US20050112641A1

US20050112641A1 - Polypeptides implicated in the expression of resistance to glycopeptides, in particular in Gram-positive bacteria, nucleotide sequence coding for these polypeptides and use for diagnosis

Info

Publication number: US20050112641A1
Application number: US10/952,915
Authority: US
Inventors: Michel Arthur; Sylvie Dukta-Malen; Catherine Molinas; Patrice Courvalin
Original assignee: Institut Pasteur de Lille
Current assignee: Institut Pasteur de Lille
Priority date: 1990-10-31
Filing date: 2004-09-30
Publication date: 2005-05-26
Also published as: US6916906B1

Abstract

The invention relates to a composition of polypeptides, characterized in that it contains at least one protein or part of a protein selected from the sequences of amino acids identified in the list of the sequences as SEQ ID NO 1 (VanH), SEQ ID NO 2 (VanA), SEQ ID NO 3 (VanX) or SEQ ID NO 19 (VanC), or any protein or part of a protein recognized by the antibodies directed against VanH, VanA, VanX or VanC, or any protein or part of a protein encoded in a sequence hybridizing with one of the nucleotide sequences identified in the list of the sequences as SEQ ID NO 8, SEQ ID NO 9 or SEQ ID NO 10 or with one of the following sequences V1 or V2 under stringent or only slightly stringent conditions: V1: GGX GAA GAT GGX TCX TTX CAA GGX G C AG C G A V2: AAT ACX ATX CCX GGX TTT AC C T C C

The invention also relates to the nucleotide sequences coding for these polypeptides as well as their utilization for the diagnosis of resistance to the glycopeptides.

Description

The invention relates to the polypeptides associated with the expression of resistance to antibiotics of the glycopeptide family, in particular in Gram-positive bacteria, in particular in the family of the Gram-positive cocci. The invention also relates to a nucleotide sequence coding for these polypeptides. It also relates to the use of these polypeptides and their nucleotide sequence as agents for the in vitro detection of resistance to glycopeptides. Among the Gram-positive cocci, the invention relates most particularly to the enterococci, the streptococci and the staphylococci which are of particular importance for the implementation of the invention.
The glycopeptides, which include vancomycin and teicoplanin are antibiotics which inhibit the synthesis of the bacterial cell wall. These antibiotics are very much used for the treatment of severe infections due to Gram-positive cocci (enterococci, streptococci and staphylococci), in particular in the of allergy and resistance to the penicillins. In spite of long clinical usage of vancomycin, this antibiotic has remained active towards almost all of the strains up to 1986, the date at which the first resistant strains were isolated Since then, resistance to the glycopeptides has been detected by many microbiologists in Europe and in the United States, in particular in strains isolated from immunodepressive patients, making necessary a systematic evaluation of the sensitivity of the microbes in hospital environments.
The activity of the glycopeptides depends on the formation of a complex between the antibiotic and the precursors of the peptidoglycan, more than on the direct interaction with enzymes of cell wall metabolism. In particular, it has been observed that the glycopeptides bind to the terminal D-alanyl-D-alanine residues (D-ala-D-ala) of the precursors of the peptidoglycan.
The recent emergence of resistance to the glycopeptides, in particular in the enterococci, has led to certain results being obtained with regard to knowledge of the factors conferring this resistance.
For example it has been observed in a particular strain of enterococci, Enterococcus faecium BM4147, that the determinant of resistance to the glycopeptides is localized on a plasmid of 34 kb, the plasmid pIP816. This determinant has been cloned in E. coli (Brisson Noel et al., 1990, Antimicrob Agents Chemother 34, 924-927).
According to the results hitherto obtained, the resistance to the glycopeptides is associated with the production of a protein of molecular weight of about 40 kDa, the synthesis of this protein being induced by sub-inhibitory concentrations of certain glycopeptides such as vancomycin.
By carrying out a more detailed study of the resistance of certain strains of Gram-positive cocci towards glycopeptides, in particular vancomycin or teicoplanin, the inventors have observed that this resistance might be linked to the expression of several proteins or polypeptides encoded in sequences usually borne by plasmids in the resistant strains. The recent results obtained by the inventors also make it possible to distinguish the genes coding for two phenotypes of resistance, on the one hand strains highly resistant to the glycopeptides, and, on the other, strains with a low level of resistance.
By strain with a high level of resistance is meant a strain of bacteria, in particular a strain of Gram-positive cocci, for which the minimal inhibitory concentrations (MIC) of vancomycin and teichoplaninare higher than 32 and 8 μg/ml, respectively. The MIC of vancomycin towards strains with low-level resistance are included between 16 and 32 μg/ml. These strains are apparently sensitive to teicoplanin.
The inventors have isolated and purified, among the components necessary for the expression of the resistance to the glycopeptides, a particular protein designated VANA or VanA which exhibits a certain homology with D-alanine-D-alanine ligases. VanA is nonetheless functionally distinct from the ligases.
In principle, a gene sequence will be designated by “van . . . and an amino acid sequence by “Van . . . .”
The invention relates to polypeptides or proteins implicated in the expression of resistance to antibiotics of the glycopeptide family and, in particular, to vancomycin and/or teicoplanin as well as to the nucleotide sequences coding for such complexes.
The invention also relates to nucleotide probes which can be used for the detection of resistance to the glycopeptides, in particular by means of the polymerase chain reaction (PCR), or by tests involving antibodies.
The invention relates to a composition of polypeptides, characterized in that it contains at least one protein or part of a protein selected from the amino acid sequences identified in the list of the sequences as SED ID NO 1 (VanH), SEQ ID NO 2 (VanA), SEQ. ID NO 3 (VanX) or SEQ ID NO 19 (VanC), or any protein or part of a protein recognized by the antibodies directed against VanH, VanA, VanX or VanC, or any protein or part of a protein encoded in a sequence hybridizing with one of the nucleotide sequences identified in the list of the sequences as SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10 or SEQ ID NO 21 or with one of the following sequences V1 or V2 under stringent or only slightly stringent conditions:

V1: GGX GAA GAT GGX TCX TTX CAA GGX

G C AG C G

A

V2: AAT ACX ATX CCX GGX TTT AC

C T C

C
A first particular composition according to the invention implicated in the expression of the resistance to the glycopeptides is characterized in that it comprises at least 3 proteins or any part of one or more of these proteins necessary to confer to Gram-positive bacteria the resistance to antibiotics of the glycopeptide family, in particular to vancomycin and/or teicoplanin or to promote this resistance, in particular in strains of the family of the Gram-positive cocci, these proteins or parts of proteins being

a) recognized by antibodies directed against one of the sequences identified in the list of the sequences as SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 31
b) or encoded in genes containing a sequence identified as SEQ ID NO 8, SEQ ID NO 9 or SEQ ID NO 10 or hybridizing with one of these sequences or its complementary sequence or with the sequences V1 or V2, under stringent or only slightly stringent conditions.

These sequences are also designated, respectively, by ORF3, ORF1 containing the gene VanH, vanA (or ORF2); they characterize the proteins responsible for resistance as obtained from the strain Enterococcus faecium BM4147 described by Leclerq et al (N. Engl. J. Med. 319:157-161).
Another protein, VanC, related to the D-Ala-D-Ala ligases but of different specificity has been characterized in Enterococcus gallinarum BM4173; the vanC gene possesses domains having sufficient homology with the vanA gene for probes corresponding to defined regions of vanA to make possible its detection.
E. gallinarum is a constitutive isolate resistant to low level of vancomycin (Dutka-Malen et al., Antimicrob. Agents Chemother 34 (1990b) 1875-1879).
By the expression “polypeptides” is meant any sequence of amino acids constituting proteins or being of a size less than that of a protein.
The stringent conditions mentioned above are defined according to the usual conditions pertaining to the hybridization of nucleotide sequences. As an example, in the case of the sequences which hybridize with the sequence of the vanA gene (SEQ ID NO 8) it will be possible to apply the following conditions:

- for hybridization under conditions of high stringency:
  - a reaction temperature of 65° C. overnight in a solution containing 0.1% SDS, 0.7% skimmed milk powder, 6×SSC (1×SSC 0.15 M NaCl and 0.015 M sodium citrate at pH=7.0)
  - washes at 65° C. in 2×SSC-0.1% SDS;
- for hybridization under slightly stringent conditions, the hybridization temperature is 60° C. overnight and the temperature of the washings is 45° C.

The expression of resistance to glycopeptides may be expresses by the persistence of an infection due to microbes usually sensitive to the glycopeptides.
A polypeptide or a protein is necessary for the expression of resistance to the glycopeptides, if its absence makes the strain which contains this polypeptide or this protein more sensitive to the glycopeptides and if this polypeptide or protein is not present in sensitive strains.
Different levels of resistance to the glycopeptides exist in the strains of Gram-positive cocci, in particular.
According to a preferred embodiment of the invention, the polypeptides included in the composition defined above correspond to the combination of the proteins identified in the list of the sequences as SEQ ID NO 1 (VanH), SEQ ID NO 2 (VanA), SEQ ID NO 3 (VanX).
The inventors have thus observed that the expression of resistance to the glycopeptides in Gram-positive bacteria requires the expression of at least three proteins or of polypeptides derived from these proteins.
According to a first particular embodiment of the invention, the polypeptides of the composition are also characterized in that the amino acid sequences necessary for the expression of resistance to antibiotics of the glycopeptide family are under the control of regulatory elements, in particular of the proteins corresponding to the sequences designated by SEQ ID NO 4 and SEQ ID NO 5 in the list of the sequences, and which correspond to a regulatory sequence R and to a sensor sequence S, respectively.
VanS and VanR constitute a two-component regulatory system, VanR being an activator of transcription and VanS stimulating the transcription dependent on VanR. VanS is capable of modulating the level of phosphorylation of VanR in response to the vancomycin present in the external medium and is thus involved in the control of the transcription of the genes for resistance to vancomycin.
These regulatory sequences are in particular capable of increasing the level of resistance, to the extent to which they promote the expression of the proteins responsible for resistance comprised in the polypeptides of the invention.
According to another advantageous embodiment of the invention the polypeptides of the above composition are encoded in the sequence SEQ ID NO 6 identified in the list of the sequences, which represents the sequence coding for the 5 proteins previously described.
Another sequence according to the invention is designated by SEQ ID NO 11 which contains the sequence SEQ ID NO 6 as well as a sequence upstream from SEQ ID NO 6 coding for a transposase (encoded in the (−) strand of the sequence, and a sequence downstream from SEQ ID NO 6 corresponding to the genes vanY and vanZ and at each end reverse repeated sequences of 38 bp. SEQ ID NO 11 constitutes a transpose, the genes of which are implicated at different levels in the establishment of resistance to the glycopeptides.
The invention also relates to the purified proteins belonging to the composition and to the polypeptides described previously. In particular, the invention relates to the purified protein VanA, characterized in that it corresponds to the amino acid sequence SEQ ID NO in the list of the sequences or a protein VanC, encoded in a gene capable of hybridizing with the vanA gene.
The protein VanA contains 343 amino acids and has a calculate molecular mass of 37400 Da. The protein VanC contains 343 amino acids and has a calculated molecular mass of 37504 Da.
Other interesting proteins in the framework of the invention correspond to the sequences identified as SEQ ID NO 1 (VanH), SEQ ID NO 3 (VanX), SEQ ID NO 4 (VanR), SEQ ID NO 5 (VanS) in the list of the sequences.
The sequence identified by the abbreviation SEQ ID NO 1 contains the protein VanH encoded in the gene vanH, this protein contains 322 amino acids and begins with a methionine. This protein is an enzyme implicated in the synthesis of the peptidoglycan and has a molecular mass of 35,754 kDA. VanH exhibits some similarities to dehydrogenases which catalyze the NAD⁺-dependent oxidation of 2-hydroxy carboxylic acids to form the corresponding 2-keto-carboxylic acids. In fact, the VanH protein might use NADP⁺rather than NAD⁺. The VanH protein also contains several residues of reactive sites which probably participate directly in the binding of the substrate and in catalysis. VanH might be implicated in the synthesis of a substrate of the ligase VanA. This substrate of VanA might be a D-α-hydroxy-carboxylic acid, which might be condensed by VanA with D-alanine in the place of a D-amino acid, which might affect the binding of the precursor of the peptidoglycan with vancomycin, as a result of the loss of a hydrogen bond because one of the hydrogen bonds formed between vancomycin and N-acetyl-D-Ala-D-Ala occurs with the NH group of the terminal D-alanine residue. Let it be recalled that “Ala” is the abbreviation for “alanine”.
The inventors have been able to detect some interactions between the proteins VanA and VanH and have in particular been able to describe the following: the nature of the VanA protein (D-alanine: D-alanine ligase with reduced specificity for its substrate) which has made possible resistance to glycopeptides, implies the biosynthesis by VanA of a novel compound different from D-Ala-D-Ala, a peptide which may be incorporated into the peptidoglycans but which is not recognized by vancomycin. In particular, the observation of similarities between the product of the vanH gene and the D-specific α-keto-acid reductases has made it possible to determine that this compound cannot be a D-amino acid but is a D hydroxy acid which when it is bound to D-alanine by VanH, can generate the novel depsipeptide precursor of the peptidoglycan.
The invention also relates to any combination of these different proteins in a resistance complex, as well as to hybrid proteins comprising one or several of the above proteins, or part of these proteins, in combination with a defined amino acid sequence.
Also included in the framework of the invention are nucleotide sequences coding for one of the amino acid sequences described above.
A particular sequence is the nucleotide sequence of about 7.3 kb, corresponding to the HindIII-EcoRI restriction fragment, such as that obtained starting from the plasmid pIP816 described in the publication of Leclerq et al —1988, cited above.
This sequence of 7.3 kb comprises the nucleotide sequence coding for the 3 resistance proteins and the 2 regulatory proteins referred to above. This coding sequence is included in an internal BglII-XbaI fragment. It also comprises a part of the sequences coding for the transposase and the resolvase.
The invention also relates to any nucleotide fragment comprising the above-mentioned restriction fragment as well as any part of the HindIII-EcoRI fragment, in particular the EcoRI-XbaI fragment of about 3.4 kb coding for the 3 resistance proteins or the EcoRV-SacII fragment of about 1.7 kb coding for VanA or also HindIII-EcoRI fragment of about 3.3 kb coding for the 2 regulatory proteins VanR and VanS.
Another definition of a nucleotide sequence of the invention corresponds to a nucleotide fragment containing the following restriction sites in the following order, such as obtained starting from pIP816 mentioned above:

HindIII, BglII, BglII, EcoRI, BamHI, XbaI, EcoRI.

Another nucleotide sequence according to the invention is characterized in that it corresponds to a sequence selected from the sequences identified as SEQ ID NO 7, SEQ ID NO 6, SEQ ID NO 11 or SEQ 20′ ID NO 22, or in that it includes this sequence or any part of this sequence, or also any sequence or part of the sequence of the complementary DNA or any sequence of RNA corresponding to one of these DNAs, capable,

- either of constituting a hybridization probe for the detection of resistance to antibiotics of the glycopeptide family, in particular to vancomycin and/or teicoplanin in particular in strains of the family of the Gram-positive cocci,
- or of coding for a sequence necessary or associated with the expression of resistance to antibiotics of the glycopeptide family, in particular to vancomycin and/or teicoplanin., in particular in strain of the family of the Cram-positive cocci.

The sequence SEQ ID NO 7 codes for the 3 resistance proteins VanH, VanA and VanX.
The sequence SEQ ID NO 22 and the sequence SEQ ID NO 11 include a transposon shown in FIG. 7 a; this transposon contains the genes necessary for the expression of resistance to the glycopeptides as well as the genes associated with this resistance implicated, for example, in the regulation of the expression of the genes necessary to produce the resistance phenotype or implicated in the amount of resistance polypeptide produced.
A specific sequence corresponding to the above definition is one of the following sequences:

V1: GGX GAA GAT GGX TCX TTX CAA GGX

G C AG C G

or A

V2: AAT ACX ATX CCX GGX TTT AC

C T T

C
V1 and V2 make possible the constitution of probes, if necessary, in combination with other nucleotides, depending on the degree of specificity desired in order to detect vanA and vanC and may also be used as primers in polymerase chain reactions.
Other preferred nucleotide sequences are the sequences SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10, SEQ ID NO 21, SEQ ID NO 12 (transposase), SEQ ID NO 13 (resolvase), SEQ ID NO 14 (vanY), SEQ ID NO 15 (vanZ), SEQ ID NO 23 (vanR), SEQ ID NO 24 (vanS) or a variant of one of these sequences provided that it codes for a protein having immunological and/or functional properties similar to those of the proteins encoded in the sequences SEQ ID NO 8 (vanA), SEQ ID NO 9 (vanH), SEQ ID NO 10 (vanX), or SEQ ID NO 21 (vanC), SEQ ID NO 12 (transposase), SEQ ID NO 13 (resolvase), SEQ ID NO 14 (vanY), SEQ ID NO 15 (vanZ), SEQ ID NO 23 (vanR), SEQ ID NO 24 (vanS) or in that it makes possible the detection of strains resistant to antibiotics of the glycopeptide family.
Variants include all of the fragments of the sequences having the following properties.
These sequences code for the resistance proteins VanH, VanA and VanX.
The nucleotide sequence designated by SEQ ID NO 8 corresponds to a DNA fragment of 1029 bp situated between the ATG codon at position 377 and the TGA codon at position 1406 on the plasmid pAT214<FIG. 6).
The invention also relates to a nucleotide sequence coding for the sequence SEQ ID NO 6 corresponding to the sequence coding for the 5 proteins (2 regulatory proteins and 3 resistance proteins), and also comprising the flanking sequences associated with these coding sequences, or comprising this sequence.
Also included in the framework of the invention is a sequence modified with respect to SEQ ID NO 6, characterized in that it lacks the flanking sequences. These flanking sequences are the sequences shown in the following pages and defined as follows:

- sequence upstream from the sequence coding for R: between the bases 1 and 1476 of the sequence shown in FIG. 5,
- sequence between the sequence coding for the sensor protein S and ORF1: between the bases 3347 and 3500 of the sequence shown in FIG. 5,
- sequence downstream from the sequence coding for ORF3: between the bases 6168 and 7227 of the sequence shown in FIG. 5.

