ l of cell suspension of the bacteria S. massilia.
FIG. 16 obtained with the pair of primers DDNI + / DDNS- shows that none DNA fragment is only amplified in the absence of bacteria added to the mixture reaction (track 1 to 3) and this, whatever the concentration of fish chromosomal DNA.
If we add the bacteria S. massilia in the reaction mixture (tracks 4 to 6) a DNA fragment specific is amplified. The same results were obtained for the pairs of primers BN6 + BN2-, BN6 + BN4- or BC1 + BC2-, and thus allow to conclude that the presence of DNA
foreign in the PCR reaction mixture does not interfere with the specificity of primers molecular according to the invention.
These important results therefore make it possible to exclude the possibility of a amplification no specific which would be due to the chromosomal DNA of the fish.
(2) Extraction of total DNA from fish flesh and validation of the technical PCR.
Traditionally, microorganism detection tests, during control microbiological food, require a first step of pre-enrichment of bacteria on a selective medium.
With a view to a rapid freshness test, the technique of PCR was applied directly to fish samples. Different conditions extraction the total DNA of altered fish flesh (bacterial DNA + DNA
fish) have therefore been tested.
First, a rapid technique for extracting DNA from cells has been tested by treating fish samples with NaOH / SDS (25). This technique allows lysis cell and therefore the extraction of total chromosomal DNA. The preparation is done from about 25 mg of flesh taken from a fish (red mullet) kept 12 hours at ambient temperature. After 10 min of incubation at 95 ° C, 5 w1 of the mixture fish flesh homogenized are used directly in the PCR reaction mixture.
However, by this rapid technique of extracting total DNA, the whole of PCR reactions performed with the different molecular primers did not allowed to amplify a specific DNA fragment. The presence of several substances that inhibit the PCR
contained in fish flesh could explain this result. In effect of compounds present in large quantities in certain food products (fats, proteins ...) can influence the efficiency of PCR (26). In order to verify this hypothesis, the same PCR tests were carried out by adding to the fish flesh samples

5 ng of DNA
chromosome of S. massilia. However, as before, no fragments no DNA
was amplified by PCR, whatever the molecular primers used.
These results allow us to conclude that the PCR reaction is inhibited by some compounds found in fish flesh.
The technique of purifying total chromosomal DNA from Pisces, used according to the present invention, must therefore make it possible to eliminate all the compounds PCR inhibitors.
A quick DNA extraction kit (about 1 hour), based on the fixation acids nucleic acid to silica beads, was thus tested (kit sold under The denomination "High Pure PCR Template Preparation Kit" by Boehringer Mannheim / Roche).
After fixing DNA, this technique makes it possible to eliminate salts, proteins and others cellular impurities by a simple washing step.
Total DNA extraction (bacterial DNA + fish DNA) was carried out out of four different species of fish in process of decomposition (kept 12 h at ambient temperature) 2 species from the Mediterranean Sea (red mullet and sea bream), and 2 species from the Atlantic (sole and cod). From approximately 50 mg of flesh of these fish (taken under the skin), 1 to 4 pg of total chromosomal DNA were thus purified. About 25 ng of this DNA
are then added to the PCR reaction mixture (final volume 50 p1). For the group reactions PCR, the hybridization temperature is brought to 55 ° C., and 100 pmol of each leader molecules are added to the reaction mixture.
FIG. 17 groups together the results obtained using the pairs of primers DDN1 + / DDNS-, BN6 + / BN2- and BN6 + / BN4-. For all fish species tested, a single DNA fragment is amplified, and this with each of the pairs of primers used.
The size of the amplified DNA fragment corresponds to the size expected for a amplification from the torA gene. This result indicates that the different DNA fragments have have been amplified from the torA gene of bacteria present on fish meat.
Similarly, the tests carried out on two other species of fish in the process of putrefaction (7 days under melting ice), mackerel (fatty fish) and whiting, have allowed to put in evidence of the torA gene in spoilage bacteria.
Thus, the use of the pairs of molecular primers DDNl + / DDNS-, BN6 + / BN2-and BN6 + / BN4- can be considered for the application of the PCR test to flesh of Pisces.
In addition, the use of a silica bead filter is a method adequate to purify the total DNA contained in fish flesh. This technique is very fast (about one hour), and very easy to implement compared to an extraction isoamylalcohol / chloroform which requires several steps to eliminate contained proteins in fish flesh (28).
Amplification carried out under the same conditions with the pair of primers BC1 + / BC2 has also given encouraging results on the total DNA of flesh of a red mullet in the process of putrefaction (Figure 18). Indeed, despite a background noise important, agarose gel electrophoresis of PCR products reveals the presence of fragment of 177 base pairs.
The application of the technique to a follow-up of cod alteration made it possible to to show that the technique is in direct correlation with the freshness quality of the flesh of fish during their conservation over time.
III) Conclusion The pairs of molecular primers according to the invention therefore make it possible to detect the gene torA in marine bacteria, but also in bacteria relatively distant phylogenetically, namely enterobacteria and bacteria in water brackish.
Application of the PCR test to fish flesh gives results encouraging S since the molecular primers according to the invention make it possible to detect the torA gene from bacteria on putrefying fish. Amplification PCR cannot be applied directly to fish flesh samples, an extraction total DNA
of this flesh is necessary beforehand.
In addition, the technique is directly linked to the freshness quality of the flesh fish and is more sensitive than conventional techniques like ABVT.
B) MATERIALS AND METHODS
1) Isolation of bacterial strains, media and growth conditions The fish used for the isolation of the bacterial strains Shewanella c and Shewanella massilia corresponds to a red mullet (Mullus surmuletus) caught in the sea Mediterranean at off Marseille, and kept at room temperature for 48 hours in a sterile tube containing seawater. The seawater has been previously sterilized at through a filter millipore (0.45 pM).
Shewanella strains are grown anaerobically or aerobically to 30 ° C in LB medium (“Luria Broth”: yeast extract 10 g / 1, bacto-peptone Sg / 1 and NaCI Sg / 1). The E. coli and Salmonella typhimurium bacteria are grown at 37 ° C on middle LB. Strain Photobacterium phosphoreum was obtained by ATCC (n ° 11040) and does not not push on conventional media such as LB medium. It is grown at 15 ° C
on marine environment (marine broth; Difco).
2) PCR techniques a) Standard PCR
Standard PCR amplifications are carried out under the conditions described in the reference (6). The reaction mixture (50 μl) contains 10 ng of DNA
chromosomal, 0.2 ~ g of each of the oligonucleotide probes, 100 µM of each of the four deoxyribonucleotides triphosphates (dXTPs), 10 mM Tris / HCl (pH 8.3), 1.5 mM MgCl2, 50 mM
KCI, 0.01 gelatin and one unit of DNA polymerase (Taq polymerase, Boehringer Mannheim / Roche).
The reaction mixture is finally covered with 50 ~ 1 of mineral oil in order to prevent evaporation during the PCR process.
Amplification, carried out in a thermal cycler (MJ Research), highlights Thu 30 reaction cycles, a cycle comprising a denaturation step at 94 ° C for 30 seconds, a hybridization step at 55 ° C for 30 seconds and a elongation step at 72 ° C
for about 45 seconds (lkb / min). DNA is previously denatured during 1.5 mins to 94 ° C before starting the first cycle. 10 ~, 1 of each product amplification are then analyzed by electrophoresis on an agarose gel.
b) PCR conditions with degenerate molecular primers according to the invention The molecular primers according to the invention, in particular the primers called "DDN", "BN" or "BC" are as defined above.
The reaction mixture (50 μl) contains either 10 ng of chromosomal DNA from the bacteria, either 5 ~ l of cell suspension (DO6oo = 0.5-1) or 25 ng of DNA
total extracted at from fish, 100 µM of each of the four deoxyribonucleotides triphosphates (dXTPs), 0.8 ~, g of each of the primer mixtures according to the invention, 10 mM Tris-HCl (pH
8.3), 1.5 mM
MgCl2, 50 mM KCI, 0.01% gelatin and one unit of Taq polymerase.
The PCR reaction involves 30 successive reaction cycles consisting of of a stage of denaturation at 94 ° C for 30 seconds followed by a step hybridization at SS ° C or 45 ° C
for 30 seconds, then an elongation step at 72 ° C for 45 seconds. DNA is previously denatured for 1.5 min at 94 ° C before starting the first cycle.
3) Preparation of chromosomal DNA from bacteria The chromosomal DNA preparation of bacteria is carried out from 10 ml of culture (D06oo ~ 1). After centrifugation of the cells at 10,000 rpm for 10 min, the base is resuspended in 1 ml of 10 mM EDTA (pH 8). Cell lysis is obtained by addition of SDS (0.5% final). This solution is mixed in an equal volume isoamyl alcohol / chloroform (1:24). After centrifugation for 20 min at 7,000 rpm, the upper phase is recovered.
This operation is repeated several times in order to eliminate the greatest amount of protein residual. The DNA is finally precipitated by adding NaCl (0.3 M final) and ethanol (2.2 Flight).

4) Preparation of total chromosomal DNA from the flesh of fish in the process of putrefaction for PCR
a) Rapid preparation using a kit based on the attachment of DNA to marbles sillice (High pure PCR template Preparation Kit, Boehringer Mannheim / Roche) 25 mg of fish flesh are incubated for 45 min at 55 ° C. in 200 p1 of lysis pad (4 M urea, 200 mM Tris, 20 mM NaCI and 200 mM EDTA, pH 7.4) in the presence of 40 p1 of proteinase K (0.8 mg). In order to facilitate cell lysis, the sample is previously ground using a scalpel. 200 ~ 1 fixation buffer (guanidine-HCl 6 M, urea 10 mM, Tris-HCl mM and Triton 20% v / v, pH 4.4) are added to the sample which is then incubated for 10 10 min at 72 ° C. This solution is mixed with 100 ~ 1 of isopropanol then centrifuged at 8,000 rpm for 1 min through a filter based on sillice beads (High Pure tube Filter + tube collector). Residual impurities are removed by two stages of washing (buffer washing: 20 mM NaCl, 2 mM Tris-HCl, 80% v / v ethanol, pH 7.5). DNA is then elected in an elution buffer (10 mM Tris, pH 8.5).
b) DNA extraction from cells by NaOA / SDS treatment mg of fish flesh is taken from a putrefying fish (after 16 hours of incubation in a sterile tube kept at room temperature), and placed in 50 p1 0.05 M NaOH solution, 0.25% SDS. After grinding the cells, the sample is incubated 15 min at 95 ° C. 450 ~, 1 of sterile water is added to the mixture and cellular debris are 20 then eliminated by a centrifugation step of 2 min at 8000 g. 5 ~, 1 of supernatant obtained are directly used for the PCR test.
c) IsoamylalcooUchloroform extraction Preparation of total chromosomal DNA by extraction isoamyl alcohol / chloroform is made from 25 mg of contaminated fish flesh as described previously 25 for bacterial DNA. The number of extraction steps is however increased in order to ability to remove large amounts of impurities from samples of fish flesh.