The sequence designated by SEQ ID NO 6 is also characterized by the fragment bearing the restriction sites in the following order:

- BglIII-EcoRI-BamHI-EcoRI

The location of the regulatory proteins and the resistance proteins is shown in FIG. 3.
The inventors have identified upstream and downstream from 1 genes vanR, vans, vanH, vanA and vanX, which are necessary for or associated with the expression of resistance to glycopeptides at a given level, genes coding for a transposase and a resolvase (upstream from the group previously mentioned) and genes vanY and vanz, downstream from this group. The genes for the transposase and resolvase might be implicated in transposition functions and the vanY gene coding for a D,D-carboxy peptidase might be implicated in the metabolism of the peptidoglycan, and might contribute to resistance to the glycopeptide in E. faecium BM4147 even though vanR, vanS, vanH, vanA and vanX born by a plasmid in a high number of copies, alone confer a high level of resistance.
Let it be noted that the sequence coding for the transposase is located on the (−) strand of the sequence ID NO 22 which codes for vanR, vanS, vanH, vanA, vanX, vanY, vanZ and the resolvase.
The invention relates not only to the DNA sequences identified in the list of the sequences but also to the complementary DNA sequence and the corresponding RNA sequences. The invention concerns in addition sequences which are equivalent to the former, either in terms of expression of proteins, polypeptides or their fragments described above or in terms of the capacity to detect, for example by chain polymerization procedures, strains of Gram-positive bacteria exhibiting resistance to antibiotics of the glycopeptide family such as vancomycin or teicoplanin.
Recombinant sequences characterized in that they comprise one of the above nucleotide sequences also form part of the invention.
The invention also relates to a recombinant vector characterized in that it includes one of the above nucleotide sequence at a site inessential for its replication, under the control of regulatory elements likely to be implemented in the expression of the resistance to antibiotics of the glycopeptide family, in particular to vancomycin or teicoplanin in a defined host.
Particularly advantageous recombinant vectors for the implementation of the invention are the following vectors: pAT214 containing the EcoRV-SacII fragment of 1761 bp containing a nucleotide sequence coding for the VanA protein; in these vectors the sequences of the invention are advantageously placed under the control of promoters such as the lac promoter.
The invention also relates to a recombinant cell host containing a nucleotide sequence such as that previously described or a vector such as that described above under conditions which make possible the expression of resistance to antibiotics of the glycopeptide family, in particular resistance to vancomycin and/or this host being for example selected from the bacteria, in particular the Gram-positive cocci.
In certain applications it is also possible to use yeasts, fungi, insect or mammalian cells.
The invention also relates to a nucleotide probe characterize in that it is capable of hybridizing with a sequence previously described, this probe being labelled if necessary. These probes may or may not be specific for the proteins of resistance to glycopeptides.
Labels which can be used for the requirements of the invention are the known radioactive labels as well as other labels such as enzymatic labels or chemoluminescent labels.
Probes thus labelled may be used in hybridization tests in order to detect resistance to glycopeptides in Gram-positive bacteria. In this case, conditions of low stringency will be used.
Nucleotide probes according to the invention may be characterized in that they are specific in Gram-positive bacteria for the sequences coding for a resistance protein to the glycopeptides, in particular to vancomycin and/or teicoplanin these probes being in addition universal among these sequences.
By these specific probes is meant any oligonucleotide hybridizing with a nucleotide sequence coding for one of the proteins according to the invention, such as described in the preceding pages, and not exhibiting a cross hybridization reaction or amplification reaction (PCR) with sequences present in all of the sensitive strains.
The universal character of the oligonucleotide which can be used in PCR is defined by their capacity to promote specifically the amplification of a nucleotide sequence implicated in resistance in any one strain of Gram-positive bacteria, resistant to the antibiotics of the glycopeptide family.
The size of the nucleotide probes according to the invention may vary depending on the use desired. For the oligonucleotides which are used in PCR, recourse will be had to fragments of a length which is usual in this procedure. In order to construct probes, it is possibly to take any part of the sequences of the invention, for example probe fragments of 200 nucleotides.
According to a particular embodiment of the invention, a nucleotide probe is selected for its specificity towards a nucleotide sequence coding for a protein necessary for the expression in Gram-positive bacteria of a high level of resistance to antibiotics of the glycopeptide family, in particular to vancomycin and teicoplanin.
As examples, useful probes may be selected from the intragenic part of the vanA gene.
Other useful probes for carrying out the invention are characterized by their universal character, according to the preceding definition, but are not specific for the resistance genes. They may also be used as primers in PCR, and are for example:

V1: GGX GAA GAT GGX TCX TTX CAA GGX

G C AG C G

A

V2: AAT ACX ATX CCX GGX TTT AC

C T C

C
V1 and V2 hybridize with vanA and vanC and are capable of leading to the detection of proteins associated with resistance to glycopeptides in other micro-organisms.
Other particular probes of the invention have the specific character of a nucleotide sequence coding for a protein necessary for the expression in Gram-positive bacteria of a low level of resistance to antibiotics of the glycopeptide family, in particular to vancomycin in Gram-positive bacteria.
It should also be mentioned that oligonucleotide probes which might be derived from the sequence of the vanA gene coding for the VanA protein may be used indiscriminantly to detect high-level or low-level resistance.
In a particularly preferred manner, a probe of the invention is characterized in that it hybridizes with a chromosomal or non-chromosomal nucleotide sequence of a Gram-positive strain resistant to glycopeptides, in particular to vancomycin and/or teicoplanin, in particular in that it hybridizes with a chromosomal or non-chromosomal nucleotide sequence of a strain of Gram-positive cocci, for example an enterococcal strain and preferably E. faecium 4147 or E. gallinarum.
In order to distinguish strains with a high level of resistance from strains with a low level of resistance it is possible to carry out a hybridization test using conditions of high stringency.
The oligonucleotides of the invention may be obtained from the sequences of the invention by cutting with restriction enzymes, or by chemical synthesis according to the standard methods.
Furthermore, the invention relates to polyclonal or monoclonal antibodies, characterized in that they recognize the polypeptide(s) described above or an amino acid sequence described above.
These antibodies may be obtained according to standard method for antibody production. In particular, in the case of the preparation of monoclonal antibodies, recourse will be had to the method of Köhler and Milstein according to which monoclonal antibodies are prepared by cell fusion between myeloma cells and mouse spleen cells previously immunized with a polypeptide or a composition according to the invention, in conformity with the standard procedure.
The antibodies of the invention can advantageously be used for the detection of the presence of proteins characteristic of resistance to the glycopeptides, in particular to vancomycin and teicoplanin.
Particularly useful antibodies are polyclonal or monoclonal antibodies directed against the protein VanA or VanC. Such antibodies advantageously make it possible to detect strains of bacteria, in particular Gram-positive cocci, exhibiting high-level resistance to the antibiotics of the glycopeptide family. If necessary, a step entailing lysis of the cells of the sample undergoing detection is performed prior to the placing in contact of the sample with the antibodies.
In order to carry out this detection, recourse will advantageously be had to antibodies labelled for example with a radioactive substance or other type of label.
Hence, tests for the detection in Gram-positive bacteria of resistance to the glycopeptides, in particular tests making use of the ELISA procedures, are included in the framework of the invention.
A kit for the in vitro diagnosis of the presence of Gram-positive strains, resistant to the glycopeptides, in particular to vancomycin and/or teicoplanin these strains belonging in particular to the Gram-positive cocci for example enterococci, for example E. faecium or E. gallinarum is characterized in that it comprises:

- antibodies corresponding to the above definition, labelled if necessary,
- a reagent for the detection of an immunological reaction of the antigen-antibody type,
- if necessary, reagents to effect the lysis of the cells of the sample to be tested.

Furthermore, the agents developed by the inventors offer the very useful advantage of being suitable for the development of a rapid and reliable test or kit for the detection of Gram-positive strains resistant to the glycopeptides by means of the polymerase chain reaction (PCR). Such a test makes it possible to improve the sensitivity of the existing tests which remain rather unreliable and, in certain cases, may make possible the detection of all of the representatives of the family of the genes coding for resistance proteins to the glycopeptides in Gram-positive bacteria.
The carrying out of a test by means of the method of amplification of the genes of these proteins is done by the PCR procedure or by the RPCR procedure (RPCR: abbreviation for reverse polymerase chain reaction).
The RPCR technique makes possible the amplification of the NH₂and COOH terminal regions of the genes it is desired to detect.
Some specific primers make it possible to amplify the genes of the strains with low-level resistance. These primers are selected, for example, from the sequence coding for the resistance protein VanA.
As examples, the following sequences-can be used as primers for the preparation of probes for the detection of an amplification by means of the PCR or RPCR method.

V1: GGX GAA GAT GGX TCX TTX CAA GGX

G C AG C G

A

V2: AAT ACX ATX CCX GGX TTT AC

C T C

C
X represents one of the bases A, T, C or G or also corresponds in all cases to inosine.
Naturally, the invention relates to the complementary probes of the oligonucleotides previously described as well as possibly to the RNA probes which correspond to them.
A kit for the in vitro diagnosis of the presence of strains of Gram-positive bacteria resistant to the glycopeptides, in particular resistant to vancomycin and/or teicoplanin these strains belonging in particular to the Gram-positive cocci, in particular that they are strains of enterococci, for example E. faecium or E. gallinarum, is characterized in that it contains:

- a nucleotide probe complying with the above specifications and if necessary,
- oligonucleoside triphosphates in an amount sufficient to make possible the amplification of the desired sequence,
- a hybridization buffer,
- a DNA polymerization agent.

The invention also relates to a procedure for the in vitro detection of the presence of Gram-positive strains resistant to the glycopeptides, in particular to vancomycin and/or teicoplanin these strains belonging in particular to the family of the Gram-positive cocci, in particular in that they are strains of enterococci, for example E. faecium or E. gallinarum, characterized in that it comprises

a) the placing of a biological sample likely to contain the resistant strains in contact with a primer constituted by a nucleotide sequence described above, or any part of a sequence previously described, capable of hybridizing with a desired nucleotide sequence necessary for the expression of resistance to the glycopeptides, this sequence being used as matrix in the presence of the 4 different nucleoside triphosphates and a polymerization agent under conditions of hybridization such that for each nucleotide sequence which has hybridized with a primer, an elongation product of each primer complementary to the matrix is synthesized,
b) the separation of the matrix from the elongation product obtained, this latter then also being capable of behaving as a matrix,
c) the repetition of step a) so as to produce a detectable amount of the desired nucleotide sequences,
d) the detection of the product of amplification of the nucleotide sequences.

The detection of the elongation products of the desired sequence may be carried out by a probe identical with the primers used to carry out the PCR or RPCR procedure, or also by a probe different from these primers, this probe being labelled if necessary.
Details relating to the implementation of the PCR procedures may be obtained from the patent applications EP 0229701 and EP 0200362.
Other advantages and characteristics of the invention will become apparent in the examples which follow and from the figures.

FIGURES

FIG. 1: electrophoresis on SDS-polyacrylamide gel (SDS-PAGE) of the proteins of the membrane fractions line 1 and line 4, molecular weight standards; line 2, E. faecium BM4147 placed in culture in the absence of vancomycin; line 3, BM4147 placed in culture in the presence of 10 μg/ml of vancomycin. The head of the arrow indicates the position of the VanA protein.
FIG. 2:
A: Restriction maps of the inserts of the plasmids pAT213 and pAT214. The vector and the DNA insert are distinguished by light and dark segments, respectively. The open arrow represents the vanA gene.
B: Strategy for the nucleotide sequencing of the insert of 1761 bp in the plasmid pAT214. The arrows indicate the direction and extent of the sequencing reactions by the dideoxy method. The synthetic oligonucleotide primer (5′ ATGCTCCTGTCCTTTC 3′ OH) is complementary to the sequence between the positions 0.361 and 378. Only the pertinent restriction sites are given.
FIG. 3: position of the sequences R, S, ORF1, ORF2, ORF3.
FIG. 4: representation of SEQ ID NO 6.
FIG. 5: representation of SEQ ID NO 6 and the corresponding protein.
FIG. 6: sequence of the vanA gene and the corresponding protein.
FIG. 7:
(a): Localization of the genes vanR, vanS, vanH, vanA, vanX, vanY, vanZ of the gene for the transposase and of the gene for the resolvase as well as the repeated reverse terminal sequences of 38 bp at the end of the transposon.
(b): Mapping of the plasmids. (A) Polylinker pAT29 and derivatives constructed in this study. The arrow labelled P2 indicates the position and orientation of the P2 promoter of aphA-3 (Caillaud et al., 1987, Mol. Gen. Genet. 207:509-513). (B) Insert pAT80. The white rectangles indicate the DNA of pAT29 but they are not shown to scale. The rectangles terminating in an arrow indicate the coding sequences. The arrows shown in vertical and horizontal full lines indicate the position and orientation, respectively, of the apha-1 gene in the derivatives of pAT80. Restriction sites: Ac, AccI; B, BamHI; Bg, BglII; Bs, BssHII E, EcoRI; H, HindIII; Hc, HincII; K, KDnI; P, PstI; S, SmaI; SI, SacI, SII, SacII; Sa, SalI; Sp, SphI; Xb, XbaI. (C) Inserts in pAT86, pAT87, pAT88 and pAT89. The inserts are shown by full lines and the corresponding vectors are indicated in parentheses.
FIG. 8: nucleotide sequence of the transposon shown in FIG. 7 and amino acid sequence of the corresponding proteins. The nucleotide sequence is shown for the (+) strand and for the (−) strand (corresponding to the complementary sequence of the (+) strand for the positions 1 to 3189) on which the coding sequence of the transpose is located.
FIG. 9: Nucleotide sequence of the SacI-PstI fragment of 1347 bp of the plasmid pAT216 containing the vanC gene. The numbering start at the first base G of the SacI restriction site. The potential RBS sequence upstream from the initiation codon ATG of translation at position 215 is underlined. The STOP codon (TGA) is indicated by *. The region coding for the vanC and the deduced amino acid sequence are indicated in bold characters. Sequential overlapping clones were generated by restriction fragments of subcloning of pAT216 in the bacteriophage M13 mp10 (Amersham, England). The universal primer (New England Biolabs Beverly Mass.) was used to sequence the insert in the recombinant phages. The sequencing was performed by the enzymatic dideoxy nucleotide method (Sanger et al., 1977 PNAS 74: 5463-5467) by using the T7 DNA polymerase (Sequenase US B CORP, Cleveland, Ohio) and [∝-³⁵S] dATP (Amersham, England). The reaction products were loaded onto 6% denaturing polyacrylamide gels.
FIG. 10: alignment of the amino acid sequences of VanC, VanA, DdlA and DdlB. The identical (I) amino acids and the conservative (C) substitutions in the 4 sequences are indicated in the alignment. In order to classify the conservative substitutions, the amino acids were grouped as follows: RK, LFPMVI, STQNC, AGW, H, ED and Y. The regions of high homology corresponding to the domains 1, 2, 3 and 4 are underlined. The sequences corresponding to the peptides 1 and 2 are indicated by the arrows.
FIG. 11: description of the oligonucleotides V1 and V2 (A): Amino acid sequence of the peptides 1 and 2 of VanA and of the D-Ala-D-Ala ligases. The number of amino acids between the N-terminus and peptide 1, between the peptides 1 and 2 and the peptide 2 and the C-terminus is indicated. The identical amino acids between at least 2 of the 3 sequences are indicated in bold characters.
(B): Target peptides and deduced nucleotide sequence. X represents any base of the DNA. Peptide 2 in DdlB differs from the target peptide at 2 positions (*).
(C): Nucleotide sequence of V1 and V2. Alternate nucleotides and deoxyinosine (I) which may correspond to any base in the DNA, were used at the positions at which the nucleotide sequences coding for the target peptides vary. The arrows indicate the direction of DNA synthesis. The oligonucleotides were synthesized by the methoxy-phosphoramidite method with a Biosystem DNA 380B machine (Applied Biosystem, Foster City, Calif.). The DNA was isolated from bacterial lysate by extraction with hexadecyl trimethyl ammonium bromide (Inst. biotechnologies, Inc., New Haven, Colo.) (Le Bouguénec et al., 1990, J. Bacteriol. 172:727-734) and used as matrix for the amplification by means of PCR with a controlled heating system “Intelligent Heating Block” IBH101 (Hybarid Ltd., GB) according to the description of Mabila et al. (1990, Plasmid 23:27-34). The amplification products were revealed by electrophoresis on a 0.8% gel, after staining with ethidium bromide.
FIG. 12: Inactivation by insertion of vanC. The vanC gene is shown by an open arrow and the internal EcoRI-HincII fragment of 690 bp is hatched. The DNA of pAT114 is shown by a thin line; the chromosomal DNA of PM4174 by a thick line; the arrows indicate the genes for resistance to the antibiotics: aphA-3 is the gene coding for the 3′-aminoglycoside phosphotransferase; erm is the gene coding for the ER^Rmethyl transferase.
(A): The plasmid pAT217 was constructed by ligation of the EcoRI-Hinc fragment of pAT216 to the suicide vector pAT114 (Trieu-Cuot et al., 1991, Gene 106:21-27), digested with EcoRI and SmaI.
(B): vanC region of the chromosomal DNA of BM4174.
(C): vanC region after integration of pAT217.
FIG. 13: Southern blot analysis of the integration of pAT217 into the vanC gene of BM4174.
(left hand side): Total DNA of BM4175 (line 2) and BM4174 (line 3) digested with EcoRI and resolved by means of electrophoresis on a 1% agarose gel. The DNA of the bacteriophage lambda digested with PstI was used as molecular mass standard (line 1). The DNA was transferred under vacuum to a Nytran membrane (Schleicher and Schül, Germany) by using a Trans-Vac TE80 apparatus (Höfer Scientific Instruments, San Francisco, Calif.) and bound to the membrane through the intermediary of UV light. The hybridization was carried out with the probe C (Middle or the probe aphA-3 specific for pAT114 (Lambert et al., 1985, Annales de l'Institut Pasteur/Microbiol. 136(b): 135-150).
(right hand side): the probes were labelled with ³²P by nick translation. The molecular masses (kb) are indicated.
FIG. 14: alignment of the deduced amino acid sequences of VanS derived from E. faecium BM4147 and of PhoR and EnvZ from E. coli. The numbers on the left refer to the position of the first amino acid in the alignment. The numbers on the right refer to the position of the last amino acid of the corresponding line. The identical amino acids are placed in boxes. The dotted lines indicate gaps introduced in order to optimize their similarity. The dashes indicate the positions of the amino acid residues conserved in other HPK. The histidine residues in bold characters in section 1 are potential sites of auto-phosphorylation.
FIG. 15: alignment of the deduced amino acid sequences of VanR from E. faecium BM4147, OmpR and PhoB from E. coli as well as that of CheY from Salmonella typhimurium. The numbers on the right indicate the position of the last amino acid of the corresponding line. The identical amino acids are placed in boxes. The dotted lines indicate the gaps introduced in order to optimize the homologies. The residues in bold characters correspond to the amino acids strongly conserved in the effector domains of other RR. The aspartic acid residue 57 of CheY is phosphorylated by the HPK associated with CheA.

I—Identification of vanA

Materials and Methods for the Identification and Characterization of the vanA Gene
Bacterial Strains and Plasmids

The origin of the plasmids used is given in the table below.



	Strain or plasmid	Source or reference

	Escherichia coli
	JM83	Messing (1979)
	AR1062	Rambach and Hogness (1977)
	JM103	Hannshan (1983)
	ST640	Lugtenberg and van Schijndel
		van-Dam (1973)
	Enterococcus faecium
	BM4147	Leclercq et al (1988)
	Plasmid pUC18	Norrander et al (1983)
	pAT213	Brisson-Noel et al (1990)
	pAT214	Described in this text

Preparation of the Enterococcal Membranes

Enterococcus faecium BM4147 was cultivated in 500 ml of heart brain broth (BHI broth medium) until the optical density (OD₆₀₀) reaches 0.7. Induction was effected with 10 μg/ml of vancomycin (Eli Lilly Indianapolis Ind.). The subsequent steps were performed at 4° C. The cells were recovered by centrifugation for 10 minutes at 6000 g, washed with a TE buffer (0.01 M TRIS-HCl, 0.002 M EDTA, pH 7.0) and lysed by glass beads (100 μm in diameter) in a Braun apparatus for 2 minutes. The cell debris were separated by centrifugation for 10 minutes at 6000 g. The membranes were collected by centrifugation for 1 hour at 65000 g and resuspended in 0.5 ml of TE buffer.
Preparation of the Minicells
Plasmids were introduced by transformation into the strain E. coli AR1062 prepared in the form of bacterial vesicles. The bacteria vesicles were recovered on sucrose gradients and the proteins were labelled with 50 μCi of [³⁵S]-L-methionine (Amersham, Great Britain) according to the method of Rambach and Hogness (1977, P.N.A.S. USA, 74; 5041-5045).
Preparation of the Membrane Fractions and the Cytoplasmic Fractions of E. coli
E. coli JM83 and strains derived from it were placed in culture in BHI medium until an optical density (OD₆₀₀) of 0.7 was attained, washed and suspended in a TE buffer. The cell suspension was treated by sonication (ultrasound) for 20 seconds at doses of 50 W in a cell fragmentation apparatus in a Branson B7 sonication apparatus and the intact cells were removed by centrifugation for 10 minutes at 6000 g. The supernatant was fractionated into membrane and cytoplasmic fractions by means of centrifugation for 1 hour at 100,000 g.
Electrophoresis on SDS-polyacrylamide Gel (SDS-PAGE)
The proteins from the bacterial fractions were separated by means of SDS-PAGE on linear gradients of polyacrylamide gels (7.5%-15%) (Laemli 1970, Nature 227: 680-685). The electrophoresis was carried out for 1 hour at 200 V, then for 3 hours at 350 V. The gels were stained with Coomassie blue. The proteins of the extracts were separated on 10% polyacrylamide gels and visualized by means of autoradiography.
Purification of the Protein Band and Determination of the N-Terminal Sequence
The proteins of the membrane fractions of an induced culture of E. faecium BM4147 were separated by means of SDS-PAGE. The gel was electrotransferred for 1 hour at 200 mA to a polyvinylidene difluoride membrane (Immobilon Transfer, Millipore) by using a transfer apparatus (Electrophoresis Unit LKB 2117 Multiphor II) in accordance with the instructions of the manufacturer. The transferred proteins were stained with Ponceau red. The portion of membrane bearing the protein of interest was excised, centered on a Teflon filter and placed in the cartridge of a sequencer (Sequencer Applied Biosystems model 470A). The protein was sequenced by means of the automated Edman degradation (1967, Eur. J. Biochem. 1; 80-81).
Construction of Plasmids
The plasmid pAT213 (Brisson-Noel et al., 1990, Antimicrob. Agents Chemother., 34; 924-927) consists of a EcoRI fragment of DNA of 4.0 kb of the enterococcal plasmid pIP816 cloned at the EcoRI site of a Gram-positive-Gram-negative shuttle vector pAT187 (Trieu-Cuot et al., 1987, FEMS Microbiol. Lett. 48; 289-294). In order to construct pAT214, the EcoRV-SacII DNA fragment of 1761 bp of pAT213 was purified, treated with the Klenow fragment of the DNA polymerase I of E. coli and ligated to the DNA of pUC18 which had previously been digested with SmaI and dephosphorylated (FIG. 2). The cloning (Maniatis et al., 1982 Cold Spring Harbor Laboratory Press) was carried out with restriction endonucleases (Boehringer Mannheim and Pharmacia), with the T4 DNA ligase (Pharmacia) and alkaline phosphatase (Pharmacia) according to the instructions of the manufacturer.
Subcloning in M13 and Nucleotide Sequence
The DNA restriction fragments were subcloned in the polylinker of the replicative forms of the derivatives mp18 and mp19 of the bacteriophage M13 (Norrander et al., 1983, Gene 26; 101-106), obtained from Pharmacia P-L Biochemicals. E. coli JM103 was transfected with recombinant phages and the single-stranded DNA was prepared. The nucleotide sequencing was carried out by the enzymatic di-deoxy nucleotide method (Sanger et al., 1977, P.N.A.S. USA 74; 5463-5467) by using a T7 DNA polymerase (Sequenase, United States Biochemical Corporation, Cleveland, Ohio) and [∝-³⁵S] dATP (Amersham, Great Britain) The reaction products were revealed on 6% polyacrylamide gels containing a denaturing buffer.
Data-Processing Analysis and Data on the Sequence
The complete DNA sequence was assembled by using the computer programs DBCOMP and DBUTIL (Staden, 1980, Nucleic Acids Res 8; 3673-3694). The protein data bank PSEQIP of the Pasteur Institute was screened using an algorithm developed by Clayerie (1984, Nucleic Acids Res 12; 397-407). The alignments between the pairs of amino acid sequences were constructed using the algorithm of Wilbur et al (1983, P.N.A.S. USA 80; 726-730). The statistical significance of the homology was evaluated with the algorithm of Lipman and Pearson (1985, Science 227; 1435-1440).
For each comparison 20 amino acid sequences were used to calculate the mean values and the standard deviations of the random results.
Genetic Complementation Tests
The plasmids were introduced by transformation into E. coli ST640, a temperature-sensitive mutant with an unmodified D-ala-D-ala ligase (Lugtenberg et al 1973, J. Bacteriol 110; 26-34). The transformants were selected at 30° C. on plates containing 100 μg/ml of ampicillin and the presence of the plasmid DNA of the expected size and the restriction maps were verified. Single colonies grown at 30° C. in BHI broth medium containing ampicillin were placed on a BHI agar medium containing both 100 μg/ml of ampicillin and 50 μM of isopropyl-1-thio-β-D-galacto-pyranoside (IPTG) and the plates were incubated at a permissive temperature of 30° C. and at a non-permissive temperature of 42° C. The complementation test was considered to be positive if the colonies were present after 18 hours of incubation at 42° C.

Results

Identification of the VanA Protein and its N-Terminal Sequence
The membrane fractions of the E. faecium BM4147 cells placed in culture, on the one hand, under conditions of induction, and, on the other, in the absence of induction, were analysed by means of SDS-PAGE. The sole difference which could be detected, related to the exposure to sub-inhibitory concentrations of vancomycin, was the marked intensification of a band which corresponded to a protein of an estimated molecular weight of about 40 kDa. In the induced cells and in the non-induced cells, the protein band represents the same protein because this band is absent from membranes of a derivative of BM4147 which has lost the pIP816 plasmid. The inducible protein, designated as VanA, was purified after SDS-PAGE and automated Edman degradation was carried out on a 50 pmol. sample. Nine amino acids of the N-terminal sequence of VanA were identified: Met Asn Arg Ile Lys Val Ala Ile Leu.
Sub-Cloning of the vanA Gene
The insert of 4.0 kb of the plasmid pAT213 bears the determinant for resistance to the glycopeptides of E. faecium BM4147. Various restriction fragments of this insert were subcloned in pUC18 and the recombinant plasmids specific for vanA in E. coli were identified by SDS-PAGE analysis of the proteins of the cytoplasmic and membrane fractions or of the extracts of the bacterial vesicles. This approach was used since E. coli is intrinsically resistant to the glycopeptide. The EcoRV-SacII insert of the pAT214 plasmid (FIG. 2) codes for a unique polypeptide of 40 kDa which migrates together with VanA, derived from the membrane preparations of E. faecium BM4147.
Nucleotide Sequence of the Insert in pAT214 and Identification of the vanA Coding Sequence
The nucleotide sequence of the EcoRV-SacII insert of 1761 bp in pAT214 was determined on both strands of the DNA according to the strategy described in FIG. 2. The location of the termination codons (TGA, TAA, TAG) in three reading frames on each DNA strand showed the presence of a unique open reading frame (ORF) which was sufficiently long to code for the VanA protein. This reading frame ORF is located between the TAA codon at position 281 and the TAG codon at position 1406. The amino acid sequence deduced for ORF was compared with that of the N-terminus of VanA. The nine amino acids identified by protein sequencing are encoded in the nucleotide sequence beginning with the ATG (methionine) codon at position 377 (FIG. 3). This codon for the initiation of translation is preceded by a sequence (TGAAAGGAGA), characteristic of a ribosomal binding site (RBS) in Gram-positive bacteria which is complementary to the 8 bases of the rRNA of the 16S subunit of Bacillus subtilis in its sequence (3′OH UCUUUCCUCC 5′) (Moran et al., 1982, Mol. Gen. Genet. 186; 339-346). In this ORF, there is no other ATG or GTG initiation codon between the positions 281 and 377. The sequence of 1029 bp which-extends from the ATG codon at position 377 to the TGA codon at position 1406 codes for a protein containing 343 amino acid residues. The calculated molecular weight of this protein is 37400 Da, which is in agreement with the estimation of 40 kDa obtained by SDS-PAGE analysis.
Homology of the Amino Acid Sequences of VanA and the D-ala-D-ala Ligase Enzymes
The screening of the protein data bank PSEQIP has shown the existence of a sequence homology between VanA and the D-ala-D-ala ligases of E. coli (ECOALA, Robinson et al., 1986, J. Bacteriol. 167; 809-817) and of Salmonella typhimurium (DALIG, Daub et al., 1988, Biochemistry 27; 3701-3708). The calculated percentage of homology between pairs of proteins was included between 28% and 36% for the identical amino acids and between 48% and 55% by taking into consideration homologous amino acids. VanA and DALIG are more closely related. The statistical significance of these similarities wa evaluated by aligning VANA and sequences containing the same composition of amino acids as DALIG or ECOALA (Lipman and Pearson, 1985, Science 227; 1435-1440).
Genetic Complementation Test for the Activity of D-ala-D-ala Ligase
The E. coli strain ST640 is a thermosensitive mutant exhibiting a deficient D-ala-D-ala ligase activity (lugtenberg et al., 1973, J. Bacteriol. 113: 96-104). The plasmids pUC18 and pAT214 were introduced into E. coli ST640 by transformation. The strains ST640 and ST640 (pUC18 grew normally only at the permissive temperature (30° C.) whereas E. coli ST640 (pAT214) grew both at the permissive temperature and at the non-permissive temperature (42° C.).
This test shows that VANA is functionally related to the D-Ala-D-Ala ligases in E. coli and is probably capable of catalysing the same ligation reaction as DALIG.
II—VanS-VanR Two-Component Regulation System for the Control of the Synthesis of Depsipeptides of the Precursor of Peptidoglycans
Materials and Methods
Strains, Plasmids and Conditions of Culture
The restriction fragments of pIP816 (Tra⁻, Mob⁺, Vm^r) were cloned in derivatives of the vector pAT29 which constitutes a shuttle vector between the Gram-positive and Gram-negative bacteria (oriR pAMβ1, oriR pUC, oriT RK2, spc, lacZ) (Trieu-Cuot et al., 1990, Nucleic Acids Res. 18:4296). This vector was constructed by the inventors and used to transform the strain E. coli JM103 ((lac-proAB), supE, thi, strA, sbcB15, endA, hspR4, F traD36, proAB, LacI^q, lacZ M15) (Messing et al., 1983, Methods Enzymol. 101:20-78). The plasmid DNA was prepared by an alkaline lysis protocol on a small scale (Sambrook et al., 1982, Molecular cloning, a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor N.Y.) and introduced by electroporation (Cruz-Rodz A. L. et al., 1990, Mol. Gen. Genet. 224: 152-154) in E. faecalis JH2-2 (Fus^R, Rif^R) (Jacob A. E. et al., 1974, J. Bacteriol. 117: 360-372), by using a Gene Pulser apparatus (Bio-Rad laboratories, Richmond, Calif.). The restriction profiles of the purified plasmids from E. faecalis and E. coli were compared in order to detect possible rearrangements of DNA.
The integrative plasmid pAT113 (Mob⁺, Em^R, Km^R, oriR PACYC184 attTn1545, lacZ) (Trieu-Cuot et al., Gene 106: 21-27) carries the joined ends of the transposon Tn1545. This vector does not replicate in Gram-positive bacteria but is integrated into the chromosome of the host by illegitimate recombination mediated by the integrase of Tn1545 or of Tn916 (Trieu-Cuot et al. previously mentioned). The integrative plasmids were introduced into E. faecalis BM4148 (strain JH2-2::Tn916) by means of electroporation. This strain is modified by the transposon Tn917 described by Franque A. E. et al. (1981, J. Bacteriol. 145: 494-502).
The cultures were grown in brain-heart broth (BHI—Brain Heart Infusion Broth) or on agar at 37° C. The method of Steers et al (Antibiot. Chemother. Basel. 9: 307-311) was used to determine the minimal inhibitory concentrations (MICs) of the antibiotics on a Mueller-Hinton gelose agar medium.
Recombinant DNA Procedures
The cleavage of DNA with restriction endonucleases (Boehringer Mannheim and Pharmacia), the purification of the DNA restriction fragments from agarose gels, the conversion of the cohesive ends to blunt ends with the Klenow fragment of the DNA polymerase I of E. coli (Boehringer Mannheim), the dephosphorylation of the ends of the DNA with calf intestinal phosphatase (Boehringer Mannheim), the ligation of the DNA fragments with the T4 DNA ligase (Amersham) were carried out according to the standard methods of Sambrook et al (1982, Molecular Cloning, a Laboratory Manual. Cold Spring Harbor Laboratory. Cold Spring Harbor N.Y.).
Construction of Plasmids
The origin of the vectors and the inserts used for the recombinant plasmids constructed here is the following:

- (i) vector pAT78 for the recognition of the promoter: the amplified DNA of the cat gene for chloramphenicol acetyltransferase of the plasmid pC194 of Staphylococcus aureus (Horinouchi et al., 1982, J. Bacteriol. 150: 815-825) was inserted between the PstI and ShI restriction sites of the shuttle vector pAT29. Amplification by means of the polymerase chain reaction was carried out by means of primers A1 and A2 which were synthesized by the methoxy phoshoramidite method (Mabilat et al., 1990, Plasmid 23: 27-34). The sequence of the primer A1 (5′ GCTGCAGATAAAAATTTAGGAGG) is composed of a PstI recognition site (underlined) and 18 bases (positions 6 to 23) of pC194 which include the ribosomal binding site (RBS; AGGAGG positions 18 to 23) of the cat gene. The sequence of the primer A2 (5′CGCATGCTATTATAAAA GCCAGTC) contains the SphI cleavage site (underlined) and is complementary (positions 8 to 24) to 17 bases at the 3′ end of the cat gene. The triplet ATT at positions 9 to 11 correponds to the TAA stop codon of cat. The DNA fragments amplified with the primers A1 and A2 hence consist of an open reading frame (orf) and a ribosomal binding site for CAT (positions 1234 to 1912 according to the numbering of Horinouchi et al. (1982, J. Bacteriol. 150: 815-825) flanked by the PstI and SphI sites. The position 1234 is located at the interior of the loop of the secondary structure of the mRNA which blocks translation in the absence of chloramphenicol. Thus, the amplified sequence does not contain the cat promoter nor the sequence complementary to the RBS which is essential for the regulation of translation Ambulos, N. P. et al., 1984, Gene 28: 171-176).