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(3) McEwan AG, Ferguson SJ and Jackson JB (1983) Electron flow to dimethylsulphoxide or trimethylamine N oxide generates a membrane potential in Rhodopseudomonas capsulata. Arch.Microbiol. 136, 300-305.
(4) Barrett EL and Kwan HS (1985) Bacterial reduction of trimethylamine oxide.
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(8) Knablein J., Mann K., Ehlert S., Fonstein M., Huber R. and Schneider F.
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(9) Ujüye T., Yamamoto L, Nakama H., Okubo A., Yamazaki S. and Satoh T. (1996) Nucleotide sequence of the genes, encoding the pentaheme cytochrome (dmsC ~ and tea transmembrane protein (dmsB), involved in dimethyl sulfoxide respiration from Rhodobacter sphaeroides f. sp. denitrificans. Biochim.Biophys.Acta 1277, 1-5.

(10) Mouncey NJ, Choudhary M. and Kaplan S. (1997) Characterization of genes encoding dimethyl sulfoxide reductase of Rhodobacter sphaeroides 2.4.1T: an essential metabolic Bene function encoded on chromosome II. J. Bacteriol. 179, 7617-7624.

(11) Malle P. (1994) Bacterial microflora from marine fish and evaluation of the alteration. Med Rec. Vet. 170, 147-157.

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(13) Dalgaard P., Gram L. and Huss HH (1993) Spoilage and shelf life of cod little girls packed in vacuum or modified atmospheres. Int.J. Food Microbiol. 19, 283-294.

(14) Malle P. and Poumeyrol M. (1989) A new chemical criterion for the quality control of fish: Trimethylamine / Total Volatile Basic Nitrogen (%). J. Food Prot. 52, 419-423.

(15) Large PJ and MacDougall H. (1975) An enzymatic method for the microestimation of trimethylamine. Anal.Biochem. 64, 304-310.

(16) Ritskes TM (1975) Gas chromatographic determination of trimethylamine and 1 S dimethylamine in fish, fishery products, and other funds. .LFood Technol.
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(1998) Crystal structure of oxidized trimethylamine N oxide reductase from Shewanella massilia at 2.5 F ~ Resolution. .l. Mol. Biol. 284, 434-447.

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LIST OF SEQUENCES
<110> CNRS
<120> NUCLEOTIDE SEQUENCES FROM GENES ENCODING THE
TRIMETHYLAMINE N-OXIDE REDUCTASE, AND THEIR
USES, PARTICULARLY FOR THE DETECTION OF BACTERIA
<130> WOB 99 AX CNR DORA
<140>
<141>
<150> FR9911543 <151> 1999-09-15 <160> 27 <170> PatentIn Ver. 2.1 <210> 1 <211> 2487 <212> DNA
<213> Shewanella massilia <400> 1 atgaacagaa gagacttttt aaagggtatc gcctcatcct ctttcgttgt cttaggtggc 60 agctcagtgt taacgccctt aaatgcctta gccaaagcgg gcatcaatga agatgaatgg 120 ctaaccacag gttcacactt cggcgccttt aaaatgaagc gcaaaaacgg cgtcattgcc 180 gaagtgaaac ccttcgactt agataagtat ccaacggata tgattaacgg catccgcggc 240 atggtctaca atccatcgcg tgtacgttac cctatggtgc gcttagattt tttactcaaa 300 ggtcataaga gtaataccca tcaacggggt gatttccgct ttgttcgcgt aacgtgggac 360 aaggcattaa cactgtttaa gcattcatta gatgaagtcc aaacccaata cggtccatca 420 ggtctgcatg cggggcaaac cggttggcgc gccactggtc aactgcattc cagcacgagt 480 catatgcaac gtgcggtggg gatgcacggc aactatgtta agaaaatcgg cgactactcc 540 acaggtgcag gccaaactat tctgccctac gtgttaggtt caaccgaagt gtatgcccag 600 ggcacttcat ggccgctgat cttagaacac agcgacacta tcgtgctgtg gtcgaacgat 660 ccgtacaaga acctgcaagt gggttggaat gcggaaaccc atgaatcttt tgcttatctt 720 gcgcagttaa aagagaaagt gaagcaaggc aagatccgcg tgatcagtat cgaccctgtg 780 gtgactaaga cccaagccta tttgggctgc gagcaactct acgttaaccc acagacagat 840 gtgaccttaa tgctggccat cgcccacgag atgatcagca aaaagctcta cgacgataaa 900 tttatccaag gctacagctt aggttttgaa gagtttgtgc cctatgtgat gggtactaaa 960 gatggcgtag ccaaaacccc agaatgggcc gcgcctatct gtggtgttga agçccatgtt 1020 atccgcgact tggctaaaac cttagtcaag ggccgcactc agttcatgat gggctggtgt 1080 atccagcgcc agcaacacgg cgaacaaccc tattggatgc cggcggtact ggcgaccatg 1140 atcggccaaa tcggtctacc cggtggtggc atcagctatg gtcaccacta ctcgagtatc 1200 ggcgtgcctt catcgggtgc cgctgcgcca ggtgccttcc cccgtaactt ggacgaaaat 1260 caaaagccac tctttgatag ctcagacttc aagggcgcga gtagcacgat tccggttgcc 1320 cgctggattg atgcgattct cgaacccggt aaaaccattg atgctaacgg ctcgaaagtg 1380 gtttatcccg atatcaagat gatgattttc tcgggtaata atccttggaa ccatcaccaa 1440 gacagaaacc gcatgaagca agccttccat aagcttgagt gtgtggtcac tgtcgatgtg 1500 aactggacgg caacttgccg cttctcggat atcgtactgc ccgcttgtac tacctatgag 1560 cgcaacgata tcgacgtgta cggcgcctat gctaaccgcg gtattttagc catgcagaaa 1620 atggttgagc cactgtttga tagcttgtcg gattttgaaa ttttcactcg ctttgccgcc 1680 gtactcggca aagagaaaga atacacccgt aacatgggcg aaatggagtg gttagaaacc 1740 ctctataacg aatgtaaagc cgccaacgcg ggcaagtttg agatgcctga ctttgcgact 1800 ttctggaaac aaggttatgt gcattttggt gacggtgaag tctggacgcg ccatgcagac 1860 tttagaaacg atcctgaaat caatccacta ggcacgcctt caggtttgat tgaaatcttt 1920 agccgtaaga ttgatcaatt cggttacgat gactgtaaag gtcacccaac gtggatggag 1980 aaaaccgagc gtagtcatgg cggccctggc tctgacaagc atccgatttg gttgcagtca 2040 tgccatccag acaaacgttt acactcgcag atgtgtgagt cgcgagaata ccgcgagact 2100 tacgcagtca atggccgtga gcctgtgtat atcagccctg tcgacgcaaa agcacgtggc 2160 atcaaagatg gcgatatagt gcgagtcttt aacgaccgtg gccaactgtt ggcgggtgct 2220 gtggtatcgg acaacttccc taaagggatt gtgcgaattc acgaaggcgc gtggtatggg 2280 ccagtaggta aagatggtag cactgaaggt ggtgctgaag tcggcgccct gtgtagttat 2340 ggcgatccta acaccctcac tttagacata ggcacctcta aacttgccca agcttgctca 2400 gcctatactt gcttagtcga gtttgaaaaa taccaaggca aagtgcctaa ggtcagctcc 2460 ttcgatggcc cgatcgaagt cgaaatc 2487 <210> 2 <211> 2486 <212> DNA
<213> Shewanella putrefaciens <400> 2 atgaacagaa gagacttttt aaaaggctta gcctcaacct ctttcgttgc tttaggtggc 60 agctcagtac tagcgccctt aaatgcgctg gccaatactg gcctgaatga aaacgaatgg 120 ctgaccactg gctcccactt cggtgccttt aaaatcaagc gtaaaaacgg catgattgcc 180 gaagtcaaag ccttcgattt agataaatat ccaacggata tgattaacgg tatccggggt 240 atggtctata acccatcccg cgtgcgttac ccgatggttc gcttagactt tttactaaaa 300 ggccataaga gtaataccca gcagcggggg gatttccgct ttgttcgtgt gacctgggat 360 aaagcattaa agctgtttaa acactcactc gatgaggtcc aaaccaagta cggtccatcg 420 ggcttacacg caggacaaac tggttggcgc gccacggggc aactgcattc cagcaccagc 480 catatgcagc gcgcggtggg gatgcacggt aattttgtga aaaaaatcgg cgactactcc 540 accggtgcag gccaaccatt ctgccctatg tattaggctc aaccgaagta tatgcccaag 600 gcacctcttg gccactgatc ttagaaaaca gcaacacgat tgtgctgtgg tcaaacaatc 660 cttacaaaaa cctgcaagtg ggctggaacg ctgaaaccca tgaggccttt gcgtacctcg 720 cgcaattaaa agagaaggtc aaacagggta aaatccgcgt gatcagtatc gaccctgtgg 780 tgactaaaac ccaggcttac cttggctgtg agcagttgta tgtgaaccca cagactgacg 840 tgacgctgat gctggccatc gcccatgaga tgatcaccca aaagctacac gatgagaaat 900 tcatccaagg ttacagctta ggctttgaag agtttgtgcc ttacgtgatg ggcactaaag 960 atggtatcgc caaaacccct gagtgggcgg cgcctatctg cggtgttgaa ccacacatta 1020 tccgcgatct ggccaaaacc ttagttaagg gccgtaccca aataatgatg ggttggtgta 1080 ttcagcgcca acaacacggt gagcaacctt actggatggc cgcagtactt gcgaccatga 1140 taggccaaat cggcttaccc ggcggtggta ttagctatgg tcaccactac tccagtattg 1200 gtgtgccggc gaccacagct gcagctccgg gcgctttccc acgtaactta gacgagaatc 1260 aaaaaccgct gtttgacagc acagacttta aaggcgcaag cagcacgatt cccgttgccc 1320 gctggattga tgcgattctc gaacccggca aaaccattga tgccaacggt tctaaagtgg 1380 tatatcccga tattaagatg atgattttct cgggtaataa cccatggaac caccatcaag 1440 accgtaaccg catgaagcaa gccttccaaa agcttgaatg cgtggtctct attgatgtga 1500 actggacagc cacttgtcgt ttctccgaca tcgtgttgcc agcttgcacc acctatgagc 1560 gtaacgatat cgacgtgtac ggtgcctatg ccaaccgcgg tattctggcg atgcagaaaa 1620 tggtcgagcc tttgtttgaa agcttgtctg actttgagat ctttactcgt tttgccgcgc 1680 tgctgggtaa agagaaagaa tacacccgca atatgagcga aatggagtgg atagaaaccc 1740 tctataacga gtgtaaagcc gctaacgccg gtaagtatga gatgcctgac tttgccacct 1800 tctggaagca aggttatgta cactttggtg aaggcgaatt atggacgcgc cacgctgact 1860 ttagaaacga tcctgaaatc aacccattag gcacgccttc cggtttgatt gaaatcttca 1920 gtcgtaagat tgaacaattt ggctatgatg actgccaagg ccatcctatg tggatggaaa 1980 aagctgaacg cagccatggt ggtccaggtt cgaataaata tcctatgtgg ttgcaatctt 2040 gccatccgga tcaccgctta cactcacaaa tgtgtgagtc aaaggaatac cgcgaaacct 2100 acacagttaa tggtcgcgaa cctgtgtata ttagccctga agatgctaaa acccgtggca 2160 ttaaagatgg cgatatcgtg cgggtcttta acgaccgagg tcaactgtta gctggcgcag 2220 tggtatcgga tcgtttccct aaaggtgtag tgcgaattca tgaaggtgca tggtatggcc 2280 cagtgggtaa agatggcagc gttgaaggcg gagccgaaat cggtgccctg tgcagctatg 2340 gtgaccctaa taccctaacc ttagacattg gtacctctaa gttggctcaa gcttgctcag 2400 cctatacatg tctggttgag tttgaaaaat accaaggtaa agcacctaag gttagctcct 2460 tcgatggtcc tatcgaagtt gaaatc 2486 <210> 3 <211> 2487 <212> DNA