(ii) expression vector pAT79: the ClaI-BssHII fragment of 243 bp bearing the P2 promoter of the aphA-3 gene of the enterococcal plasmid pJH1 (Caillaud et al., 1987, Mol. Gen. Genet. 207: 509-513) was inserted between the EcoRI and SacI restriction sites of pAT78.
(iii) plasmid pAT80 and its derivatives: the BglII-XbaI fragment of 5.5 kb of pIP816 was inserted between the BamHI and XbaI sites of pAT78. The resulting plasmid, designated as pAT80 was partially digested with HincII and ligated with the EcoRV fragment containing a gene related to the apha-I gene of the transposon Tn903 (Oka A. et al., 1981, J. Mol. Biol. 147:217-226. This fragment contains the aphA-I gene which codes for the 3′aminoglycoside phosphotransferase of type I conferring resistance to kanamycin. The insertion of aphAI was carried out at three different sites in pAT80, generating the plasmids pAT81, pAT83 and pAT85. The cassettes BamHI and EcoRI containing aphA-1 were inserted at the BamHI (to form the plasmid pAT84) and EcoRI (to form the plasmid pAT82) sites of pAT80.
(iv) plasmids pAT86, pAT87, pAT88 and pAT89: the plasmid pAT86 was constructed by cloning the EcoRI-SacII fragment of 2.803 bp of pAT80 coding for VanH and VanA at a SmaI site of pAT79. pAT87 was obtained by inserting the EcoRI-XbaI fragment of 3.4 kb of pAT80 upstream from the cat gene of the detection vector of promoter pAT78. The plasmid pAT88 resulted from the ligation of pAT78 digested with EcoRI and BamHI to the EcoRI-BamHI fragment of 1.731 bp of pAT80. The BglII-AccI fragment (positions 1 to 2356) of pAT80 was inserted into the polylinker of the integrative vector pAT113, generating pAT89.
Sub-Cloning in M13 and Sequencing
The DNA restriction fragments were subcloned in a polylinker of replicative derivatives of the bacteriophage M13, these derivatives being called mp18 and mp19 (Norrander et al., 1983, Gene 26:101-106). E. coli JM103 was transfected with the recombinant phages and a single-stranded DNA was prepared. The sequencing of the nucleotides was carried out according to the conditions described by Sanger et al. (Proc. Natl. Acad. Sci. USA, 1977, 74: 5463-5467) by using the modified T7 DNA polymerase (Sequenase, United States, Biochemical Corporation Cleveland Ohio) and [∝-³⁵S] dATP (Amersham). The reaction products were resolved on gradient gels of polyacrylamide in a 6% buffer.
Enzymatic Test
The JH2-2 derivatives of E. faecalis were grown to an optical density OD₆₀₀of 0.7 in a BHI broth supplemented with spectinomycin (300 μg/ml). The cells were treated with lysozyme, lysed by sonication and the cell debris were centrifuged for 45 minutes at 100,000 g according to the description given by Courvalin et al. (1978, Antimicrob. Agents Chemother. 13:716-725). The formation of 5-thio-2-nitrobenzoate was measured at 37° C. in the presence and in the absence of chloramphenicol and the specific CAT activity was expressed in micromole per minute and per milligram of proteins (Shaw et al., 1975, Methods Enzymol. 43:737-755).

Results

The vanH and vanA genes of pIP816 were cloned in a plasmid pAT79 under the control of the heterologous promoter P2 (Caillaud et al., 1987, Mol. Gen. Genet. 207:509-513) and the plasmid pAT86 formed did not confer resistance to vancomycin on the strain E. faecalis JH2-2. These genes are thus not sufficient for the synthesis of peptoglycan in the absence of the antibiotic. Different restriction fragments of pIP816 were cloned in the vector pAT78. The BglII-XbaI fragment of 5.5 kb of pAT80 is the smallest fragment obtained which conferred resistance to vancomycin.
Nucleotide Sequence of the vanR and vanS Genes
The sequence of the insert in pAT80 was determined on both strands of the DNA from the BglII site to the ATG initiation codon for the translation of VanH. Two open reading frames (orf) were detected within the sequence of 2475 bp: the first open reading frame extends from the nucleotide 386 to the nucleotide 1123; at position 431 a sequence characteristic of the RBS sequences in Gram-positive bacteria is found, 6 base pairs upstream from the ATG initiation codon for translation (TGAAAGGGTG); the other initiation codons for translation in this orf are not preceded by this type of sequence. The sequence of 693 bp extending from the ATG codon at position 431 to the TAA codon at position 1124 is capable of coding for a protein of 231 amino acids with a molecular mass of 26,612 Da which is designated as VanR.
In the case of the second open reading frame (from nucleotide 1089 to nucleotide 2255) the amino acid sequence deduced from the first initiation codon in phase (TTG at position 1104) would code for a protein of 384 amino acids having a molecular mass of 43,847 Da and designated as VanS. The TTG codon at position 1116 and the ATG codon at position 1164 are in-phase initiation codons for translation preceded by sequences with low complementarity with the 3′OH terminus of the 16S sub-unit of the rRNA of B. subtilis (GGGGGGTTGG-N-8-TTG and AGAACGAAAA-N-6-ATG, respectively).
Between the last codon of vanS and the initiation codon ATG for the translation of vanH a sequence of 217 bp is to be observed which contains a repeated reverse sequence of 17 bp. This sequence does not function as a terminator of strong transcription.
The comparison of the sequences obtained with data bases has shown that the conserved amino acid residues identified by Stock et al. (1989, Microbiol. Rev. 53:450-490) in the kinase domain of 16 HPK (Histidine Protein Kinase) were detected in the C-terminal part of VanS. VanS possesses two groups of hydrophobic amino acids in the N-terminal region. The histidine residue 164 of VAnS is aligned with the residue His216 of PhoR (Makino et al., 1986, J. Mol. Biol. 192: 549-556) and His 243 of EnvZ (Comeau et al., 1985, 164:578-584) which are presumed sites of autophosphorylation in these proteins.
Similarly, the amino acids 1 to 122 of VanR exhibit similarity with the effector domains of response regulators RR. The aspartic acid 53 of VanR might be a phosphorylation site because this residue is aligned with Asp 57 of Che Y which is phosphorylated by HPK associated with CheA and corresponds to an invariant position in other proteins of the RR type (Stock et al previously mentioned). VanR might belong to the sub-class OmpR-PhoB of RR which activates the initiation of transcription mediated by the RNA polymerase containing the 70S factor of E. coli (Stock et al. previously mentioned).
Inactivation of the van Genes by Insertion
Cassettes of resistance to kanamycin inserted in the group of van genes in the plasmid pAT80 have shown the following: the insertion in vanR suppresses resistance to vancomycin and chloramphenicol; VanR is an activator of transcription necessary for the expression of the genes for resistance to vancomycin. The inactivation of vanS leads to a two-fold reduction of the minimal inhibitory concentration (MIC) of chloramphenicol and to a three-fold reduction of the specific CAT activity but the minimal inhibitory concentration of vancomycin remains unchanged. Hence, VanS is necessary to produce a high level of transcription of the genes for resistance to vancomycin although it is not required for the expression of the phenotype of resistance to vancomycin.
Derivatives of pAT80 bearing insertions in vanH (pAT83), vanA (pAT84) or in the region 1.0 kb downstream from vana (pAT85) have made it possible to obtain resistance to chloramphenicol but not to vancomycin. This dissociated phenotype corresponds to the inactivation of genes coding for enzymes which synthesize the depsipeptide precursors necessary for the assembly of the bacterial cell walls in the presence of vancomycin.
Downstream from the vanA gene the presence of an inactivated orf has been detected in pAT85 in the region of the sequence of 365 bp after the TGA codon of vanA and before the SacII site and this orf contains an in-phase ATG initiation codon preceded by a RBS-like sequence. This sequence codes for a protein necessary for resistance to the glycopeptide, designated as VanX and which comprises maximally about 330 amino acids.
Trans-Activation of the Transcription of the van Genes
The integrative plasmid pAT89 coding for VanR and VanS was introduced into the chromosome of E. faecalis BM4138. The plasmid pAT87 bearing the genes vanH, vanA and vanX cloned upstream from the cat gene lacking the promoter for pAT78 conferred resistance to vancomycin on this strain but not to E. faecalis JH2-2. The level of expression of the cat gene of pAT87 in the strains BM4138::pAT89 and JH2-2 indicated that VanR activates the transcription of the reporter gene localized at the 3′ end of the group of van genes. Similar levels of CAT synthesis were observed for pAT88 which bears a transcription fusion between the 5′ parts of vanA and the cat gene. These results show that in E. faecalis BM4138::pAT89 (pAT87) VanR and VanS encoded in the chromosome activate in a trans manner the transcription of vanA, vanH and vanX of pAT87 making possible the production of resistance to vancomycin.
Moreover, it has been observed that the expression of the gene was essentially constitutive when vanR and vanS were borne by a multicopy plasmid pAT80 and weakly inducible by vancomycin when the genes for the regulatory proteins were present on the chromosome of the host.
III—Characterization of the Sequence of the vanC Gene of Enterococcus gallinarum BM4174
Definition and Use of Universal Primers for the Amplification of Genes Coding for D-Ala-D-Ala Ligases and Related Proteins Implicated in Resistance to Vancomycin
The protein VanA necessary for the expression of a high level of resistance to the glycopeptides in E. faecium BM4147 shares a similarity of about 28 to 36% as regards its amino acids with the D-Ala-D-Ala ligases of E. coli but possesses a different substrate specificity from that of these ligases. Peptides designated as 1 and 2 which are conserved in the sequences of the DdlA and DdlB ligases (Zawadzke, 1991 Biochemistry 30:1673-1682) of E. coli and in the protein VanA were selected in order to synthesize universal primers intended to amplify internal fragments of genes coding for D-Ala-D-Ala ligases or related enzymes. The peptide targets GEDG(S/T) (I/L)QG and NT(I/L)PGFT were translated back as is shown in FIG. IV.1 in order to obtain degenerate oligonucleotides V1 and V2. As the peptides 1 and 2 of VanA, DdlA and DdlB are separated by amino acid sequences of similar length, the predicted size for the amplification product was about 640 bp.
Amplification by means of PCR with the DNA of E. coli JM83 and of E. faecium BM4147 made it possible to amplify products corresponding to the expected size which have then been purified and cloned in the bacteriophage M13 mp10 (Norrander et al., 1983, Gene 26:101-106). The sequencing of the insert obtained with E. coli JM83 has shown that the product of PCR was an internal fragment of ddlA. A probe generated starting from a recombinant phage obtained with the amplification fragment of BM4147 was used for the Southern blot analysis of a DNA of BM4147 and BM4147-1 which is a derivative of BM4147 sensitive to vancomycin and which lacks the plasmid pIP816 (Leclercq et al., 1988, N. Engl. J. Med. 319:157-161). The probe hybridized with the EcoRI DNA fragment of 4 kb from BM4147 but not with the DNA from E. faecium BM4147-1. As the vanA gene is borne by the EcoRI fragment of 4 kb from pIP816, these results indicate that the primers also make possible the amplification of a part of vanA. Thus the oligonucleotides V1 and V2 may amplify fragments of genes coding for different proteins related to the D-Ala-D-Ala ligases, and may do this in different species.
Amplification, Cloning and Sequencing of the vanC Gene
Amplification by means of PCR was carried out on the total DNA of E. gallinarum BM4174 and the amplification product obtained of about 640 bp was cloned in the bacteriophage M13 mp10. The single-stranded DNA isolated from the recombinant phage was used to construct a probe C (Hu et al., 1982, Gene 17:2171-2177). In Southern analysis the probe hybridized with a PstI fragment of 1.7 kb from BM4174 but not with the DNA of BM4147 and BM4147-1.
The DNA of BM4174 was digested with PstI and fragments of 1.5 and 2 kb were purified by electrophoresis on agarose gel and cloned in pUC18 (Norrander et al., 1983, mentioned previously). The recombination plasmids were introduced into E. coli JM83 by transformation and screens by hybridization on colonies (Sambrook et al., 1989, Molecular cloning a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.) by using the probe C. A homology was detected with a transformant harbouring a plasmid called pAT216 which contained a PstI insert of 1.7 kb. The sequence of the SacI-PstI part of 1347 bp of the insert of pAT216 was determined on both strands of the DNA. The location of the termination codons in the three reading frames of each strand of DNA revealed the presence of an ORF phase located between the TGA codons at positions 47 and 1244. The initiation codon of transcription ATG at position 215 is preceded by a sequence GAAAGGAAGA characteristic of the RBS sequences complementary to the RNA of the 16S subunit of B. subtilis (Moran et al., 1982, Mol. Gen. Genet. 186:339-346). The sequence of 1029 bp which extends from the ATG codon at position 215 to the TGA codon at position 1244 might code for a protein of 343 amino acids having a calculated molecular mass of 37504 Da designated as VanC. A sequence homology was detected between VanC, VanA and the D-Ala-D-Ala ligases of E. coli. In particular, four domains of strong homology previously found between VanA and the D-Ala-D-Ala ligases of the enterobacteria are also present in VanC. The percentage of identical amino acids calculated for these proteins taken two at a time varied between 29 and 38%. The alignment of the four sequences revealed the presence of 57 invariant amino acids which include the conserved residues of the peptides 1 and 2 used to define the oligonucleotide probes V1 and V2.
Inactivation of the vanC Gene by Insertion
In order to evaluate the contribution of vanC to resistance to vancomycin in E. gallinarum BM4174, the vanC gene was inactivated by insertion. A EcoRI-HincII fragment of 690 bp, internal to vanC was cloned in pAT114 which does not replicate in Gram-positive bacteria. The resulting pAT217 plasmid was introduced into BM4174 by electroporation (Cruz-Rodz et al., 1990, Mol. Gen. Genet. 224:152-154) and the clones supposed to result from a homologous recombination leading to the integration of pAT217 into vanC were selected on erythromycin. The clone BM4175 was compared with BM4174 by Southern hybridization using the probe C and aphA-3 specific for pAT114. The two probes hybridized with the EcoRI fragment of 8.6 kb from BM4175. The probe C hybridized with a fragment of 2.5 kb from BM4174 whereas no signal was observed with the probe aphA-3. The results indicate that the plasmid pAT217 of 6.1 kb was integrated into the vanC gene. The determination of the minimal inhibitory concentration of vancomycin for BM4174 (16 mg/l) and BM4175 (2 mg/l) indicated that the inactivation by insertion in vanC abolishes resistance to vancomycin.
VanC is thus required for resistance to vancomycin. It may thus be supposed that this protein synthesizes a dipeptide or a depsipeptide which is incorporated into the precursors of peptido-glycans and is not recognized by vancomycin.
The sequences which are the object of the invention are given in the following pages after the list of the sequences containing the description of these sequences. In this list of the sequences, the proteins are identified with respect to the position of the nucleotide bases corresponding to the amino acids of the extremities of the proteins.

List of the Sequences

(contained in the sequences I (Ia, Ib), II presented below or in the sequence shown in FIG. 5).
Amino Acid Sequences
SEQ ID NO 1 (VanH): sequence of the first resistance protein, corresponding to the amino acid sequence of the open reading frame No. 3, starting at the base 3501 and terminating at the base 4529, containing the sequence coding for the vanH gene between the bases 3564 and 4529 with respect to the sequence shown in FIG. 5 or corresponding to the sequence between the positions of the nucleotides 6018 and 6983 of the sequence Ia.
SEQ ID NO 2 (VanA): sequence of the VanA protein, corresponding to the amino acid sequence of the open reading frame No. 1, starting at the base 4429 and terminating at the base 5553 with respect to the sequence shown in FIG. 5 or corresponding to the sequence between the positions of the nucleotides 6977 and 7807 of the sequence Ia.
SEQ ID NO 3 (VanX) sequence of the third resistance protein, corresponding to the amino acid sequence of the open reading frame No. 3, starting at the base 5526 and terminating at the base 6167 with respect to the sequence shown in FIG. 5 or corresponding to the sequence between the positions of the nucleotides 7816 and 8621 of the sequence Ia.
SEQ ID NO 4 (VanR): sequence of the regulatory protein R, corresponding to the amino acid sequence of the open reading frame No. 1, starting at the base 1477 and terminating at the base 2214 with respect to the sequence shown in FIG. 5 or corresponding to the sequence between the positions of the nucleotides 3976 and 4668 of the sequence Ia.
SEQ ID NO 5 (VanS): sequence of the sensor protein S, corresponding to the amino acid sequence of the open reading frame No. 2, starting at the base 2180 and terminating at the base 3346 with respect to the sequence shown in FIG. 5 or corresponding to the sequence between the positions of the nucleotides 4648 and 5800 of the sequence Ia.
SEQ ID NO 16: sequence of the transposase corresponding to the amino acids included between the nucleotides 150 and 3112 of the sequence Ib.
SEQ ID NO 17: sequence of the resolvase comprising the amino acids situated between the positions of the nucleotides 3187 and 3759 of the sequence Ia.
SEQ ID NO 18: VanY sequence comprising the amino acids situated between the positions of the nucleotides 9046 and 9960 of the sequence Ia.
SEQ ID NO 19: VanZ sequence comprising the amino acids situated between the positions of the nucleotides 10116 and 10598 of the sequence Ia.
SEQ ID NO 20: VanC amino acid sequence shown in list II.
Nucleotide Sequences
SEQ ID NO 6: nucleotide sequence containing the sequence coding for the 5 proteins as well as the flanking sequences, shown in FIG. 5.
SEQ ID NO 7: sequence containing the sequence coding for the 3 resistance proteins as well as the flanking sequences and starting at the base 3501 and terminating at the base 6167, shown in FIG. 5.
SEQ ID NO 8: sequence of the vanA gene, starting at the base 4429 and terminating at the base 5553 of the sequence shown in FIG. 5, or corresponding to the nucleotide sequence situated between the nucleotides 6977 and 7807 of the sequence Ia.
SEQ ID NO 9: sequence coding for the first resistance protein called VanH, starting at the base 3501 and terminating at the base 4529, in particular the sequence vanH, the coding sequence of which is located between the bases 3564 and 4529 of the sequence shown in FIG. 5, or corresponding to the nucleotide sequence situated between the nucleotides 6018 and 6983 of the sequence Ia.
SEQ ID NO 10 sequence coding for the third resistance protein VanX, starting at the base 5526 and terminating at the base 6167 of the sequence shown in FIG. 5, or corresponding to the nucleotide sequence situated between the nucleotides 7816 and 8621 of the sequence Ia.
SEQ ID NO 11: sequence of the transposon coding for the transposase, the resolvase, vanR, VAnS, VanH, VanA, VanX, VanY and VanZ and containing the repeated reverse sequence of 38 bp at its N- and C-termini and corresponding to the sequence Ia.
SEQ ID NO 12: sequence coding for the transposase, starting at the base 150 and terminating at the base 3112 of the sequence Ib.
SEQ ID NO 13: sequence coding for the resolvase, starting at the base 3187 and terminating at the base 3759 of the sequence Ia.
SEQ ID NO 14 sequence coding for VanY, starting at the base 9046 and terminating at the base 9960 of the sequence Ia.
SEQ ID NO 15: sequence coding for VanZ, starting at the base 10116 and terminating at the base 10598 of the sequence Ia.
SEQ ID NO 21: sequence coding for VanC, shown in the list II in relation to the protein VanC.
SEQ ID NO 22 complete sequence Ia of the transposon of E. faecium, starting at the base 1 and terminating at the base 10851.
SEQ ID NO 23 sequence coding for the protein VanR, starting at the base 3976 and terminating at the base 4668 of the sequence Ia.

SEQ ID NO 24: sequence coding for the protein VanS, starting at the base 4648 and terminating at the base 5800 of the sequence Ia.