<213> Schewanella c <900> 3 atgaacagaa gagacttttt aaagggtatc gcctcatcct ctttcgttgt cttaggtggc 60 agctcagtgt tagcgccctt aaatgcctta gccaaaacgg gcatcaatga agacgaatgg 120 ctaaccacag gttcacactt cggcgccttt aaaatgaagc gcaaaaacgg cgtcattgcc 180 gaagtgaaac ccttcgactt agataagtat ccaacggata tgattaacgg catccgcgac 240 atggtctaca atccatcgcg tgtacgttac cctatggtgc gcttagattt tttactcaaa 300 ggtcataaga gtaataccca tcaacggggt gatttccgct ttgttcgtgt aacatgggac 360 aaggcattaa cactgtttaa gcattcatta gatgaagtcc aaacccaata cggtccatca 420 ggtctgcatg cgggtcaaac tggttggcgc gccacgggtc aactgcattc cagcacgagt 980 catatgcaac gtgcggtggg gatgcacggc aactatgtga agaaaatcgg cgactactcc 540 acaggtgcag gccaaacaat tctgccctac gtgttaggtt caaccgaagt gtatgcccaa 600 ggcacttcat ggccgctgat cttagaacac agcgacacta tcgtgctctg gtcgaacgat 660 ccgtacaaga acctgcaagt gggttggaat gcggaaaccc atgaatcttt tgcttatctt 720 gcgcagttaa aagagaaagt gaagcaaggc aagatccgtg ttatcagtat cgaccctgtg 780 gtgactaaga cccaagccta tttgggctgt gagcaactct acgttaaccc acagacagac 840 gtgactttaa tgctggccat cgcccacgag atgatcagca aaaagctcta cgacgataaa 900 tttatccaag gctacagctt aggttttgaa gagtttgtgc cctatgtgat gggtactaaa 960 gatggcgtag ccaaaacccc agaatgggcc gcgcctatct gtggtgttga agcccatgtt 1020 atccgcgact tggctaaaac cttagtcaag ggccgcactc agttcatgat gggctggtgt 1080 atccagcgcc agcaacacgg cgaacaaccc tattggatgg cggcggtact ggcgaccatg 1140 atcggccaaa tcggtctacc cggtggtggc atcagttatg gtcaccacta ctcgagtatc 1200 ggcgtgcctt catcgggtgc cgcggcgcca ggtgctttcc cccgtaactt ggacgaaaat 1260 caaaagccac tatttgatag ctcagacttc aagggcgcga gcagcacaat tccggttgcc 1320 cgctggattg atgcgattct cgaacctggt aaaaccattg atgctaacgg ctcgaaagtg 1380 gtttatcccg atatcaagat gatgattttc tcgggtaata atccttggaa ccatcaccaa 1440 gacagaaacc gtatgaagca agccttccat aagcttgagt gtgtggtcac tgttgatgtg 1500 aactggacgg caacttgccg cttctcggat atcgtactac ccgcttgtac tacctatgag 1560 cgcaacgata tcgacgttta cggcgcctat gctaaccgcg gtattttagc catgcagaaa 1620 atggttgagc cactgtttga tagcttgtcg gattttgaaa ttttcactcg ctttgccgcc 1680 gtacttggta aagagaaaga atacacccgt aacatgggcg aaatggagtg gctagaaacc 1740 ctctataacg aatgtaaagc cgccaacgcg ggcaagtttg agatgcctga ctttgcgact 1800 ttctggaaac aaggttatgt gcattttggt gacggtgaac tctggacgcg ccatgcagac 1860 tttagaaacg atcctgaaat caatccacta ggcacgcctt caggtttgat tgaaatcttt 1920 agccgtaaga ttgatcaatt cggttacgat gactgtaaag gtcacccaac ttggatggag 1980 aaaaccgagc gtagccatgg cggccctggt tctgacaagc atccgatttg gttgcagtca 2040 tgccacccag acaaacgctt acactcgcaa atgtgtgagt cgcgagaata ccgcgagacc 2100 tacgcagtca atggccgtga gcctgtgtat atcagccctg tcgacgcaaa agcgcgcggc 2160 ataaaagatg gcgatatagt gcgagtcttt aacgaccgtg gccaactgtt ggcgggtgca 2220 gtggtatcgg acaacttccc tactggtatt gtgcggattc acgaaggcgc atggtatggg 2280 ccagtaggta aagatggtag cactgaaggt ggggctgaag tcggcgccct gtgcagttat 2340 ggcgatccta acaccctcac tttagacata ggcacatcta aacttgccca agcttgctca 2400 gcctatactt gcttagtcga gtttgagaaa taccaaggca aagtgcctaa ggtcagctcc 2460 ttcgatggcc ctatcgaagt cgaaatc 2487 <210> 4 <211> 1080 <212> DNA
<213> Artificial sequence <220>
<223> Description of the artificial sequence: sequence partial coding for the protein TorA of Photobacterium phosphoreum <400> 4 acaatactga aagattgtaa gacattgata tggtggtcaa atgatccgat taaaaacagt 60 caggttggct ggcagtgtga gactcatggt tcttatgagt attatgcgca attaaagcag 120 aaggtcgcag atggtgggat ccgtatgatc tcggtcgatc ctgtagtgtc gaaatcgcaa 180 aaatatttta actgtgagca ccaatacgtc aatcctcaaa ctgacgttcc tttcatgctt 240 gctattgcgc atacattgta taaagaagat ctgtacgata aacaatttct ggaaacttac 300 actttaggct tcaatgaatt cttgccttac ttattgggta caggcaaaga taaaatagcc 360 aaaacgccag aatgggcaga gccaatttgt ggcgttaaag cagaggctat tcgagaattt 420 gctcgcggat tagttaaaaa ccgtacgatg ataatgtttg gttgggctgt acagcgtcaa 480 caacacggtg agcagcctta ttggatggga gcagtgctgg cttcgatgtt aggccaaata 540 ggcttacctg gtggagggat ttcctattct cacttttaca gtggcgttgg gttacctttc 600 agtactgcag ctgggccggg gggatttccg cgtaatgttg atgaaggcca ~ acagccgatt 660 tggaataata acgattttaa aggctacagt tcgacaattc cggtcgcaag atggattgat 720 gcgatcatgg aaccaggtaa aaaaattcaa tataacggcg ctaatgtggt gttgcctgat 780 attaagatga tggtctttag tggttgtaat ccgtggaatc atcatcaaca acgtaatcgt 890 atgaaacaag catttagaaa gctgcaaacc gtggttaata ttgattatac atggacacca 900 acctgtcgtt tttccgatat tgtattacct gcttgtaccc aatttgagcg tagtgattta 960 gatcaatatg gtacttattc aactagcggt attttagcga tgcataagct aattgatccg 1020 ctttatcaat caaaaacaga ctttcagata tttactgaat taaccgaacg ctttgggaaa 1080 <210> 5 <211> 392 <212> PRT
<213> Shewanella massilia <400> 5 Met Lys Trp Leu Thr Asn Leu Trp Arg Thr Leu Asn Lys Pro Thr Lys Ala Leu Thr Leu Gly Ala Val Ser Ile Ser Ala Phe Ile Met Gly Ile Ile Phe Trp Gly Gly Phe Asn Thr Ala Leu Glu Ala Thr Asn Thr Glu Ala Phe Cys Ile Ser Cys His Ser Met Glu Ser Lys Pro Tyr Gln Glu Leu Gln Glu Thr Val His Trp Ser Asn His Phe Gly Val Arg Ala Thr Cys Pro Asp Cys His Val Pro His Asn Trp Ser Arg Lys Ile Ala Arg Lys Met Glu Ala Ser His Asp Val Trp Gly Trp Leu Phe Asn Thr Val Asn Thr Pro Glu Lys Phe Glu Ala Lys Arg Leu Glu Met Ala Ser Arg Glu Trp Lys Arg Phe Asp Arg Asp Asn Ser Leu Ala Cys Lys Asn Cys His Asn Tyr Asn Ser Met Lys Trp Glu Ala Met Ser Pro Leu Ala Gln Lys Gln Met Lys Arg Ala Ala Glu Ile Asp Gln Ser Cys Ile Asp Cys His Lys Gly Ile Ala His His Leu Pro Glu Met Gly Thr Ala Arg Ala Pro Glu Leu Ile Ala Glu Val Gly Ala Gly Val Ser Ser Val Glu Thr Asn Gln Thr Tyr Tyr Ser Ala Leu Thr Lys Pro Leu Phe Phe Thr Asp Lys Gly Asp Val Glu Ala Gly Thr Leu Asn Val Ala Thr Lys Val Lys Val Leu Glu Thr Gln Gly Lys Arg Ile Lys Ile Gly Ile Asp Gly Trp Arg Lys Lys Ile Gly Ala Gly Arg Val Ile Tyr Met Asp Phe Gly Val Asn Ile Leu Ser Ala Gln Leu Thr Lys Asp Ala Ala Glu Thr Gly Gly Val Ile Gln Thr Phe Glu Glu Lys Glu Asp Pro Met Thr Gly Leu Lys Trp Gln Arg Ile Glu Ala Gln Ile Trp Thr Asp Lys Asp Tyr Leu Leu Thr Glu Leu Gln Pro Leu Trp Gly Tyr Ala Arg Asp Thr Phe Arg Ser Ser Cys Ser Val Cys His Thr Gln Pro Asp Glu Ala His Phe Asp Ala Asn Thr Trp Pro Gly Met Phe Gln Gly Met Leu Ala Phe Val Asn Met Asp Gln Asp Thr Gln Ala Leu Val Gln Lys Tyr Leu Gln Glu His Ser Ser Thr Phe Val Lys Lys Glu His 385,390 <210> 6 <211> 2523 <212> DNA