1

GGG

GTA

GCG

TCA

GGA

AAA

TGC

GGA

TTT

ACA

ACG

CTA

AGC

CTA

TTT

TCC

TGA

CGA

ATC

CCT

61

CGT

TTT

TAA

CAA

CGT

TAA

GAA

AGT

TTT

AGT

GGT

CTT

AAA

GAA

TTT

AAT

GAG

ACT

TTC

121

TCT

GAG

TTA

AAA

TGG

TAT

TCT

CCT

AGT

AAA

TTA

ATA

TGT

TCC

CAA

CCT

AAG

GGC

GAC

ATA

181

TGG

TGT

AAC

AAA

TCT

TCA

TTA

AAG

CTA

CCT

GTC

CGT

TTT

TTA

TAT

TCA

ACT

GCT

GTT

241

AGG

TGG

AGA

GTA

TTC

CAA

ATA

CTT

ATA

GCA

TTG

ATA

ATT

ATG

TTT

AAA

GCA

CTG

GCT

CTT

301

TGC

AAT

TGA

TGC

TGT

ATG

GTG

CGT

TCT

CTA

AGC

TCA

CCT

TGT

TTT

CCG

AAG

AAA

ATA

GCT

361

CTT

GCC

AAT

CCA

TTC

ATG

GCT

TCT

CCT

TTA

TTC

AAT

CCT

CTT

TGT

ATT

TTT

CTT

AAT

421

GAT

TCA

TCC

GAT

ATA

TAA

TTC

AAA

ATA

AAG

ATC

GTT

TTT

TCT

ATT

CGG

CCC

ATC

TCA

CGT

481

AAG

GCT

GTA

GCT

AAG

CTG

TTT

TGT

CTT

GAA

TAG

GAA

CCT

AGC

TTC

CCC

ATA

AGG

GAT

541

GCT

GAA

ACT

GTT

CCC

TCC

CTT

ATA

GAA

TGA

GCT

AAT

CGC

AAA

ACA

TCC

TCA

TAA

TTT

TCT

601

TTA

ATG

ACC

TTT

GTA

TTT

ATT

TGT

CCA

CGT

AAA

ATG

GCT

TCT

AGT

TTT

GGA

TAC

TCA

CTT

661

GCT

TTA

TCT

ATC

GTA

AAT

TTT

GAG

TCC

GAT

AAA

TCC

CTT

ATT

CTT

GGG

GCA

AAT

TTA

721

AAT

CCT

AAT

AAA

TGA

GTC

AGT

CCG

AAT

ATT

TGG

TCA

GTG

TAA

CCG

GCA

GTG

TCT

GTA

TAA

781

TGT

TCC

TCT

ATG

TTT

AGA

TCC

GTC

TCA

TGA

TGT

AAC

AAA

CCA

TCC

AAA

ACA

TGA

ATC

GCA

841

TCT

CTT

GAA

TTA

GTA

TGA

ATA

ATC

TTT

GTG

TAG

TAA

GAA

GAG

AAT

TGA

TCA

CTT

GTA

AAT

901

CGG

TAG

ATG

GTG

GCT

CCT

TTT

CCA

GTT

CCA

TAA

TGT

GGA

TTT

GCA

TCT

GCA

TGT

AGT

GAT

961

GAA

ACA

CCT

AGC

TGC

ATT

CTC

ATA

CCA

TCT

GAC

GAA

GAT

GTT

GTA

CCG

TCG

CCC

CAA

TAG

1021

AAA

GGC

AAT

TGT

AAT

TTA

TGA

AAG

TTT

ACT

AAT

ATG

GCT

TGG

GCT

TTA

TTC

ATG

GCA

1081

TCT

TCA

TAC

ATG

CGC

CAT

TGA

GAT

ACA

TTG

GCT

AGT

TGC

TTA

TAT

GTA

AGT

CCG

GGT

GTG

1141

GCT

TCG

GCC

ATC

TTG

CTC

AAG

CCA

ATA

TTC

ATT

CCC

ATT

CCT

AAA

AGG

GCA

GCC

ATG

ATA

1201

ATG

ATT

GTT

TCT

TCC

TTA

TCT

GGT

TTT

CGA

TTA

TTG

GAA

GCA

TGA

GTG

AAT

TGC

TCA

TGA

1261

AAT

CCT

GTT

ATA

TGG

GCC

ACA

TCC

ATG

AGT

AAA

TCA

GTT

AAT

TTT

ATT

CTT

GGT

AGC

ATC

1321

TGA

TAA

AGG

CTT

GCA

CTA

AAT

TTT

GCT

TCT

GGA

ACA

TCT

TTT

TCT

AAG

CGT

GCA

1381

AGT

GAT

AGC

TTT

CCT

TTT

TCA

AGA

GAA

ACC

CCA

TCT

AAC

TTA

TTG

GAA

TTG

GCA

GCT

AAC

1441

CAC

TTT

AAC

CTT

TCA

TTA

AAG

CTG

GTT

CTC

TCC

GTT

ATA

TAA

TCT

TCG

AAT

GAT

AAA

1501

CTA

ACT

GAT

AAT

CTC

GTA

TTC

CCC

TTC

GAT

TGA

TTC

CAT

GTA

TCT

TCC

GAA

AAC

AAA

TAT

1561

TCC

TCA

AAA

TCC

CTA

TAT

TGT

CTG

CCA

ACA

ATG

GAA

ACA

TCT

CCT

GCC

CGA

ACA

TGC

1621

TCC

CGA

AGT

TCT

GTT

AAA

ACA

GCC

ATT

TCA

TAG

TAA

TGA

CGA

TTA

ATT

GTT

GTA

CCA

TCA

1681

TCC

TCG

TAT

AAA

TGT

CTT

TTC

CAT

CGT

TTT

GAA

ATA

AAA

TCC

ACA

GGT

GAG

TCA

GGC

1741

ACT

TTT

CGC

TTT

CCA

GAT

TCG

TTC

ATT

CCT

CGG

ATA

ATC

TCA

ACA

GCT

TGT

AAA

AGT

GGC

1801

TCA

TTT

GCC

TTT

GTA

GAA

TGA

AAT

TCC

AAT

ACT

CTT

AAT

AGC

GTT

GGC

GTA

TAT

TTT

CTT

1861

AGT

GAA

TAA

AAC

CGT

TTT

TGC

AGT

AAG

TCT

AAA

TAA

TCA

TAG

TCG

GCA

GGA

CGT

GCA

AGT

1921

TCC

TGA

GCC

TCT

ACT

GAA

GAG

ACA

AAG

GTA

TTC

CAT

TCA

ATA

ACC

GAT

TCT

AAA

ACC

1981

TTA

AAA

ACG

TCT

AAT

TTT

TCC

TCT

CTT

GCT

TTA

ATT

AAT

GCT

TGT

CCG

ATG

TTC

GTA

AAG

2041

TGT

ATA

ACT

TTC

TCA

TTT

AGC

TTT

TTA

CCG

TTT

TGT

TTC

TGG

ATT

TCC

TCT

TGA

GCC

TTA

2101

CGA

CCT

TTT

GAT

AAC

AAA

CTA

AGT

ATT

TGC

CTA

TCA

TGA

ATT

TCA

AAC

GCT

TTA

TCC

GTT

2161

AGC

TCC

TGA

GTA

AGT

TGT

AAT

AAA

TAG

ATG

GTT

AAT

ATC

GAA

TAA

CGT

TTA

TTT

TCT

TGA

2221

AAG

TCA

CGG

AAT

GCA

TAC

GGC

TCG

TAT

CTT

GAG

CCT

AAG

CGA

GAC

AGC

TGC

AAC

AGG

CGG

2281

TTA

CGG

TGC

AAA

TGA

CTA

ATT

TGC

ACT

GTT

TCT

AAA

TCC

ATT

CCT

CGT

ATG

TAT

TCG

AGT

2341

CGT

TCT

ATT

TTT

AGA

AAA

GTT

TCG

GGT

GAA

GGA

TGA

CCC

GGT

GGC

TCT

TTT

AAC

CAA

2401

CCC

AAT

ATC

GTT

TTA

TTG

GAT

TCG

GAT

GGA

TGC

GAG

GTA

ATA

ATC

CCT

TCA

AGC

TTT

2461

TCT

TTT

TGC

TCA

TTT

GTT

AGA

GAT

TTA

CTA

ACC

GTA

TTA

AAT

AGC

TTC

TTT

TCA

GCC

ATT

2521

GCC

CTT

GCT

TCC

CAC

ACC

ATT

CTT

TCA

AGT

GTA

GTG

ATA

GCA

GGC

AGT

ATA

ATT

TTG

TTT

2581

TTT

CTT

AGA

AAA

TCT

ATG

CAT

TCA

TGC

AGT

AGA

TGA

ATG

GCA

TCA

CCA

TTT

TCC

AAA

GCT

2641

AAT

TGA

AGG

TAC

TTA

AAT

GTC

ATT

CGA

TAT

TCA

CTC

AGG

GTA

AAA

GTT

ACA

AAG

TCG

2701

TAT

TCA

CTT

CGA

ATT

TCT

TTC

AAA

TGA

TCC

CAA

AGT

GTA

TTT

TCC

CTT

TGA

GGA

TAA

TGA

2761

TCA

AGC

GAG

GAT

GGA

CTA

ACA

CCA

ATC

TGT

TTC

GAT

ATA

TAT

TGT

ATG

ACC

GAA

TCT

GGG

2821

ATG

CTT

TTG

ATA

TGA

GTG

TAT

GGC

CAA

CCG

GGA

TAC

CGA

AGA

ACA

GCT

AAT

TGA

ACA

GCA

2881

AAT

CCT

AAA

CGG

TTT

TCT

TCC

CTC

CTT

CGC

TTA

ACT

ATT

TCT

AAA

TCC

CGT

TTG

GAA

2941

AAA

GTG

AAG

TAG

GTC

CCC

AGT

ATC

CAT

TCA

TCT

TCA

GGG

ATT

TGC

ATA

AAA

GCC

TGT

CTC

3001

TGT

TCC

GGT

GTA

AGC

AAT

TCT

CTA

CCT

CTC

GCA

ATT

TTC

ATT

CAG

TAT

CAT

TCC

ATT

TCT

3061

GTA

TTT

TCA

ATT

TAT

TAG

TTC

AAT

TAT

ATA

TCA

ATA

GAG

TGT

ACT

CTA

TTG

ATA

CAA

ATG

3121

TAG

ACT

GAT

AAA

ATC

ATA

GTT

AAG

AGC

GTC

TCA

TAA

GAC

TTG

TCT

CAA

AAA

TGA

GGT

3181

resolvase

LEU

ARG

LYS

ILE

GLY

TYR

ILE

ARG

VAL

SER

THR

ASN

GLN

ASN

PRO

SER

ARG

GAT

ATT

TTG

CGG

AAA

ATC

GGT

TAT

ATT

CGT

GTC

AGT

TCG

ACT

AAC

CAG

AAT

CCT

TCA

AGA

3241

GLN

PHE

GLN

LEU

ASN

GLU

ILE

GLY

MET

ASP

ILE

TYR

GLU

LYS

VAL

SER

GLY

CAA

TTT

CAG

TTG

AAC

GAG

ATC

GGA

ATG

GAT

ATT

ATA

TAT

GAA

GAG

AAA

GTT

TCA

GGA

3301

ALA

THR

LYS

ASP

ARG

GLU

GLN

LEU

GLN

LYS

VAL

LEU

ASP

LEU

GLN

GLU

ASP

ILE

GCA

ACA

AAG

GAT

CGC

GAG

CAA

CTT

CAA

AAA

GTG

TTA

GAC

GAT

TTA

CAG

GAA

GAT

GAC

ATC

3361

ILE

TYR

VAL

THR

ASP

LEU

THR

ARG

ILE

THR

ARG

SER

THR

GLN

ASP

LEU

PHE

GLU

LEU

ILE

ATT

TAT

GTT

ACA

GAC

TTA

ACT

CGA

ATC

ACT

CGT

AGT

ACA

CAA

GAT

CTA

TTT

GAA

TTA

ATC

3421

ASP

ASN

ILE

ARG

ASP

LYS

ALA

SER

LEU

LYS

SER

LEU

LYS

ASP

THR

TRP

LEU

ASP

LEU

GAT

AAC

ATA

CGA

GAT

AAA

AAG

GCA

AGT

TTA

AAA

TCA

CTA

AAA

GAT

ACA

TGG

CTT

GAT

TTA

3481

SER

GLU

ASP

ASN

PRO

TYR

SER

GLN

PHE

LEU

ILE

THR

VAL

MET

ALA

GLY

VAL

ASN

GLN

LEU

TCA

GAA

GAT

AAT

CCA

TAC

AGC

CAA

TTC

TTA

ATT

ACT

GTA

ATG

GCT

GGT

GTT

AAC

CAA

TTA

3541

GLU

ARG

ASP

LEU

ILE

ARG

MET

ARG

GLN

ARG

GLU

GLY

ILE

GLU

LEU

ALA

LYS

GLU

GLY

GAG

CGA

GAT

CTT

ATT

CGG

ATG

AGA

CAA

CGT

GAA

GGG

ATT

GAA

TTG

GCT

AAG

AAA

GAA

GGA

3601

LYS

PHE

LYS

GLY

ARG

LEU

LYS

TYR

HIS

LYS

ASN

HIS

ALA

GLY

MET

ASN

TYR

ALA

VAL

AAG

TTT

AAA

GGT

CGA

TTA

AAG

TAT

CAT

AAA

AAT

CAC

GCA

GGA

ATG

AAT

TAT

GCG

GTA

3661

LYS

LEU

TYR

LYS

GLU

GLY

ASN

MET

THR

VAL

ASN

GLN

ILE

CYS

GLU

ILE

THR

ASN

VAL

SER

AAG

CTA

TAT

AAA

GAA

GGA

AAT

ATG

ACT

GTA

AAT

CAA

ATT

TGT

GAA

ATT

ACT

AAT

GTA

TCT

3721

ARG

ALA

SER

LEU

TYR

ARG

LYS

LEU

SER

GLU

VAL

ASN

AGG

GCT

TCA

TTA

TAC

AGG

AAA

TTA

TCA

GAA

GTG

AAT

TAG

CCA

TTC

TGT

ATT

CCC

CTA

3781

ATG

GGC

AAT

ATT

TTT

AAA

GAA

AAG

GAA

ACT

ATA

AAA

TAT

TAA

CAG

CCT

AGC

GAT

3841

GCC

GAA

AAG

CCC

TTT

GAT

AAA

AGA

ATC

TTA

AGA

AAT

TCT

TAG

TCA

TTT

ATT

3901

ATG

TAA

ATG

CTT

ATA

AAT

TCG

GCC

CTA

TAA

TCT

GAT

AAA

TTA

AGG

GCA

AAC

TTA

TGT

3961

VanR

MET

SER

ASP

LYS

ILE

LEU

ILE

VAL

ASP

GLU

HIS

GLU

ILE

ALA

GAA

AGG

GTG

ATA

ACT

ATG

AGC

GAT

AAA

ATA

CTT

ATT

GTG

GAT

GAA

CAT

GAA

ATT

GCC

4021

ASP

LEU

VAL

GLU

LEU

TYR

LEU

LYS

ASN

GLU

ASN

TYR

THR

VAL

PHE

LYS

TYR

THR

ALA

GAT

TTG

GTT

GAA

TTA

TAC

TTA

AAA

AAC

GAG

AAT

TAT

ACG

GTT

TTC

AAA

TAC

TAT

ACC

GCC

4081

LYS

GLU

ALA

LEU

GLU

CYS

ILE

ASP

LYS

SER

GLU

ILE

ASP

LEU

ALA

ILE

LEU

ASP

ILE

MET

AAA

GAA

GCA

TTG

GAA

TGT

ATA

GAC

AAG

TCT

GAG

ATT

GAC

CTT

GCC

ATA

TTG

GAC

ATC

ATG

4141

LEU

PRO

GLY

THR

SER

GLY

LEU

THR

ILE

CYS

GLN

LYS

ILE

ARG

ASP

LYS

HIS

THR

TYR

PRO

CTT

CCC

GGC

ACA

AGC

GGC

CTT

ACT

ATC

TGT

CAA

AAA

ATA

AGG

GAC

AAG

CAC

ACC

TAT

CCG

4201

ILE

MET

LEU

THR

GLY

LYS

ASP

THR

GLU

VAL

ASP

LYS

ILE

THR

GLY

LEU

THR

ILE

GLY

ATT

ATC

ATG

CTG

ACC

GGG

AAA

GAT

ACA

GAG

GTA

GAT

AAA

ATT

ACA

GGG

TTA

ACA

ATC

GGC

4261

ALA

ASP

TYR

ILE

THR

LYS

PRO

PHE

ARG

PRO

LEU

GLU

LEU

ILE

ALA

ARG

VAL

LYS

ALA

GCG

GAT

TAT

ATA

ACG

AAG

CCC

TTT

CGC

CCA

CTG

GAG

TTA

ATT

GCT

CGG

GTA

AAG

GCC

4321

GLN

LEU

ARG

TYR

LYS

PHE

SER

GLY

VAL

LYS

GLU

GLN

ASN

GLU

ASN

VAL

ILE

VAL

CAG

TTG

CGC

CGA

TAC

AAA

TTC

AGT

GGA

GTA

AAG

GAG

CAG

AAC

GAA

AAT

GTT

ATC

GTC

4381

HIS

SER

GLY

LEU

VAL

ILE

ASN

VAL

ASN

THR

HIS

GLU

CYS

TYR

LEU

ASN

GLU

LYS

GLN

LEU

CAC

TCC

GGC

CTT

GTC

ATT

AAT

GTT

AAC

ACC

CAT

GAG

TGT

TAT

CTG

AAC

GAG

AAG

CAG

TTA

4441

SER

LEU

THR

PRO

THR

GLU

PHE

SER

ILE

LEU

ARG

ILE

LEU

CYS

GLU

ASN

LYS

GLY

ASN

VAL

TCC

CTT

ACT

CCC

ACC

GAG

TTT

TCA

ATA

CTG

CGA

ATC

CTC

TGT

GAA

AAC

AAG

GGG

AAT

GTG

4501

VAL

SER

GLU

LEU

PHE

HIS

GLU

ILE

TRP

GLY

ASP

GLU

TYR

PHE

SER

LYS

SER

ASN

GTT

AGC

TCC

GAG

CTG

CTA

TTT

CAT

GAG

ATA

TGG

GGC

GAC

GAA

TAT

TTC

AGC

AAG

AGC

AAC

4561

ASN

THR

ILE

THR

VAL

HIS

ILE

ARG

HIS

LEU

ARG

GLU

LYS

MET

ASN

ASP

THR

ILE

ASP

ASN

AAC

ACC

ATC

ACC

GTG

CAT

ATC

CGG

CAT

TTG

CGC

GAA

AAA

ATG

AAC

GAC

ACC

ATT

GAT

AAT

4621

PRO

LYS

TYR

ILE

LYS

THR

VAL

TRP

GLY

VALGLYTYRLYSILEGLULYS

CCG

AAA

TAT

ATA

AAA

ACG

GTA

TGG

GGG

GTTGGTTATAAAATTGAAAAAT

AAA

AAC

GAC

VanS

LEUVALILELYSLEULYSASN

LYS

ASN

ASP

4682

TYR

SER

LYS

LEU

GLU

ARG

LYS

LEU

TYR

MET

TYR

ILE

VAL

ALA

ILE

VAL

ALA

ILE

TAT

TCC

AAA

CTA

GAA

CGA

AAA

CTT

TAC

ATG

TAT

ATC

GTT

GCA

ATT

GTT

GTG

GTA

GCA

ATT

4742

VAL

PHE

VAL

LEU

TYR

ILE

ARG

SER

MET

ILE

ARG

GLY

LYS

LEU

GLY

ASP

TRP

ILE

LEU

SER

GTA

TTC

GTG

TTC

TAT

ATT

CGT

TCA

ATG

ATC

CGA

GGG

AAA

CTT

GGG

GAT

TGG

ATC

TTA

AGT

4802

ILE

LEU

GLU

ASN

LYS

TYR

ASP

LEU

ASN

HIS

LEU

ASP

ALA

MET

LYS

LEU

TYR

GLN

TYR

SER

ATT

TTG

GAA

AAC

AAA

TAT

GAC

TTA

AAT

CAC

CTG

GAC

GCG

ATG

AAA

TTA

TAT

CAA

TAT

TCC

4862

ILE

ARG

ASN

ILE

ASP

ILE

PHE

ILE

TYR

VAL

ALA

ILE

VAL

ILE

SER

ILE

LEU

ILE

LEU

ATA

CGG

AAC

AAT

ATA

GAT

ATC

TTT

ATT

TAT

GTG

GCG

ATT

GTC

ATT

AGT

ATT

CTT

ATT

CTA

4922

CYS

ARG

VAL

MET

LEU

SER

LYS

PHE

ALA

LYS

TYR

PHE

ASP

GLU

ILE

ASN

THR

GLY

ILE

ASP

TGT

CGC

GTC

ATG

CTT

TCA

AAA

TTC

GCA

AAA

TAC

TTT

GAC

GAG

ATA

AAT

ACC

GGC

ATT

GAT

4982

VAL

LEU

ILE

GLN

ASN

GLU

ASP

LYS

GLN

ILE

GLU

LEU

SER

ALA

GLU

MET

ASP

VAL

MET

GLU

GTA

CTT

ATT

CAG

AAC

GAA

GAT

AAA

CAA

ATT

GAG

CTT

TCT

GCG

GAA

ATG

GAT

GTT

ATG

GAA

5042

GLN

LYS

LEU

ASN

THR

LEU

LYS

ARG

THR

LEU

GLU

LYS

ARG

GLU

GLN

ASP

ALA

LYS

LEU

ALA

CAA

AAG

CTC

AAC

ACA

TTA

AAA

CGG

ACT

CTG

GAA

AAG

CGA

GAG

CAG

GAT

GCA

AAG

CTG

GCC

5102

GLU

GLN

ARG

LYS

ASN

ASP

VAL

MET

TYR

LEU

ALA

HIS

ASP

ILE

LYS

THR

PRO

LEU

THR

GAA

CAA

AGA

AAA

AAT

GAC

GTT

ATG

TAC

TTG

GCG

CAC

GAT

ATT

AAA

ACG

CCC

CTT

ACA

5162

SER

ILE

GLY

TYR

LEU

SER

LEU

ASP

GLU

ALA

PRO

ASP

MET

PRO

VAL

ASP

GLN

LYS

TCC

ATT

ATC

GGT

TAT

TTG

AGC

CTG

CTT

GAC

GAG

GCT

CCA

GAC

ATG

CCG

GTA

GAT

CAA

AAG

5222

ALA

LYS

TYR

VAL

HIS

ILE

THR

LEU

ASP

LYS

ALA

TYR

ARG

LEU

GLU

GLN

LEU

ILE

ASP

GLU

GCA

AAG

TAT

GTG

CAT

ATC

ACG

TTG

GAC

AAA

GCG

TAT

CGA

CTC

GAA

CAG

CTA

ATC

GAC

GAG

5282

PHE

GLU

ILE

THR

ARG

TYR

ASN

LEU

GLN

THR

ILE

THR

LEU

THR

LYS

THR

HIS

ILE

ASP

TTT

GAG

ATT

ACA

CGG

TAT

AAC

CTA

CAA

ACG

ATA

ACG

CTA

ACA

AAA

ACG

CAC

ATA

GAC

5342

LEU

TYR

MET

LEU

VAL

GLN

MET

THR

ASP

GLU

PHE

TYR

PRO

GLN

LEU

SER

ALA

HIS

GLY

CTA

TAC

TAT

ATG

CTG

GTG

CAG

ATG

ACC

GAT

GAA

TTT

TAT

CCT

CAG

CTT

TCC

GCA

CAT

GGA

5402

LYS

GLN

ALA

VAL

ILE

HIS

ALA

PRO

GLU

ASP

LEU

THR

VAL

SER

GLY

ASP

PRO

ASP

LYS

LEU

AAA

CAG

GCG

GTT

ATT

CAC

GCC

CCC

GAG

GAT

CTG

ACC

GTG

TCC

GGC

GAC

CCT

GAT

AAA

CTC

5462

ALA

ARG

VAL

PHE

ASN

ILE

LEU

LYS

ASN

ALA

TYR

SER

GLU

ASP

ASN

SER

ILE

GCG

AGA

GTC

TTT

AAC

ATT

TTG

AAA

AAC

GCC

GCT

GCA

TAC

AGT

GAG

GAT

AAC

AGC

ATC

5522

ILE

ASP

ILE

THR

ALA

GLY

LEU

SER

GLY

ASP

VAL

SER

ILE

GLU

PHE

LYS

ASP

THR

GLY

ATT

GAC

ATT

ACC

GCG

GGC

CTC

TCC

GGG

GAT

GTG

TCA

ATC

GAA

TTC

AAG

AAC

ACT

GGA

5582

SER

ILE

PRO

LYS

ASP

LYS

LEU

ALA

ILE

PHE

GLU

LYS

PHE

TYR

ARG

LEU

ASP

ASN

ALA

AGC

ATC

CCA

AAA

GAT

AAG

CTA

GCT

GCC

ATA

TTT

GAA

AAG

TTC

TAT

AGG

CTG

GAC

AAT

GCT

5642

ARG

SER

ASP

THR

GLY

ALA

GLY

LEU

GLY

LEU

ALA

ILE

ALA

LYS

GLU

ILE

VAL

CGT

TCT

TCC

GAT

ACG

GGT

GGC

GCG

GGA

CTT

GGA

TTG

GCG

ATT

GCA

AAA

GAA

ATT

GTT

5702

GLN

HIS

GLY

GLN

ILE

TYR

ALA

GLU

SER

ASN

ASP

ASN

TYR

THR

PHE

ARG

VAL

GLU

CAG

CAT

GGA

GGG

CAG

ATT

TAC

GCG

GAA

AGC

AAT

GAT

AAC

TAT

ACG

TTT

AGG

GTA

GAG

5762

LEU

PRO

ALA

MET

PRO

ASP

LEU

VAL

ASP

LYS

ARG

SER

CTT

CCA

GCG

ATG

CCA

GAC

TTG

GTT

GAT

AAA

AGG

TCC

TAA

GA

GAT

GTA

TAT

AAT

TTT

5821

TTA

GGA

AAA

TCT

CAA

GGT

TAT

CTT

TAC

TTT

TTC

TTA

GGA

AAT

TAA

CAA

TTT

AAT

ATT

AAG

5881

AAA

CGG

CTC

GTT

CTT

ACA

CGG

TAG

ACT

TAA

TAC

CGT

AAG

AAC

GAG

CCG

TTT

TCG

TTC

5941

AGA

GAA

AGA

TTT

GAC

AAG

ATT

ACC

ATT

GGC

ATC

CCC

GTT

TTA

TTT

GGT

GCC

TTT

CAC

AGA

6001

VanH

MET

ASN

ILE

GLY

ILE

THR

VAL

TYR

GLY

CYS

GLU

GLN

ASP

GLU

AAGGGTTGG

TCT

TAA

TT

ATG

AAT

AAC

ATC

GGC

ATT

ACT

GTT

TAT

GGA

TGT

GAG

CAG

GAT

GAG

6063

ALA

ASP

ALA

PHE

HIS

ALA

LEU

SER

PRO

ARG

PHE

GLY

VAL

MET

ALA

THR

ILE

ASN

ALA

GCA

GAT

GCA

TTC

CAT

GCT

CTT

TCG

CCT

CGC

TTT

GGC

GTT

ATG

GCA

ACG

ATA

ATT

AAC

GCC

6123

ASN

VAL

SER

GLU

SER

ASN

ALA

LYS

SER

ALA

PRO

PHE

ASN

GLN

CYS

ILE

SER

VAL

GLY

HIS

AAC

GTG

TCG

GAA

TCC

AAC

GCC

AAA

TCC

GCG

CCT

TTC

AAT

CAA

TGT

ATC

AGT

GTG

GGA

CAT

6183

LYS

SER

GLU

ILE

SER

ALA

SER

ILE

LEU

ALA

LEU

LYS

ARG

ALA

GLY

VAL

LYS

TYR

ILE

AAA

TCA

GAG

ATT

TCC

GCC

TCT

ATT

CTT

GCG

CTG

AAG

AGA

GCC

GGT

GTG

AAA

TAT

ATT

6243

SER

THR

ARG

SER

ILE

GLY

CYS

ASN

HIS

ILE

ASP

THR

ALA

LYS

ARG

MET

GLY

ILE

TCT

ACC

CGA

AGC

ATC

GGC

TGC

AAT

CAT

ATA

GAT

ACA

ACT

GCT

AAG

AGA

ATG

GGC

ATC

6303

THR

VAL

ASP

ASN

VAL

ALA

TYR

SER

PRO

ASP

SER

VAL

ALA

ASP

TYR

THR

MET

LEU

ILE

ACT

GTC

GAC

AAT

GTG

GCG

TAC

TCG

CCG

GAT

AGC

GTT

GCC

GAT

TAT

ACT

ATG

CTA

ATT

6363

LEU

MET

ALA

VAL

ARG

ASN

VAL

LYS

SER

ILE

VAL

ARG

SER

VAL

GLU

LYS

HIS

ASP

PHE

ARG

CTT

ATG

GCA

GTA

CGC

AAC

GTA

AAA

TCG

ATT

GTG

CGC

TCT

GTG

GAA

AAA

CAT

GAT

TTC

AGG

6423

LEU

ASP

SER

ASP

ARG

GLY

LYS

VAL

LEU

SER

ASP

MET

THR

VAL

GLY

VAL

GLY

THR

GLY

TTG

GAC

AGC

GAC

CGT

GGC

AAG

GTA

CTC

AGC

GAC

ATG

ACA

GTT

GGT

GTG

GGA

ACG

GGC

6483

GLN

ILE

GLY

LYS

ALA

VAL

ILE

GLU

ARG

LEU

ARG

GLY

PHE

GLY

CYS

LYS

VAL

LEU

ALA

TYR

CAG

ATA

GGC

AAA

GCG

GTT

ATT

GAG

CGG

CTG

CGA

GGA

TTT

GGA

TGT

AAA

GTG

TTG

GCT

TAT

6543

SER

ARG

SER

ARG

SER

ILE

GLU

VAL

ASN

TYR

VAL

PRO

PHE

ASP

GLU

LEU

GLN

ASN

SER

AGT

CGC

AGC

CGA

AGT

ATA

GAG

GTA

AAC

TAT

GTA

CCG

TTT

GAT

GAG

TTG

CTG

CAA

AAT

AGC

6603

ASP

ILE

VAL

THR

LEU

HIS

VAL

PRO

LEU

ASN

THR

ASP

THR

HIS

TYR

ILE

SER

HIS

GLU

GAT

ATC

GTT

ACG

CTT

CAT

GTG

CCG

CTC

AAT

ACG

GAT

ACG

CAC

TAT

ATT

ATC

AGC

CAC

GAA

6663

GLN

ILE

GLN

ARG

MET

LYS

GLN

GLY

ALA

PHE

LEU

ILE

ASN

THR

GLY

ARG

GLY

PRO

LEU

VAL

CAA

ATA

CAG

AGA

ATG

AAG

CAA

GGA

GCA

TTT

CTT

ATC

AAT

ACT

GGG

CGC

GGT

CCA

CTT

GTA

6723

ASP

THR

TYR

GLU

LEU

VAL

LYS

ALA

LEU

GLU

ASN

GLY

LYS

LEU

GLY

ALA

LEU

ASP

GAT

ACC

TAT

GAG

TTG

GTT

AAA

GCA

TTA

GAA

AAC

GGG

AAA

CTG

GGC

GGT

GCC

GCA

TTG

GAT

6783

VAL

LEU

GLU

GLY

GLU

PHE

TYR

SER

ASP

CYS

THR

GLN

LYS

PRO

ILE

ASP

ASN

GTA

TTG

GAA

GGA

GAG

GAA

GAG

TTT

TTC

TAC

TCT

GAT

TGC

ACC

CAA

AAA

CCA

ATT

GAT

AAT

6843

GLN

PHE

LEU

LYS

LEU

GLN

ARG

MET

PRO

ASN

VAL

ILE

THR

PRO

HIS

THR

ALA

TYR

CAA

TTT

TTA

CTT

AAA

CTT

CAA

AGA

ATG

CCT

AAC

GTG

ATA

ATC

ACA

CCG

CAT

ACG

GCC

TAT

6903

TYR

THR

GLU

GLN

ALA

LEU

ARG

ASP

THR

VAL

GLU

LYS

THR

ILE

LYS

ASN

CYS

LEU

ASP

PHE

TAT

ACC

GAG

CAA

GCG

TTG

CGT

GAT

ACC

GTT

GAA

AAA

ACC

ATT

AAA

AAC

TGT

TTG

GAT

TTT

6963

VanA

METASN

ARG

ILE

LYS

VAL

ALA

ILE

LEU

PHE

GLY

CYS

SER

GAA

AGG

AGA

CAG

GAG

CATGAAT

AGA

ATA

AAA

GTT

GCA

ATA

CTG

TTT

GGG

GGT

TGC

TCA

GLU	ARG	ARG	GLN	GLU	HISGLU

7021

GLU

HIS

ASP

VAL

SER

VAL

LYS

SER

ALA

ILE

GLU

ILE

ALA

ASN

ILE

ASN

LYS

GLU

GAG

CAT

GAC

GTA

TCG

GTA

AAA

TCT

GCA

ATA

GAG

ATA

GCC

GCT

AAC

ATT

AAT

AAA

GAA

7081

LYS

TYR

GLU

PRO

LEU

TYR

ILE

GLY

ILE

THR

LYS

SER

GLY

VAL

TRP

LYS

MET

CYS

GLU

LYS

AAA

TAC

GAG

CCG

TTA

TAC

ATT

GGA

ATT

ACG

AAA

TCT

GGT

GTA

TGG

AAA

ATG

TGC

GAA

AAA

7141

PRO

CYS

ALA

GLU

TRP

GLU

ASN

ASP

ASN

CYS

TYR

SER

ALA

VAL

LEU

SER

PRO

ASP

LYS

CCT

TGC

GCG

GAA

TGG

GAA

AAC

GAC

AAT

TGC

TAT

TCA

GCT

GTA

CTC

TCG

CCG

GAT

AAA

7201

MET

HIS

GLY

LEU

VAL

LYS

ASN

HIS

GLU

TYR

GLU

ILE

ASN

HIS

VAL

ASP

VAL

ALA

ATG

CAC

GGA

TTA

CTT

GTT

AAA

hAG

AAC

CAT

GAA

TAT

GAA

ATC

AAC

CAT

GTT

GAT

GTA

GCA

7261

PHE

SER

ALA

LEU

HIS

GLY

LYS

SER

GLY

GLU

ASP

GLY

SER

ILE

GLN

GLY

LEU

PHE

GLU

LEU

TTT

TCA

GCT

TTG

CAT

GGC

AAG

TCA

GGT

GAA

GAT

GGA

TCC

ATA

CAA

GGT

CTG

TTT

GAA

TTG

7321

SER

GLY

ILE

PRO

PHE

VAL

GLY

CYS

ASP

ILE

GLN

SER

ALA

ILE

CYS

MET

ASP

LYS

SER

TCC

GGT

ATC

CCT

TTT

GTA

GGC

TGC

GAT

ATT

CAA

AGC

TCA

GCA

ATT

TGT

ATG

GAC

AAA

TCG

7381

LEU

THR

TYR

ILE

VAL

ALA

LYS

ASN

ALA

GLY

ILE

ALA

THR

PRO

ALA

PHE

TRP

VAL

ILE

ASN

TTG

ACA

TAC

ATC

GTT

GCG

AAA

AAT

GCT

GGG

ATA

GCT

ACT

CCC

GCC

TTT

TGG

GTT

ATT

AAT

7441

LYS

ASP

ARG

PRO

VAL

ALA

THR

PHE

THR

TYR

PRO

VAL

PHE

VAL

LYS

PRO

ALA

ARG

AAA

GAT

AGG

CCG

GTG

GCA

GCT

ACG

TTT

ACC

TAT

CCT

GTT

TTT

GTT

AAG

CCG

GCG

CGT

7501

SER

GLY

SER

PHE

GLY

VAL

LYS

VAL

ASN

SER

ALA

ASP

GLU

LEU

ASP

TYR

ALA

ILE

TCA

GGC

TCA

TCC

TTC

GGT

GTG

AAA

GTC

AAT

AGC

GCG

GAC

GAA

TTG

GAC

TAC

GCA

ATT

7561

GLU

SER

ALA

ARG

GLN

TYR

ASP

SER

LYS

ILE

LEU

ILE

GLU

GLN

ALA

VAL

SER

GLY

CYS

GLU

GAA

TCG

GCA

AGA

CAA

TAT

GAC

AGC

AAA

ATC

TTA

ATT

GAG

CAG

GCT

GTT

TCG

GGC

TGT

GAG

7621

VAL

GLY

CYS

ALA

VAL

LEU

GLY

ASN

SER

ALA

LEU

VAL

GLY

GLU

VAL

ASP

GLN

ILE

GTC

GGT

TGT

GCG

GTA

TTG

GGA

AAC

AGT

GCC

GCG

TTA

GTT

GGC

GAG

GTG

GAC

CAA

ATC

7681

ARG

LEU

GLN

TYR

GLY

ILE

PHE

ARG

ILE

HIS

GLN

GLU

VAL

GLU

PRO

GLU

LYS

GLY

SER

GLU

AGG

CTG

CAG

TAC

GGA

ATC

TTT

CGT

ATT

CAT

CAG

GAA

GTC

GAG

CCG

GAA

AAA

GGC

TCT

GAA

7741

ASN

ALA

VAL

ILE

THR

VAL

PRO

ALA

ASP

LEU

SER

ALA

GLU

ARG

GLY

ARG

ILE

GLN

GLU

AAC

GCA

GTT

ATA

ACC

GTT

CCC

GCA

GAC

CTT

TCA

GCA

GAG

CGA

GGA

CGG

ATA

CAG

GAA

7801

THR

ALA

LYS

ILE

TYR

LYS

ALA

LEU

GLY

CYS

ARG

GLY

LEU

ALA

ARG

VAL

ASP

MET

PHE

ACG

GCA

AAA

ATA

TAT

AAA

GCG

CTC

GGC

TGT

AGA

GGT

CTA

GCC

CGT

GTG

GAT

ATG

TTT

7861

LEU

GLN

ASP

ASN

GLY

ARG

ILE

VAL

LEU

ASN

GLU

VAL

ASN

THR

LEU

PRO

GLY

PHE

THR

SER

TTA

CAA

GAT

AAC

GGC

CGC

ATT

GTA

CTG

AAC

GAA

GTC

AAT

ACT

CTG

CCC

GGT

TTC

ACG

TCA

7921

TYR

SER

ARG

TYR

PRO

ARG

MET

ALA

GLY

ILE

ALA

LEU

PRO

GLU

LEU

ILE

ASP

TAC

AGT

CGT

TAT

CCC

CGT

ATG

GCC

GCT

GCA

GGT

ATT

GCA

CTT

CCC

GAA

CTT

ATT

GAC

7981

ARG

LEU

ILE

VAL

LEU

ALA

LEU

LYS

GLY

CGC

TTG

ATC

GTA

TTA

GCG

TTA

AAG

GGG

TGATAAGC

ATG

GAA

ATA

GGA

TTT

ACT

TTT

TTA

GAT

	VanX	MET	GLU	ILE	GLY	PHE	THR	PHE	LEU	ASP

8043

GLU

ILE

VAL

HIS

GLY

VAL

ARG

TRP

ASP

ALA

LYS

TYR

ALA

THR

TRP

ASP

ASN

PHE

THR

GLY

GAA

ATA

GTA

CAC

GGT

GTT

CGT

TGG

GAC

GCT

AAA

TAT

GCC

ACT

TGG

GAT

AAT

TTC

ACC

GGA

8103

LYS

PRO

VAL

ASP

GLY

TYR

GLU

VAL

ASN

ARG

ILE

VAL

GLY

THR

TYR

GLU

LEU

ALA

GLU

SER

AAA

CCG

GTT

GAC

GGT

TAT

GAA

GTA

AAT

CGC

ATT

GTA

GGG

ACA

TAC

GAG

TTG

GCT

GAA

TCG

8163

LEU

LYS

ALA

LYS

GLN

LEU

ALA

THR

GLN

GLY

TYR

GLY

LEU

TRP

ASP

GLY

CTT

TTG

AAG

GCA

AAA

GAA

CTG

GCT

ACC

CAA

GGG

TAC

GGA

TTG

CTT

CTA

TGG

GAC

GGT

8223

TYR

ARG

PRO

LYS

ARG

ALA

VAL

ASN

CYS

PHE

MET

GLN

TRP

ALA

GLN

PRO

GLU

ASN

TAC

CGT

CCT

AAG

CGT

GCT

GTA

AAC

TGT

TTT

ATG

CAA

TGG

GCT

GCA

CAG

CCG

GAA

AAT

AAC

8283

LEU

THR

LYS

GLU

SER

TYR

PRO

ASN

ILE

ASP

ARG

THR

GLU

MET

ILE

SER

LYS

GLY

TYR

CTG

ACA

AAG

GAA

AGT

TAT

CCC

AAT

ATT

GAC

CGA

ACT

GAG

ATG

ATT

TCA

AAA

GGA

TAC

8343

VAL

ALA

SER

LYS

SER

HIS

SER

ARG

GLY

SER

ALA

ILE

ASP

LEU

THR

LEU

TYR

ARG

LEU

GTG

GCT

TCA

AAA

TCA

AGC

CAT

AGC

CGC

GGC

AGT

GCC

ATT

GAT

CTT

ACG

CTT

TAT

CGA

TTA

8403

ASP

THR

GLY

GLU

LEU

VAL

PRO

MET

GLY

SER

ARG

PHE

ASP

PHE

MET

ASP

GLU

ARG

SER

HIS

GAC

ACG

GGT

GAG

CTT

GTA

CCA

ATG

GGG

AGC

CGA

TTT

GAT

TTT

ATG

GAT

GAA

CGC

TCT

CAT

8463

HIS

ALA

ASN

GLY

ILE

SER

CYS

ASN

GLU

ALA

GLN

ASN

ARG

LEU

ARG

SER

ILE

CAT

GCG

GCA

AAT

GGA

ATA

TCA

TGC

AAT

GAA

GCG

CAA

AAT