<213> Rhodobacter sphaeroides <400> 6 atgccccgcc cgggacgaag cccggcaaga ccgtcatacg gaagaaagga aagccagatg 60 actaagttgt caggtcagga gctgcatgcc gaactctcgc ggcgcgcctt cctgagctat 120 acggcggctg tgggggctct cggtctctgc ggcacctcgc tcctcgcgca gggagcccgc 180 gcggaaggtc tcgccaacgg cgaggtcatg tcgggctgcc actggggcgt gttcaaggcc 240 cgggtcgaga acggccgcgc cgtggccttc gagccctggg acaaggaccc cgcgccgtcg 300 caccagctgc cgggcgtgct cgattcgatc tattcgccca cgcggatcaa atatccgatg 360 gtgcgccgcg aattcctcga gaagggcgtg aacgccgacc gctccacccg cggcaacggc 420 gacttcgtcc gcgtcacctg ggatgaagcg ctcgacctcg tggccaagga actgaagcgc 480 gttcaggaaa gctacgggcc caccggcacc ttcggcggct cctacggctg gaaaaacccg 540 ggccggctgc acaactgtca ggtcctcatg cgccgcgcgc tgaatctcgc gggcgggttc 600 gtgaactcgt cgggcgacta ttcgaccggc gccgcgcaga tcatcatgcc gcatgtcatg 660 ggcacgctcg aggtctacga gcagcagacc gcctggcccg tggtggtgga caacaccgaa 720 ctgatggtct tctgggccgc cgatccggtg aagaccaacc agatcggctg ggtggtcccc 780 gaccatggcg ccttcgcggg catgcaggca atgaaggaaa agggcaccaa ggtcatctgc 840 atcaaccccg tgcgcaccga gacggccgac tatttcggcg ccgaactcgt gtcgccgcgg 900 ccgcagaccg acgtggcgct gatgctcggc atggcgcaca cgctctacag cgaagatctg 960 cacgacaagg acttcatcga aaactgcacc tcgggcttcg acatcttcgc ggcctacctg 1020 accggcgaga gcgacggcac gcccaagacg gccgaatggg ccgccgagat ctgcggcctg 1080 ccggccgagc agatcaagga actcgcccgc cgcttcgtgg gcggccggac gatgctcgcc 1140 gcgggctggt cgatccagcg gatgcaccat ggcgaacagg cgcactggat gctcgtcacg 1200 ctggcctcga tgatcggcca gatcggtctt ccgggcggcg gcttcggcct tagctaccat 1260 tactccaacg gtggctcgcc cacgagcgac ggcccggcgc tgggcggtat ttcggacggc 1320 ggcaagccgg tcgaaggtgc ggcctggctg tcggcgagcg gcgcggcttc gatcccctgc 1380 gcccgggtgg tggacatgct gctcaatccg ggcggcgagt tccagttcaa cggtgccacg 1440 gcgacctatc ccgacgtgaa gctggcctac tgggtgggcg gcaacccctt cgcgcaccac 1500 caggaccgca accggatgct caaggcctgg gaaaagctcg agaccttcat cgtgcaggac 1560 ttccagtgga ccgccaccgc gcgccacgcc gacatcgtcc tgccggcgac gacctcctac 1620 gaacgcaacg acatcgagtc ggtgggcgac tattcgaacc gcgccatcct cgcgatgaag 1680 aaggtggtcg atccgctcta cgaggcccgg tcggactacg acatcttcgc agccctgacg 1740 gagcgtctgg gcaagggcaa ggaattcacc gaaggccgcg acgagatggg ctggatcagc 1800 tcgttctacg aggcggcggt gaagcaggcc gagttcaagc agatggagat gccgtcgttc 1860 gaggacttct ggtcggaagg gatcgtcgag ttcccgatca ccgagggcgc gaacttcgtt 1920 cgctatgccg acttccgcga ggatccgctg ttcaaccccc tcggcacgcc ctcgggcctg 1980 atcgagatct actcgaagaa catcgagaag atgggctatg acgattgccc ggcccatccg 2040 acctggatgg aaccggccga gcgtctcggc gggccggggg cgaaatatcc gctccatgtg 2100 gtggcgagcc acccgaactc gcggctgcac tcgcagctga acggcacctc gctgcgcgac 2160 ctctatgcgg tcgcggggca cgagccctgt ctcatcaacc ccgacgatgc ggccgcgcgc 2220 ggcatcgcgg acggcgatgt gctgcgggtg ttcaacgacc gcgggcagat cctcgtgggc 2280 gcgaaggtga gcgacgcggt gatgccgggc gcgatccagg tctacgaggg cggctggtac 2340 gacccgctcg acccctcgga ggaaggcacg ctcgacaaat acggcgacgt gaacgtgctg 2400 tcgctcgacg tcggcacctc gaagctggcg cagggcaact gcggccagac catcctcgcg 2460 gatgtcgaaa aatatgcggg cgcgccggtg acggtgaccg tgttcgacac gccgaaggga 2520 ccc 2523 <210> 7 <211> 2475 <212> DNA
<213> Rhodobacter capsulatus <400> 7 atgacgaagt tttccggaaa cgagctgcgc gcagagcttt accgccgcgc tttcctcagc 60 tactcggttg caccgggcgc gctgggcatg ttcggccggt cgcttctggc caagggcgcc 120 cgcgccgagg cgctggccaa tggcacggtg atgtcgggca gccactgggg cgtctttacc 180 gcgacggtcg aaaacggccg cgccaccgcc ttcaccccct gggaaaaaga cccgcatccg 240 acgccgatgc tggaaggcgt gctggactcg atctattcgc cgacgcggat caaatatccg 300 atggtgcggc gcgaattcct cgaaaaaggc gtgaatgctg atcgctccac ccgcggcaac 360 ggcgattttc gtcccgtcag ctgggatcag gcgctcgatc tgcatggtcg cgg: cgaggtc 420 aaacgggtcg aaggagacct acggcccgca ggcgtctttg gcggctccta tggctggaaa 480 agccccgggc ggctgcacaa ttgcaccacg cttctgcgcc ggatgctgac gctggcgggc 540 ggctatgtga acggcgcggg cgattattcg accggcgcgg cgcaggtgat catgccgcat 600 gtggtcggca cgctggaagt ctatgaacag cagaccgcct ggccggtgct ggcggaaaac 660 accgaagtca tggtgttctg ggccgccgat ccgatcaaga cagcagatat cggctgggtg 720 tatcccgaac atggcgccta tccggggact gaggcgctca aggccaaggg caccaaggtc 780 atcgtcatcg atccggtccg caccaagacg gtcgaattct tcggcgcgga tcacgtcacg 840 ccgaaaccgc agaccgatgt ggcgatcatg ctgggcatgg cgcatacgct ggtggccgaa 900 gacctgtatg taaaggactt catcgccaac tacacctcgg gcttcgacaa gttcctgccc 960 tatctgatgg gcgagaccga cagcacgccg aagaccgccg aatgggcgtc ggatatcagc 1020 ggcgttccgg ccgagacgat caaggaactg gcgcggctgt tcaaatcgaa acgcacgatg 1080 ctggcggcgg gctggtcgat gcagcggatg catcacggcg agcaggcgca ttggatgctg 1140 gtgacgctgg cctcgatgct gggtcagatc gggctgcggg gcggcggctt cgggctgtcc 1200 tatcactatt cgggcggtgg cacgccctcg agcagcggtc cggcgctttc gggcatcacc 1260 gatggcgggc gacgaagggg ccggaatggc tggcggcgag cggcgcttcg gtgtatcccg 1320 gtggcgcgcg tggtcgacat gctggaaaac cccggcgccg aattcgactt caacggtacg 1380 cggtcgaaat tcccggatgt gaagatggcc tattgggttg gcggaacccc ttcgtgtcac 1440 catcaggacc gcaaccgcat ggtcaaggcc tgggaaaaac tggaaacctt catcgtgcat 1500 gacttccagt ggacgcccac ggcgcggcat gccgacatcg tgctgcccgc gacgaccagc 1560 tatgaacgca acgacatcga gacgatcggc gattattcga acaccggcat cctggcgatg 1620 aagaagatcg tcgagccgct ttacgaagcc cgcagcgatt acgacatctt cgccgcggtc 1680 gccgaacggc tgggcaaggg caaggagttc accgaaggca aggacgagat gggctggatc 1740 aagtccttct acgacgatgc cgccaagcag gcaaagcggg ggtcgagatg ccccgccttc 1800 gacgccttct gggcggaagg gatcgtggaa ttcccggtca ccgacggcgc ggacttcgtg 1860 cgctatgcca gcttccggga agatccgctg ctcaacccgc tgggcacgcc gaccggcctg 1920 atcgagatct actcgaagaa catcgagaag atgggctatg acgactgccc ggcgcatccg 1980 acctggatgg aaccgcttga acggctcgac gggccggggg cgaaatatcc gctgcacatc 2040 gcggctcgca cccgttcaac cggtgtactc gcacccgttc accggctcaa cggcacggtg 2100 ctgcgcgaag gctatgcggt gcaggggcac gagccctgcc tgatgcaccc cgacgacgcc 2160 gccgcgcgcg gcatcgccga tggcgacgtg gtgcgggtgc acaatgatcg cggtcagatc 2220 ctgaccgggg tcaaggtgac cgatgcggtg atgaaggggg taatccagat ctacgaaggg 2280 ggctggtatg atccctcgga cgtgaccgag gcggggacgc tcgacaaata cggcgacgtt 2340 aacgtgctgt cggccgatat cggcatgtcg aaactggcgc agggcaactg tggtcagacc 2400 gtgctggccg aggtcgagaa atacaccggc cccgccgtca ccctgaccgg ctttggtcgc 2460 gcgaaggcgg tcgaa 2475 <210> 8 <211> 404 <212> PRT
<213> Rhodobacter sphareroides <400> 8 Met Gly Arg Ser Cys Gly Gln Ala Ser Glu Ala Lys Val Ile Gly Arg Ile Trp Lys Ala Phe Trp Arg Pro Ser Thr Lys Trp Gly Leu Gly Val Leu Leu Val Thr Gly Gly Ile Ala Gly Ala Val Gly Trp Asn Gly Phe His Tyr Val Val Glu Lys Thr Thr Thr Thr Glu Phe Cys Ile Ser Cys His Ser Met Arg Asp Asn Asn Tyr Glu Glu Tyr Lys Thr Thr Ile His Tyr Gln Asn Thr Ser Gly Val Arg Ala Glu Cys Ala Asp Cys His Val 85 90 95 ._ Pro Lys Ser Gly Trp Lys Leu Tyr Arg Ala Lys Leu Leu Ala Ala Lys Asp Leu Trp Gly Glu Arg Island Gly Thr Ile Asp Thr Arg Glu Lys Phe Glu Ala His Arg Leu Glu Met Ala Glu Thr Val Trp Ala Asp Met Lys Ala Asn Asp Ser Ala Thr Cys Arg Thr Cys His Ser Phe Glu Ala Met Asp Phe Ala His Gln Lys Pro Glu Ala Ser Lys Gln Met Gln Gln Ala Met Asn Glu Gly Gly Thr Cys Ile Asp Cys His Lys Gly Ile Ala His Lys Met Pro Asp Met Ala Ser Gly Tyr Arg Ala Leu Phe Ser Lys Leu Glu Lys Ala Ser Gln Ser Leu Lys Pro Arg Lys Gly Glu Thr Leu Tyr Pro Leu Arg Thr Ile Glu Ala Tyr Leu Glu Lys Pro Ser Gly Glu Lys Ala Lys Ala Asp Gly Arg Leu Leu Ala Ala Thr Pro Met Gln Val Val Asp Val Thr Gly Asp Trp Val Gln Val Ala Val Lys Gly Trp Gln Gln Glu Gly Ala Glu Arg Val Ile Tyr Glu Lys Gln Gly Lys Arg Ile Phe Asn Ala Ala Leu Ala Pro Ala Ala Thr Gly Ser Val Val Pro Gly Ala Ser Met Val Asp Pro Asp Thr Glu Gln Thr Trp Thr Asp Val Ser Leu Thr Ala Trp Val Arg Asn Arg Asp Leu Thr Gly Asp Gln Glu Ala Leu Trp Gln Tyr Gly Lys Gln Met Tyr Asn Gly Ala Cys Gly Met Cys His Val Leu Pro His Pro Glu His Phe Leu Ala Asn Gln Trp Ile Gly Thr Leu Asn Ala Met Lys Ser Arg Ala Pro Leu Asp Asp Glu Gln Phe Arg Leu Val Gln Arg Tyr Val Gln Met His Ala Lys Asp Val Glu Pro Glu Gly Ala Ala Glu <210> 9 <211> 2544 <212> DNA
<213> Escherichia coli <400> 9 atgaacaata acgatctctt tcaggcatca cgtcggcgtt ttctggcaca actcggcggc 60 ttaaccgtcg ccgggatgct ggggccgtca ttgttaacgc cgcgacgtgc gactgcggcg 120 caagcggcga ctgacgctgt catctcgaaa gagggcattc ttaccgggtc gcactggggg 180 gctatccgcg cgacggtgaa ggatggtcgc tttgtggcgg cgaaaccgtt cgaactggat 240 aaatatccgt cgaaaatgat tgccggattg ccggatcacg tacacaacgc ggcgcgtatt 300 cgttatccga tggtacgcgt ggactggctg cgtaagcgcc atctcagcga tacctcccag 360 cgcggtgata accgttttgt gcgcgtgagc tgggatgaag ccctcgacat gttctatgaa 420 gaactggaac gcgtgcagaa aactcacggg ccgagtgcct tgctgaccgc cagtggttgg 480 caatcgacgg ggatgttcca taacgcttcg gggatgcgtg cgaaacgtat tgccttgcat 540 ggtaatagcg ttggtacggg cggagattac tctaccggtg ctgcgcaggt gatcctgccg 600 cgcgtagtcg gttcgatgga agtgtatgaa cagcaaacct cctggccgct ggtattgcag 660 aacagcaaaa ccattgtgct gtggggctcc gatttgctga aaaaccagca agcgaactgg 720 tggtgcccgg atcacgatgt ttatgaatat tacgcgcagc taaagcgaaa gtcggccgcc 780 ggtgaaattg aggtcatcag catcgatccg gttgtcacat ccacccatga gtatctgggc 840 ggggagcatg tgaagcacat tgcggttaac ccgcaaactg acgtgccgct gcaactcgcg 900 ctggcacata cgctgtacag tgaaaacctg tacgacaaaa acttccttgc taactactgt 960 gtgggttttg aggagttcct gccgtatctg ctgggtgaga aagacggtca gccgaaagat 1020 gccgcatggg ctgaaaaact gagcggcatt gatgccgaaa ccattcgtgg gctggcgcgg 1080 cagatggcgg cgaacagaac gcaaattatt gctggctggt gcgtgcagcg tatgcagcac 1140 ggtgaacagt gggcgtggat gattgtggtt ctggcggcga tgctggggca aattggcctg 1200 ccaggtggtg gttttggttt tggctggcac tacaacggcg caggcacgcc ggggcgtaaa 1260 ggcgttattc tgagtggttt ctccggctct acgtcgattc cgcctgttca cgacaacagt 1320 gactataaag gctacagcag cactattccg attgcccgtt ttatcgatgc gatcctcgaa 1380 ccggggaaag tgatcaactg gaacggtaaa tcggtaaaac tgccgccgct gaaaatgtgt 1440 atttttgccg gaactaaccc attccatcgc catcagcaga tcaaccgcat tattgaaggc 1500 ttgcgcaacg tggaaacggt tatcgccata gataaccagt ggacctcaac ctgccgcttt 1560 gccgatatcg tactgcctgc gaccacgcag tttgagcgta acgatctcga ccagtacggc 1620 aatcactcca accgtggcat tatcgccatg aaacaggtgg tgccgccgca gttcgaggcg 1680 cgcaacgact tcgatatttt ccgcgagctg tgccgtcgct ttaatcgcga agaagccttt 1740 accgaagggc tggacgaaat gggctggctg aaacgcatct ggcaggaagg tgtacagcaa 1800 ggcaaaggac gcggcgttca tctgccagcg tttgatgact tctggaataa caaagagtac 1860 gtcgagtttg accatccgca gatgtttgtt cgccaccagg cattccgcga agatccggat 1920 ctcgaaccgc tgggcacgcc gagtggcctg attgagatct actcgaaaac tatcgccgat 1980 atgaactacg acgattgtca ggggcatccg atgtggtttg agaaaatcga acgctcccac 2040 ggtgggcctg gctcgcaaaa gtatccgttg catctgcaat ctgtgcatcc ggatttccga 2100 cttcactcgc agttatgtga gtcggaaacg ctgcgtcacg aatatacggt agcgggtaaa 2160 gagccagtat tcattaaccc gcaggatgcc agcgcgcgcg gtattcgtaa cggtgatgtg 2220 gtacgcgtct ttaacgctcg cggtcaggtg atggcagggg cagtggtttc tgaccgctat 2280 gcacccggcg tggcacgaat tcacgaaggg gcatggtacg atccagataa aggcggcgag 2340 ctgggtgcgc tgtgcaaata cggtaacccc aacgtgttga ccatcgacat cggtacatcg 2400 cagctcgcgc aggcgaccag tgcgcacact acgctggtgg aaattgagaa gtacaacgga 2460 acagtggagc aggtgacggc gtttaacggc cccgtggaga tggtggcgca gtgcgaatat 2520 gttcccgcgt cgcaggtgaa atca 2544 <210> 10 <211> 477 <212> DNA
<213> Artificial sequence <220>
<223> Description of the artificial sequence: sequence partial coding for the protein TorA of Salmonella typhimurium <400> 10 atgaaacagg tggtgtcgcc gcagtttgaa gcgcgtaacg actttgatat tttccgcgat 60 ctctgccgac gctttaaccg tgaagcggca ttcacggaag gtcttgatga aatgggctgg 120 ctgaaacgca tctggcagga agggagccag cagggaaaag gtcgcggtat ccacttaccg 180 attttcgagg tgttctggaa tcaacaggag tacatcgagt ttgatcatcc gcagatgttt 240 gtacgccatc aggctttccg tgaagatccg gacctggagc cgttgggcac gccaagcggt 300 ttgatcgaga tttactccaa aaccatcgcc gacatgcaat acgacgatgg tcagggccat 360 cccatgtggt tcgaaaaaat cgaacgctcg catggcgggc cgggatcgca gcgctggccg 420 ctgcacttac aatccgtcca ccctgatttc cgtctgcatt cccaactgtt gcgagtc 477 <210> 11 <211> 390 <212> PRT
<213> Escherichia coli <400> 11 Met Arg Lys Leu Trp Asn Ala Leu Arg Arg Pro Ser Ala Arg Trp Ser Val Leu Ala Leu Val Ala Ile Gly Ile Val Ile Gly Ile Ala Leu Ile Val Leu Pro His Val Gly Ile Lys Val Thr Ser Thr Thr Glu Phe Cys Val Ser Cys His Ser Met Gln Pro Val Tyr Glu Glu Tyr Lys Gln Ser Val His Phe Gln Asn Ala Ser Gly Val Arg Ala Glu Cys His Asp Cys His Ile Pro Pro Asp Ile Pro Gly Met Val Lys Arg Lys Leu Glu Ala Ser Asn Asp Ile Tyr Gln Thr Phe Ile Ala His Ser Ile Asp Thr Pro Glu Lys Phe Glu Ala Lys Arg Ala Leu Leu Ala Glu Arg Glu Trp Ala Arg Met Lys Glu Asn Asn Ser Ala Thr Cys Arg Ser Cys His Asn Tyr Asp Ala Met Asp His Ala Lys Gln His Pro Glu Ala Ala Arg Gln Met Lys Val Ala Ala Lys Asp Asn Gln Ser Cys Ile Asp Cys His Lys Gly Ile Ala His Gln Leu Pro Asp Met Ser Ser Gly Phe Arg Lys Gln Phe Asp Asp Val Arg Ala Ser Ala Asn Asp Ser Gly Asp Thr Leu Tyr Ser Ile Asp Ile Lys Pro Ile Tyr Ala Ala Lys Gly Asp Lys Glu Ala Ser Gly Ser Leu Leu Pro Ala Ser Glu Val Lys Val Leu Lys Arg Asp Gly Asp Trp Leu Gln Ile Glu Ile Thr Gly Trp Thr Glu Ser Ala Gly Arg Gln Arg Val Leu Thr Gln Phe Pro Gly Lys Arg Ile Phe Val Ala Ser Ile Arg Gly Asp Val Gln Gln Gln Val Lys Thr Leu Glu Lys Thr Thr Val Ala Asp Thr Asn Thr Glu Trp Ser Lys Leu Gln Ala Thr Ala Trp Met Lys Lys Gly Asp Met Val Asn Asp Ile Lys Pro Ile Trp Ala T ~ r Ala Asp Ser Leu Tyr Asn Gly Thr Cys Asn Gln Cys His Gly Ala Pro Glu Ile Ala His Phe Asp Ala Asn Gly Trp Ile Gly Thr Leu Asn Gly Met Ile Gly Phe Thr Ser Leu Asp Lys Arg Glu Glu Arg Thr Leu Leu Lys Tyr Leu Gln Met Asn Ala Ser Asp Thr Ala Gly Lys Ala His Gly Asp Lys Lys Glu Glu Lys 385,390 <210> 12 <211> 21 <212> DNA
<213> Artificial sequence <220>
<223> Description of the artificial sequence: PRIMER
PCR
<400> 12 cggvgaytac tcbachggtg c 21 <210> 13 <211> 20 <212> DNA
<213> Artificial sequence <220>
<223> Description of the artificial sequence: primer PCR
<400> 13 atygatgcga tyctcgaacc 20 <210> 14 ~ '-<211> 25 <212> DNA
<213> Artificial sequence <220>
<223> Description of the artificial sequence: primer PCR
<400> 14 cgtamwsgtc gakatcgttr cgctc 25 <210> 15 <211> 20 <212> DNA
<213> Artificial sequence <220>
<223> Description of the artificial sequence: primer PCR
<400> 15 gactcacaya wytgygagtg 20 <210> 16 <211> 20 <212> DNA
<213> Artificial sequence <220>
<223> Description of the artificial sequence: primer PCR
<400> 16 tgrccdcgrk cgttaaagac 20 <210> 17 <211> 20 <212> DNA
<213> Artificial sequence <220>
<223> Description of the artificial sequence: primer PCR
<400> 17 ccvggttcga gratcgcatc 20 <210> 18 <211> 16 <212> DNA
<213> Artificial sequence <220>
<223> Description of the artificial sequence: primer PCR
<400> 18 cbgayatcst rctgcc 16 <210> 19 <211> 20 <212> DNA
<213> Artificial sequence <220>
<223> Description of the artificial sequence: primer PCR
<400> 19 ggmgaytayt cbacmggygc 20 <210> 20 <211> 20 <212> DNA
<213> Artificial sequence <220>
<223> Description of the artificial sequence: primer PCR
<400> 20 twygarcgya acgaymtcga 20 <210> 21 <211> 20 <212> DNA
<213> Artificial sequence <220>
<223> Description of the artificial sequence: primer PCR
<400> 21 ggvycrtacc abscvccttc 20 <210> 22 <211> 20 <212> DNA
<213> Artificial sequence <220>
<223> Description of the artificial sequence: primer PCR
<400> 22 atcarrccns wvggcgtgcc 20 <210> 23 <211> 17 ' <212> DNA
<213> Artificial sequence <220>
<223> Description of the artificial sequence: primer PCR
<400> 23 gbcacrtcdg tytgygg 17 <210> 24 <211> 20 <212> DNA
<213> Artificial sequence <220>
<223> Description of the artificial sequence: primer PCR
<400> 24 acnccngara arttygargc 20 <210> 25 <211> 20 <212> DNA
<213> Artificial sequence <220>
<223> Description of the artificial sequence: primer PCR
<400> 25 tgyathgayt gycayaargg 20 <210> 26 <211> 20 <212> DNA
<213> Artificial sequence <220>
<223> Description of the artificial sequence: primer PCR
<400> 26 ccyttrtgrc artcdatrca 20 <210> 27 <211> 17 <212> DNA
<213> Artificial sequence <220>
<223> Description of the artificial sequence: primer PCR
<400> 27 ttngcrtcra artgngc 17

Claims (15)

1. Use of nucleotide sequences chosen from those comprising a sequence encoding a protein of the trimethylamine N-oxide reductase system (TMAO
reductase) in bacteria, or a fragment of this sequence such as a probe or a primer of about 15 to about 25 nucleotides, or a sequence derived by addition, deletion, and/or substitution of one or more nucleotides of this sequence coding for a protein of TMAO reductase system or a fragment thereof, said fragment and said sequence derivative being capable of hybridizing with said sequence encoding a system protein TMAO reductase, for implementing a method for detecting the presence of all bacteria involved in the process of degradation of animal flesh aquatic, in a host capable of carrying such bacteria. 2. Use according to claim 1, characterized in that the sequence coding for a protein of the TMAO reductase system is chosen from sequences coding for TMAO
reductase in bacteria, also referred to as the TorA or DorA protein, or encoding for a type c cytochrome, also called TorC or DorC protein. 3. Use according to claim 1 or claim 2, characterized in what the nucleotide sequences coding for a protein of the TMAO reductase system are chosen among those of the following bacteria:
- marine bacteria, such as those of the genus Shewanella, Photobacterium or Vibrio, said sequences being chosen in particular from the following:
* the sequence coding for the TorA protein of Shewanella massilia represented in figure 1, * the sequence coding for the TorA protein of Shewanella putrefaciens represented in figure 1, * the sequence coding for the TorA protein of Shewanella c represented on the figure 2, * the partial sequence coding for the TorA protein of Photobacterium phosphoreum shown in Figure 3, * the sequence coding for the TorC protein of Shewanella massilia represented in figure 14, - bacteria from brackish water, such as those of the genus Rhodobacter, or Roseobacter, said sequences being chosen in particular from the following:
* the coding sequence for the DorA protein of Rhodobacter sphaeroides shown in Figure 4, * the coding sequence for the DorA protein of Rhodobacter capsulatus shown in Figure 4, * the coding sequence for the DorC protein of Rhodobacter sphaeroides represented in figure 14, - Enterobacteriaceae, such as those of the genus Escherichia, or Salmonella, said sequences being chosen in particular from the following:
* the sequence coding for the protein TorA of Escherichia coli represented in Figure 4, * the partial sequence coding for the Salmonella TorA protein typhimurium shown in Figure 5, * the sequence coding for the protein TorC of Escherichia coli represented in Figure 14. 4. Use according to any one of claims 1 to 3, characterized in that the nucleotide sequences are used as primer pairs chosen from one any of the following three primer groups:
(1) the "DDN" primer group comprising:
~ the following “DDN+” nucleotide sequence compositions:
- DDN1+: 5' CGG vGA yTA CTC bAC hGG TGC 3': mixture of 54 sequences nucleotides, - DDN5+: 5' ATy GAT GCG ATy CTC GAA CC3': mixture of 4 sequences nucleotides, ~ the following "DDN-" nucleotide sequence compositions:
- DDN2-: 5'CGT Amw sGT CGA kAT CGT TrC GCT C3': mixture of 32 sequences nucleotides, - DDN3-: 5' GAC TCA CAy Awy TGy GAG TG 3': mixture of 16 sequences nucleotides, - DDN4-: 5' TGr CCd CGr kCG TTA AAG AC 3': mixture of 24 sequences nucleotides, - DDN5-: 5' CCv GGT TCG AGr ATC GCA TC 3': mixture of 6 sequences nucleotides, (2) the "BN" primer group comprising:
~ the following “BN+” nucleotide sequence compositions:
- BN1+: 5' C bGA yAT CsT rCT GCC 3': mixture of 16 nucleotide sequences, - BN3+: 5' GGm GAy TAy TCb ACm GGy GC 3': mixture of 96 sequences nucleotides, - BN6+: 5' Twy GAr CGy AAC GAy mTC GA 3': mixture of 64 sequences nucleotides, ~ the following "BN-" nucleotide sequence compositions:
- BN2-: 5' GG vyC rTA CCA bsC vCC TTC 3': mixture of 216 sequences nucleotides, - BN4-: 5' ATC Arr CCn swv GGC GTG CC 3': mix of 192 sequences nucleotides, -BN5-: 5'GbC ACr TCd GTy TGy GG 3': mixture of 72 nucleotide sequences, (3) the "BC" primer group comprising:
~ the following “BC+” nucleotide sequence compositions:
- BC1+: 5' ACn CCn GAr AAr TTy GAr GC 3': mixture of 256 sequences nucleotides, - BC2+: 5' TGy ATh GAy TGy CAy AAr GG 3': mixture of 96 sequences nucleotides, ~ the following "BC-" nucleotide sequence compositions:
- BC2-: 5' CCy TTr TGr CAr TCd ATr CA 3': mixture of 96 sequences nucleotides, - BC3-: 5' TTn GCr TCr AAr TGn GC 3': mixture of 128 sequences nucleotides, where n = (A,C,G,T), y = (C,T), r = (A,G), h = (A,C,T), d = (G,A,T), m = (A, C), w = (A,T), b = (G,T,C), s = (G,C), v = (G,A,C), and k = (G, T), the pairs of primers being chosen such that one of the primers of a pair corresponds to one of the compositions of nucleotide sequences DDN+, BN+ or BC+
mentioned above, while the other primer corresponds respectively to one compositions of nucleotide sequences DDN-, BN- or BC- mentioned above, said pairs primers being chosen in particular from any one of the following four pairs:
(a) the DDN1+ / DDN5- couple, leading to the amplification of fragments of the gene coding for the TorA protein in bacteria, with a size of about 820 pairs of bases (bp), and in particular to the amplification of an 821 bp fragment of the gene coding for the TorA protein in bacteria of the genus Shewanella, such as the 821 bp fragment demarcated by the nucleotides located at positions 620 to 1450 of the torA gene of S. massilia shown in the figure 4, (b) the BN6+ / BN2- couple, leading to the amplification of fragments of the gene coding for the TorA protein in bacteria, which is about 710 bp in size, and in particular to amplification of a 727 bp fragment of the gene encoding the TorA protein in bacteria of the genus Shewanella, such as the 727 bp fragment delimited by the nucleotides located at positions 1657 to 2403 of the torA gene of S. massilia shown in Figure 4, (c) the BN6+ /BN4- couple, leading to the amplification of fragments of the gene coding for the TorA protein in bacteria, which is about 360 bp in size, and in particular to amplification of a 355 bp fragment of the gene encoding the TorA protein in bacteria of the genus Shewanella, such as the 355 bp fragment delimited by the nucleotides located at positions 1657 to 2023 of the torA gene of S. massilia shown in Figure 4, (d) the BC1+ /BC2- couple, leading to the amplification of fragments of the gene coding for the TorC protein in bacteria, which is about 170 bp in size, and in particular to amplification of a 197 bp fragment of the gene encoding the TorC protein in bacteria of the genus Shewanella, such as the 197 bp fragment encoding the fragment polypeptide delimited by the amino acids located at positions 114 to 179 of the protein TorC of S. massilia shown in Figure 14. 5. Use according to one of claims 1 to 4, characterized in that the hosts likely to carry bacteria involved in the process of degradation of the flesh of aquatic animals, as described in claim 3, are organisms aquatic organisms, including marine organisms such as fish and shellfish, and more particularly Atlantic fish such as sole, cod, or the fish of the Mediterranean Sea such as rock mullet, sea bream, as well as some water animals fresh or brackish. 6. Use according to any one of claims 1 to 5, for setting implemented a method for detecting the presence of all bacteria involved in degradation flesh of aquatic animals, as part of a process for evaluating the the state of freshness aquatic animals from which the test sample is taken, where these last are extracted from their natural environment. 7. Nucleotide sequence corresponding to one of the following sequences - DDN1 +: 5' CGG vGA yTA CTC bAC hGG TGC 3', - DDN5 +: 5' ATy GAT GCG ATy CTC GAA CC 3', - DDN2 -: 5'CGT Amw sGT CGA kAT CGT TrC GCT C 3', - DDN3 -: 5' GAC TCA CAy Awy TGy GAG TG 3', - DDN4 -: 5' TGr CCd CGr kCG TTA AAG AC 3', - DDN5 -: 5' CCv GGT TCG AGr ATC GCA TC 3', - BN1+: 5' C bGA yAT CsT rCT GCC 3', - BN3+: 5' GGm GAy TAy TCb ACm GGy GC 3', - BN6+: 5' Twy GAr CGy AAC GAy mTC GA 3', - BN2-: 5' GG vyC rTA CCA bsC vCC TTC 3', - BN4-: 5' ATC Arr CCn swv GGC GTG CC 3', - BN5-: 5'GbC ACr TCd GTy TGy GG 3', - BC1+: 5' ACn CCn GAR AAr TTy GAR GC 3', - BC2+: 5' TGy ATh GAy TGy CAy AAr GG 3', - BC2-: 5' CCy TTr TGr CAr TCd ATr CA 3 ', - BC3-: 5' TTn GCr TCr AAr TGn GC 3', where n = (A,C,G,T), y = (C,T), r = (A,G), h = (A,C,T), d = (G,A,T), m = (A, C), w = (A,T), b = (G,T,C), s = (G,C), v = (G,A,C), and k = (G,T). 8. Composition of mixed nucleotide sequences, corresponding to one of the following compositions .cndot. the following “DDN+” nucleotide sequence compositions - DDN1+: 5' CGG vGA yTA CTC bAC hGG TGC 3': mixture of 54 sequences nucleotides, - DDN5+: 5' ATy GAT GCG ATy CTC GAA CC 3': mixture of 4 sequences nucleotides, ~ the following "DDN-" nucleotide sequence compositions:
- DDN2-: 5' CGT Amw sGT CGA kAT CGT TrC GCT C 3': mixture of 32 sequences nucleotides, - DDN3-: 5' GAC TCA CAy Awy TGy GAG TG 3': mixture of 16 sequences nucleotides, - DDN4-: 5' TGr CCd CGr kCG TTA AAG AC 3': mixture of 24 sequences nucleotides, - DDN5-: 5' CCv GGT TCG AGr ATC GCA TC 3': mixture of 6 sequences nucleotides, ~ the following “BN+” nucleotide sequence compositions:
- BN1+: 5' C bGA yAT CsT rCT GCC 3': mixture of 16 nucleotide sequences, - BN3+: 5' GGm GAy TAy TCb ACm GGy GC 3': mixture of 96 sequences nucleotides, - BN6+: 5' Twy GAr CGy AAC GAy mTC GA 3': mixture of 64 sequences nucleotides, ~ the following "BN-" nucleotide sequence compositions:
- BN2-: 5' GG vyC rTA CCA bsC vCC TTC 3': mixture of 216 sequences nucleotides, - BN4-: 5' ATC Arr CCn swv GGC GTG CC 3': mix of 192 sequences nucleotides, - BN5-: 5'GbC ACr TCd GTy TGy GG 3': mixture of 72 nucleotide sequences, ~ the following “BC+” nucleotide sequence compositions:
- BC1+: 5' ACn CCn GAr AAr TTy GAr GC 3': mixture of 256 sequences nucleotides, - BC2+: 5' TGy ATh GAy TGy CAy AAr GG 3': mixture of 96 sequences nucleotides, ~ the following "BC-" nucleotide sequence compositions:
- BC2-: 5' CCy TTr TGr CAr TCd ATr CA 3': mixture of 96 sequences nucleotides, - BC3-: 5' TTn GCr TCr AAr TGn GC 3': mixture of 128 sequences nucleotides, where n=(A,C,G,T), y=(C,T), r=(A,G), h=(A,C,T), d=(G,A,T), m=(A,C), w=(A,T), b=(G,T,C), s=(G,C), v=(G,A,C), and k=(G,T). 9. Pair of primers characterized in that it is chosen from one of the groups of the following primers:
(1) the "DDN" primer group comprising:
~ the following “DDN+” nucleotide sequence compositions:
- DDN1+: 5'CGG vGA yTA CTC bAC hGG TGC 3': mixture of 54 sequences nucleotides, - DDN5+: 5' ATy GAT GCG ATy CTC GAA CC 3': mixture of 4 sequences nucleotides, ~ the following "DDN-" nucleotide sequence compositions:
- DDN2-: 5'CGT Amw sGT CGA kAT CGT TrC GCT C 3': mixture of 32 sequences nucleotides, - DDN3-: 5'GAC TCA CAy Awy TGy GAG TG 3': mixture of 16 sequences nucleotides, - DDN4-: 5'TGr CCd CGr kCG TTA AAG AC 3': mixture of 24 sequences nucleotides, - DDN5-: 5'CCv GGT TCG AGr ATC GCA TC 3': mixture of 6 sequences nucleotides, (2) the "BN" primer group comprising:
~ the following “BN+” nucleotide sequence compositions:
-BN1+: 5'C bGA yAT CsT rCT GCC 3': mixture of 16 nucleotide sequences, -BN3+: 5'GGm GAy TAy TCb ACm GGy GC3': mixture of 96 sequences nucleotides, - BN6+: 5'Twy GAr CGy AAC GAy mTC GA 3': mix of 64 sequences nucleotides, ~ the following "BN-" nucleotide sequence compositions:
-BN2-: 5'GG vyC rTA CCA bsC vCC TTC 3': mix of 216 sequences nucleotides, -BN4-: 5'ATC Arr CCn swv GGC GTG CC 3': mix of 192 sequences nucleotides, -BN5-: 5'GbC ACr TCd GTy TGy GG3': mixture of 72 nucleotide sequences, (3) the "BC" primer group comprising:
~ the following “BC+” nucleotide sequence compositions:
- BC1+: 5'ACn CCn GAR AAr TTy GAR GC3': mixture of 256 sequences nucleotides, - BC2+: 5'TGy ATh GAy TGy CAy AAr GG3': mixture of 96 sequences nucleotides, ~ the following "BC-" nucleotide sequence compositions:
-BC2-: 5'CCy TTr TGr CAr TCd ATr CA 3': mixture of 96 sequences nucleotides, -BC3-: 5'TTn GCr TCr AAr TGn GC3': mixture of 128 nucleotide sequences, where n = (A,C,G,T), y = (C,T), r = (A,G), h = (A,C,T), d = (G,A,T), m = (A, C), w = (A,T), b = (G,T,C), s = (G,C), v = (G,A,C), and k = (G,T), the pairs of primers being chosen such that one of the primers of a pair corresponds to one of the compositions of nucleotide sequences DDN+, BN+ or BC+
mentioned above, while the other primer corresponds respectively to one compositions of nucleotide sequences DDN-, BN- or BC- mentioned above, said pairs primers being chosen in particular from any one of the following four pairs:
(a) the DDN1+ / DDN5- couple, leading to the amplification of fragments of the gene coding for the TorA protein in bacteria, with a size of about 820 pairs of bases (bp), and in particular to the amplification of an 821 bp fragment of the gene coding for the TorA protein in bacteria of the genus Shewanella, such as the 821 bp fragment demarcated by the nucleotides located at positions 620 to 1450 of the torA gene of S. massilia shown in the figure 4, (b) the BN6+ / BN2- couple, leading to the amplification of fragments of the gene coding for the TorA protein in bacteria, which is about 710 bp in size, and in particular to amplification of a 727 bp fragment of the gene encoding the TorA protein in bacteria of the genus Shewanella, such as the 727 bp fragment delimited by the nucleotides located at positions 1657 to 2403 of the torA gene of S. massilia shown in Figure 4, (c) the BN6+ /BN4- couple, leading to the amplification of fragments of the gene coding for the TorA protein in bacteria, which is about 360 bp in size, and in particular to amplification of a 355 bp fragment of the gene encoding the TorA protein in bacteria of the genus Shewanella, such as the 355 bp fragment delimited by the nucleotides located at positions 1657 to 2023 of the torA gene of S. massilia shown in Figure 4, (d) the BC1+ /BC2- couple, leading to the amplification of fragments of the gene coding for the TorC protein in bacteria, which is about 170 bp in size, and in particular to amplification of a 197 bp fragment of the gene encoding the TorC protein in bacteria of the genus Shewanella, such as the 197 bp fragment encoding the fragment polypeptide delimited by the amino acids located at positions 114 to 179 of the protein TorC of S. massilia shown in Figure 14. 10. Method for detecting all bacteria involved in degradation flesh of aquatic animals in a host likely to be a carrier of such bacteria, said method being carried out from a biological sample taken from this host, this sample biological corresponding in particular to a subcutaneous fragment of flesh of aquatic animal in question, and being characterized in that it comprises a step hybridization of at least a nucleotide sequence as defined in any one of claims 1 to 9, with fragments of the genes coding for a protein of the TMAO-reductase of bacteria involved in the degradation of aquatic animal flesh likely to be present in the biological sample taken from said host, followed by a revelation stage, in particular by electrophoresis, of the possible presence in said sample of genes encoding for a protein of the TMAO-reductase system, or fragments of these genes, whose number of copies has been amplified if necessary. 11. Detection method according to claim 10, characterized in that it understand the following steps:
- the processing of a biological sample taken from this host in order to to extract total DNA
of this host and to make the genome of these bacteria accessible to the sequences nucleotides or primers defined according to any one of claims 1 to 9, this treatment being in particular carried out using a rapid DNA extraction technique based on the binding from nucleic acids to silica beads, - the amplification of the number of copies of genes coding for the proteins of the system TMAO-reductase of bacteria involved in the degradation of flesh aquatic animals, or fragments of these genes, likely to be present in this sample, using aforementioned nucleotide sequences or primers hybridizing with the genes or fragments of aforementioned genes, - the detection of the possible presence of an amplified number of copies of coding genes for a protein of the TMAO-reductase system of the aforementioned bacteria, or fragments of these genes, and therefore of the presence of such bacteria in the sample biology studied. 12. Detection method according to claim 11, characterized in that amplification of the number of genes encoding TMAO-reductase includes the steps following:
- the predenaturation of the host's total double-stranded DNA into single-stranded DNA, from preferably in a buffer consisting of 10mM Tris-HCl pH 8.3, 50mM KCl, 1.5mM
MgCl2, 0.01% gelatin, of the 4 constituent deoxynucleotides of DNA (dCTP, dATP, dGTP, dTTP) at a concentration of 100 µM each, and primer pairs, such as defined in the claims 1 to 9, by heating between about 90°C and about 100 ° C, advantageously at 94°C, for about 1.5 minutes, - the actual amplification by addition to the medium obtained in step former DNA polymerase, for example Taq polymerase, ~ heating to about 94°C for about 30 seconds, which correspond to the actual denaturation step, ~ then heating between about 35°C to about 60°C, and especially at around 45°C
or 55°C, for about 30 seconds, which corresponds to step primer annealing with the genes encoding the proteins of the TMAO-reductase system of bacteria, or fragments of these genes, likely to be present in the sample biological studied, ~ and finally, heating to 72°C, for about 45 seconds, which correspond to the step of elongation of the primers, hybridized in the previous step, one towards the other, producing thus nucleotide sequences complementary to gene fragments coding for proteins of the TMAO-reductase system of bacteria, the latter sequences being demarcated by the nucleotides hybridizing with the aforementioned primers, - the repetition of the previous amplification step between about 15 and about 35 times, advantageously about 30 times. 13. Kit for the implementation of a detection method according to one of the claims to 12, characterized in that it comprises:
- one or more nucleotide sequences or primers defined in one of the 47~

claims 1 to 9, - a DNA polymerase, - a reaction medium advantageously consisting of 10 mM Tris-HCl pH 8.3, 50mM
KCl, 1.5mM MgCl2, 0.01% gelatin, of the 4 constituent deoxynucleotides of DNA (dCTP, dATP, dGTP, dTTP) at a concentration of 100 µM each. 14. Nucleotide sequence comprising:
- the sequence shown in Figure 2 of the torA gene coding for the protein TorA of the marine bacterium Shewanella c, * or any sequence derived from the aforementioned sequence by degeneracy of the coded genetic, and coding for the TorA protein of Shewanella c, whose sequence peptide is represented in figure 6, * or any sequence derived from the aforementioned nucleotide sequence, notably by substitution, deletion or addition of one or more nucleotides, said derived sequence preferably having about 35% to 100% homology with the sequence nucleotide aforementioned shown in Figure 2, * or any fragment of the aforementioned nucleotide sequence, or of a sequence derived from the latter as defined above, said fragment being of preferably constituted of at least about 15 nucleotides, - the partial sequence shown in Figure 3 of the gene coding for the TorA protein from the marine bacterium Photobacterium phosphoreum, * or any sequence derived from the aforementioned sequence by degeneracy of the coded genetic; and coding for the TorA protein of Photobacterium phosphoreum, of which the sequence peptide is shown in Figure 7, * or any sequence derived from the aforementioned nucleotide sequence, notably by substitution, deletion or addition of one or more nucleotides, said derived sequence preferably having about 35% to 100% homology with the sequence nucleotide aforementioned shown in Figure 3, * or any fragment of the aforementioned nucleotide sequence, or of a sequence derived from the latter as defined above, said fragment being of preferably constituted of at least about 15 nucleotides, - the partial sequence shown in Figure 5 of the gene coding for the TorA protein the marine bacterium Salmonella typhimurium, * or any sequence derived from the aforementioned sequence by degeneracy of the coded genetic, and coding for the TorA protein of Salmonella typhimurium whose sequence peptide is shown in Figure 8, * or any sequence derived from the aforementioned nucleotide sequence, notably by substitution, deletion or addition of one or more nucleotides, said derived sequence preferably having about 35% to 100% homology with the sequence nucleotide aforementioned shown in Figure 5, * or any fragment of the aforementioned nucleotide sequence, or of a sequence derived from the latter as defined above, said fragment being of preferably constituted of at least about 15 nucleotides. 15. Peptide sequence encoded by a nucleotide sequence according to the claim 14, and including - the amino acid sequence shown in Figure 6 of the TorA protein of Shewanella c.
* or a. sequence derived from the aforementioned peptide sequence, in particular by substitution, deletion or addition of one or more amino acids, said derived sequence preferably having about 35% to 100% homology with the sequence peptide aforementioned shown in Figure 6, * or a fragment of the aforementioned peptide sequence, or of a sequence derived of the latter as defined above, said fragment being of preferably consisting of at minus about 5 amino acids, - the partial amino acid sequence shown in Figure 7 of the TorA protein from Photobacterium phosphoreum, * or a sequence derived from the aforementioned peptide sequence, in particular by substitution, deletion or addition of one or more amino acids, said derived sequence preferably having about 35% to 100% homology with the sequence peptide aforementioned shown in Figure 7, * or a fragment of the aforementioned peptide sequence, or of a sequence derived of the latter as defined above, said fragment being of preferably consisting of at minus about 5 amino acids, - the partial amino acid sequence shown in Figure 8 of the TorA protein from Salmonella typhimurium, * or a sequence derived from the aforementioned peptide sequence, in particular by substitution, deletion or addition of one or more amino acids, said derived sequence preferably having about 35% to 100% homology with the sequence peptide aforementioned shown in Figure 8, * or a fragment of the aforementioned peptide sequence, or of a sequence derived of the latter as defined above, said fragment being of preferably consisting of at least about 5 amino acids.