CGC

AGA

CGT

TTG

CGC

TCC

ATC

8523

MET

GLU

ASN

SER

GLY

PHE

GLU

ALA

TYR

SER

LEU

GLU

TRP

HIS

TYR

VAL

LEU

ARG

ASP

ATG

GAA

AAC

AGT

GGG

TTT

GAA

GCA

TAT

AGC

CTC

GAA

TGG

CAC

TAT

GTA

TTA

AGA

GAC

8583

GLU

PRO

TYR

PRO

ASN

SER

TYR

PHE

ASP

PHE

PRO

VAL

LYS

GAA

CCA

TAC

CCC

AAT

AGC

TAT

TTT

GAT

TTC

CCC

GTT

AAA

TAAA

CTT

TTA

ACC

GTT

GCA

8641

CGG

ACA

AAC

TAT

ATA

AGC

TAA

CTC

TTT

CGG

CAG

GAA

ACC

CGA

CGT

ATG

TAA

CTG

GTT

CTT

8701

AGG

GAA

TTT

ATA

TAT

AGT

AGA

TAG

TAT

TGA

AGA

TGT

AAG

GCA

GAG

CGA

TAT

TGC

GGT

CAT

8761

TAT

CTG

CGT

GCG

CTG

CGG

CAA

GAT

AGC

CTG

ATA

AGA

CTG

ATC

GCA

TAG

AGG

GGT

8821

ATT

TCA

CAC

CGC

CCA

TTG

TCA

ACA

GGC

AGT

TCA

GCC

TCG

TTA

AAT

TCA

GCA

TGG

GTA

TCA

8881

CTT

ATG

AAA

ATT

CAT

CTA

CAT

TGG

TGA

TAA

TAG

TAA

ATC

CAG

TAG

GGC

GAA

ATA

ATT

GAC

8941

TGT

AAT

TTA

CGG

GGC

AAA

ACG

GCA

CAA

TCT

CAA

ACG

AGA

TTG

TGC

CGT

TTA

AGG

GGA

AGA

9001

VanY

MET

LYS

TTC

TAG

AAA

TAT

TTC

ATA

CTT

CCA

ACT

ATA

TAG

TTA

AGG

AGA

CTG

AAA

ATG

AAG

9061

LEU

PHE

LEU

PHE

LEU

ILE

TYR

LEU

GLY

TYR

ASP

TYR

VAL

ASN

GLU

TTG

TTT

TTA

TTG

TTA

TTG

TTA

TTC

TTA

ATA

TAC

TTA

GGT

TAT

GAC

TAC

GTT

AAT

GAA

9121

ALA

LEU

PHE

SER

GLN

GLU

LYS

VAL

GLU

PHE

GLN

ASN

TYR

ASP

GLN

ASN

PRO

LYS

GLU

HIS

GCA

CTG

TTT

TCT

CAG

GAA

AAA

GTC

GAA

TTT

CAA

AAT

TAT

GAT

CAA

AAT

CCC

AAA

GAA

CAT

9181

LEU

GLU

ASN

SER

GLY

THR

SER

GLU

ASN

THR

GLN

GLU

LYS

THR

ILE

THR

GLU

GLN

VAL

TTA

GAA

AAT

AGT

GGG

ACT

TCT

GAA

AAT

ACC

CAA

GAG

AAA

ACA

ATT

ACA

GAA

CAG

GTT

9241

TYR

GLN

GLY

ASN

LEU

ILE

ASN

SER

LYS

TYR

PRO

VAL

ARG

GLN

GLU

SER

VAL

LYS

TAT

CAA

GGA

AAT

CTG

CTA

TTA

ATC

AAT

AGT

AAA

TAT

CCT

GTT

CGC

CAA

GAA

AGT

GTG

AAG

9301

SER

ASP

ILE

VAL

ASN

LEU

SER

LYS

HIS

ASP

GLU

LEU

ILE

ASN

GLY

TYR

GLY

LEU

ASP

TCA

GAT

ATC

GTG

AAT

TTA

TCT

AAA

CAT

GAC

GAA

TTA

ATA

AAT

GGA

TAC

GGG

TTG

CTT

GAT

9361

SER

ASN

ILE

TYR

MET

SER

LYS

GLU

ILE

ALA

GLN

LYS

PHE

SER

GLU

MET

VAL

ASN

ASP

ALA

AGT

AAT

ATT

TAT

ATG

TCA

AAA

GAA

ATA

GCA

CAA

AAA

TTT

TCA

GAG

ATG

GTC

AAT

GAT

GCT

9421

VAL

LYS

GLY

VAL

SER

HIS

PHE

ILE

ASN

SER

GLY

TYR

ARG

ASP

PHE

ASP

GLU

GLN

GTA

AAG

GGT

GGC

GTT

AGT

CAT

TTT

ATT

AAT

AGT

GGC

TAT

CGA

GAC

TTT

GAT

GAG

CAA

9481

SER

VAL

LEU

TYR

GLN

GLU

MET

GLY

ALA

GLU

TYR

ALA

LEU

PRO

ALA

GLY

TYR

SER

GLU

HIS

AGT

GTG

CTT

TAC

CAA

GAA

ATG

GGG

GCT

GAG

TAT

GCC

TTA

CCA

GCA

GGT

TAT

AGT

GAG

CAT

9541

ASN

SER

GLY

LEU

SER

LEU

ASP

VAL

GLY

SER

LEU

THR

LYS

MET

GLU

ARG

ALA

PRO

GLU

AAT

TCA

GGT

TTA

TCA

CTA

GAT

GTA

GGA

TCA

AGC

TTG

ACG

AAA

ATG

GAA

CGA

GCC

CCT

GAA

9601

GLY

LYS

TRP

ILE

GLU

ASN

ALA

TRP

LYS

TYR

GLY

PHE

ILE

LEU

ARG

TYR

PRO

GLU

ASP

GGA

AAG

TGG

ATA

GAA

AAT

GCT

TGG

AAA

TAC

GGG

TTC

ATT

TTA

CGT

TAT

CCA

GAG

GAC

9661

LYS

THR

GLU

LEU

THR

GLY

ILE

GLN

TYR

GLU

PRO

TRP

HIS

ILE

ARG

TYR

VAL

GLY

LEU

PRO

AAA

ACA

GAG

TTA

ACA

GGA

ATT

CAA

TAT

GAA

CCA

TGG

CAT

ATT

CGC

TAT

GTT

GGT

TTA

CCA

9721

HIS

SER

ALA

ILE

MET

LYS

GLU

LYS

ASN

PHE

VAL

LEU

GLU

TYR

MET

ASP

TYR

LEU

LYS

CAT

AGT

GCG

ATT

ATG

AAA

GAA

AAG

AAT

TTC

GTT

CTC

GAG

GAA

TAT

ATG

GAT

TAC

CTA

AAA

9781

GLU

LYS

THR

ILE

SER

VAL

SER

VAL

ASN

GLY

GLU

LYS

TYR

GLU

ILE

PHE

TYR

PRO

GAA

AAA

ACC

ATT

TCT

GTT

AGT

GTA

AAT

GGG

GAA

AAA

TAT

GAG

ATC

TTT

TAT

CCT

9841

VAL

THR

LYS

ASN

THR

ILE

HIS

VAL

PRO

THR

ASN

LEU

ARG

TYR

GLU

ILE

SER

GLY

ASN

GTT

ACT

AAA

AAT

ACC

ATT

CAT

GTG

CCG

ACT

AAT

CTT

CGT

TAT

GAG

ATA

TCA

GGA

AAC

9901

ASN

ILE

ASP

GLY

VAL

ILE

VAL

THR

VAL

PHE

PRO

GLY

SER

THR

HIS

THR

ASN

SER

ARG

AAT

ATA

GAC

GGT

GTA

ATT

GTG

ACA

GTG

TTT

CCC

GGA

TCA

ACA

CAT

ACT

AAT

TCA

AGG

9961

TAA

GGA

TGG

CGG

AAT

GAA

ACC

AAC

GAA

ATT

AAT

GAA

CAG

CAT

TAT

TGT

ACT

AGC

ACT

TTT

10021

GGG

GTA

ACG

TTA

GCT

TTT

TAA

TTT

AAA

ACC

CAC

GTT

AAC

TAG

GAC

ATT

GCT

ATA

CTA

ATG

10081

VanZ

LEU

GLY

LYS

ILE

LEU

SER

ARG

GLY

LEU

ATA

CAA

CTT

AAA

CAA

+TL,13AAG

AATTAGAGG

AAA

TTA

TA

TTG

GGA

AAA

ATA

TTA

TCT

AGA

GGA

TTG

10143

LEU

ALA

LEU

TYR

LEU

VAL

THR

LEU

ILE

TRP

LEU

VAL

LEU

PHE

LYS

LEU

GLN

TYR

ASN

ILE

CTA

GCT

TTA

TAT

TTA

GTG

ACA

CTA

ATC

TGG

TTA

GTG

TTA

TTC

AAA

TTA

CAA

TAC

AAT

ATT

10203

LEU

SER

VAL

PHE

ASN

TYR

HIS

GLN

ARG

SER

LEU

ASN

LEU

THR

PRO

PHE

THR

ALA

THR

GLY

TTA

TCA

GTA

TTT

AAT

TAT

CAT

CAA

AGA

AGT

CTT

AAC

TTG

ACT

CCA

TTT

ACT

GCT

ACT

GGG

10263

ASN

PHE

ARG

GLU

MET

ILE

ASP

ASN

VAL

ILE

PHE

ILE

PRO

PHE

GLY

LEU

ASN

AAT

TTC

AGA

GAG

ATG

ATA

GAT

AAT

GTT

ATA

ATC

TTT

ATT

CCA

TTT

GGC

TTG

CTT

TTG

AAT

10323

VAL

ASN

PHE

LYS

GLU

ILE

GLY

PHE

LEU

PRO

LYS

PHE

ALA

PHE

VAL

LEU

VAL

LEU

SER

LEU

GTC

AAT

TTT

AAA

GAA

ATC

GGA

TTT

TTA

CCT

AAG

TTT

GCT

TTT

GTA

CTG

GTT

TTA

AGT

CTT

10383

THR

PHE

GLU

ILE

GLN

PHE

ILE

PHE

ALA

ILE

GLY

ALA

THR

ASP

ILE

THR

ASP

VAL

ILE

ACT

TTT

GAA

ATA

ATT

CAA

TTT

ATC

TTC

GCT

ATT

GGA

GCG

ACA

GAC

ATA

ACA

GAT

GTA

ATT

10443

THR

ASN

THR

VAL

GLY

PHE

LEU

GLY

LEU

LYS

LEU

TYR

GLY

LEU

SER

ASN

LYS

HIS

MET

ACA

AAT

ACT

GTT

GGA

GGC

TTT

CTT

GGA

CTG

AAA

TTA

TAT

GGT

TTA

AGC

AAT

AAG

CAT

ATG

10503

ASN

GLN

LYS

LEU

ASP

ARG

VAL

ILE

PHE

VAL

GLY

ILE

LEU

VAL

LEU

AAT

CAA

AAA

TTA

GAC

AGA

GTT

ATT

TTT

GTA

GGT

ATA

CTT

TTG

CTC

GTA

TTA

TTG

10563

LEU

VAL

TYR

ARG

THR

HIS

LEU

ARG

ILE

ASN

TYR

VAL

CTC

GTT

TAC

CGT

ACC

CAT

TTA

AGA

ATA

AAT

TAC

GTG

TAAG

ATG

TCT

AAA

TCA

AGC

AAT

10621

CTG

ATC

TTT

CAT

ACA

CAT

AAA

GAT

ATT

GAA

TGA

ATT

GGA

TTA

GAT

GGA

AAA

CGG

GAT

GTG

10681

GGG

AAA

CTC

GCC

CGT

AGG

TGT

GAA

GTG

AGG

GGA

AAA

CCG

GTG

ATA

AAG

TAA

AAA

GCT

TAC

10741

CTA

ACA

CTA

TAG

TAA

CAA

AGA

AAG

CCC

AAT

TAT

CAA

TTT

TAG

TGC

GGA

ATT

GGT

CTC

10801

TTT

AAT

AAA

TTT

CCT

TAA

CGT

TGT

AAA

TCC

GCA

TTT

TCC

TGA

CGG

TAC

CCC

Ib (−) Strand

(corresponds to the sequence of the strand complementary to the (+) strand from 1 to 3189.

1

CAA

AAT

ATC

ACC

TCA

TTT

TTG

AGA

CAA

GTC

TTA

TGA

GAC

GCT

CTT

AAC

TAT

GAT

TTT

ATC

61

AGT

CTA

CAT

TTG

TAT

CAA

TAG

AGT

ACA

CTC

TAT

TGA

TAT

ATA

ATT

GAA

CTA

ATA

AAT

121

Transposase

MET

LYS

ILE

ALA

ARG

GLY

ARG

GLU

LEU

THR

TGA

AAA

TAC

AGA

AAT

GGA

ATGATACTG

AA

ATG

AAA

ATT

GCG

AGA

GGT

AGA

GAA

TTG

CTT

ACA

182

PRO

GLU

GLN

ARG

GLN

ALA

PHE

MET

GLN

ILE

PRO

GLU

ASP

GLU

TRP

ILE

LEU

GLY

THR

TYR

CCG

GAA

CAG

AGA

CAG

GCT

TTT

ATG

CAA

ATC

CCT

GAA

GAT

GAA

TGG

ATA

CTG

GGG

ACC

TAC

242

PHE

THR

PHE

SER

LYS

ARG

ASP

LEU

GLU

ILE

VAL

ASN

LYS

ARG

GLU

ASN

ARG

TTC

ACT

TTT

TCC

AAA

CGG

GAT

TTA

GAA

ATA

GTT

AAT

AAG

CGA

AGG

GAA

AAC

CGT

302

LEU

GLY

PHE

ALA

VAL

GLN

LEU

ALA

VAL

LEU

ARG

TYR

PRO

GLY

TRP

PRO

TYR

THR

HIS

ILE

TTA

GGA

TTT

GCT

GTT

CAA

TTA

GCT

GTT

CTT

CGG

TAT

CCC

GGT

TGG

CCA

TAC

ACT

CAT

ATC

362

LYS

SER

ILE

PRO

ASP

SER

VAL

ILE

GLN

TYR

ILE

SER

LYS

GLN

ILE

GLY

VAL

SER

PRO

SER

AAA

AGC

ATC

CCA

GAT

TCG

GTC

ATA

CAA

TAT

ATA

TCG

AAA

CAG

ATT

GGT

GTT

AGT

CCA

TCC

422

SER

LEU

ASP

HIS

TYR

PRO

GLN

ARG

GLU

ASN

THR

LEU

TRP

ASP

HIS

LEU

LYS

GLU

ILE

ARG

TCG

CTT

GAT

CAT

TAT

CCT

CAA

AGG

GAA

AAT

ACA

CTT

TGG

GAT

CAT

TTG

AAA

GAA

ATT

CGA

482

SER

GLU

TYR

ASP

PHE

VAL

THR

PHE

THR

LEU

SER

GLU

TYR

ARG

MET

THR

PHE

LYS

TYR

LEU

AGT

GAA

TAC

GAC

TTT

GTA

ACT

TTT

ACC

CTG

AGT

GAA

TAT

CGA

ATG

ACA

TTT

AAG

TAC

CTT

542

HIS

GLN

LEU

ALA

LEU

GLU

ASN

GLY

ASP

ALA

ILE

HIS

LEU

HIS

GLU

CYS

ILE

ASP

PHE

CAT

CAA

TTA

GCT

TTG

GAA

AAT

GGT

GAT

GCC

ATT

CAT

CTA

CTG

CAT

GAA

TGC

ATA

GAT

TTT

602

LEU

ARG

LYS

ASN

LYS

ILE

LEU

PRO

ALA

ILE

THR

LEU

GLN

ARG

MET

VAL

TRP

GLU

CTA

AGA

AAA

AAC

AAA

ATT

ATA

CTG

CCT

GCT

ATC

ACT

ACA

CTT

GAA

AGA

ATG

GTG

TGG

GAA

662

ALA

ARG

ALA

MET

ALA

GLU

LYS

LEU

PHE

ASN

THR

VAL

SER

LYS

SER

LEU

THR

ASN

GLU

GCA

AGG

GCA

ATG

GCT

GAA

AAG

CTA

TTT

AAT

ACG

GTT

AGT

AAA

TCT

CTA

ACA

AAT

GAG

722

GLN

LYS

GLU

LYS

LEU

GLU

GLY

ILE

THR

SER

GLN

HIS

PRO

SER

GLU

SER

ASN

LYS

THR

CAA

AAA

GAA

AAG

CTT

GAA

GGG

ATT

ACC

TCG

CAG

CAT

CCA

TCC

GAA

TCC

AAT

AAA

ACG

782

ILE

LEU

GLY

TRP

LEU

LYS

GLU

PRO

GLY

HIS

PRO

SER

PRO

GLU

THR

PHE

LEU

LYS

ILE

ATA

TTG

GGT

TGG

TTA

AAA

GAG

CCA

CCG

GGT

CAT

CCT

TCA

CCC

GAA

ACT

TTT

CTA

AAA

ATA

842

ILE

GLU

ARG

LEU

GLU

TYR

ILE

ARG

GLY

MET

ASP

LEU

GLU

THR

VAL

GLN

ILE

SER

HIS

LEU

ATA

GAA

CGA

CTC

GAA

TAC

ATA

CGA

GGA

ATG

GAT

TTA

GAA

ACA

GTG

CAA

ATT

AGT

CAT

TTG

902

HIS

ARG

ASN

ARG

LEU

GLN

LEU

SER

ARG

LEU

GLY

SER

ARG

TYR

GLU

PRO

TYR

ALA

PHE

CAC

CGT

AAC

CGC

CTG

TTG

CAG

CTG

TCT

CGC

TTA

GGC

TCA

AGA

TAC

GAG

CCG

TAT

GCA

TTC

962

ARG

ASP

PHE

GLN

GLU

ASN

LYS

ARG

TYR

SER

ILE

LEU

THR

ILE

TYR

LEU

GLN

LEU

THR

CGT

GAC

TTT

CAA

GAA

AAT

AAA

CGT

TAT

TCG

ATA

TTA

ACC

ATC

TAT

TTA

CAA

CTT

ACT

1022

GLN

GLU

LEU

THR

ASP

LYS

ALA

PHE

GLU

ILE

HIS

ASP

ARG

GLN

ILE

LEU

SER

LEU

SER

CAG

GAG

CTA

ACG

GAT

AAA

GCG

TTT

GAA

ATT

CAT

GAT

AGG

CAA

ATA

CTT

AGT

TTG

TTA

TCA

1082

LYS

GLY

ARG

LYS

ALA

GLN

GLU

ILE

GLN

LYS

GLN

ASN

GLY

LYS

LEU

ASN

GLU

LYS

AAA

GGT

CGT

AAG

GCT

CAA

GAG

GAA

ATC

CAG

AAA

CAA

AAC

GGT

AAA

AAG

CTA

AAT

GAG

AAA

1142

VAL

ILE

HIS

PHE

THR

ASN

ILE

GLY

GLN

ALA

LEU

ILE

LYS

ALA

ARG

GLU

LYS

LEU

ASP

GTT

ATA

CAC

TTT

ACG

AAC

ATC

GGA

CAA

GCA

TTA

ATT

AAA

GCA

AGA

GAG

GAA

AAA

TTA

GAC

1202

VAL

PHE

LYS

VAL

LEU

GLU

SER

VAL

ILE

GLU

TRP

ASN

THR

PHE

VAL

SER

VAL

GLU

GTT

TTT

AAG

GTT

TTA

GAA

TCG

GTT

ATT

GAA

TGG

AAT

ACC

TTT

GTC

TCT

TCA

GTA

GAA

GAG

1262

ALA

GLN

GLU

LEU

ALA

ARG

PRO

ALA

ASP

TYR

ASP

TYR

LEU

ASP

LEU

GLN

LYS

ARG

PHE

GCT

CAG

GAA

CTT

GCA

CGT

CCT

CCC

GAC

TAT

GAT

TAT

TTA

GAC

TTA

CTG

CAA

AAA

CCC

TTT

1322

TYR

SER

LEU

ARG

LYS

TYR

THR

PRO

THR

LEU

ARG

VAL

LEU

GLU

PHE

HIS

SER

THR

LYS

TAT

TCA

CTA

AGA

AAA

TAT

ACG

CCA

ACG

CTA

TTA

AGA

GTA

TTG

GAA

TTT

CAT

TCT

ACA

AAG

1382

ALA

ASN

GLU

PRO

LEU

GLN

ALA

VAL

GLU

ILE

ARG

GLY

MET

ASN

GLU

SER

GLY

LYS

GCA

AAT

GAG

CCA

CTT

TTA

CAA

GCT

GTT

GAG

ATT

ATC

CGA

GGA

ATG

AAC

GAA

TCT

GGA

AAG

1442

ARG

LYS

VAL

PRO

ASP

SER

PRO

VAL

ASP

PHE

ILE

SER

LYS

ARG

TRP

LYS

ARG

HIS

LEU

CGA

AAA

GTG

CCT

CAT

GAC

TCA

CCT

GTG

GAT

TTT

ATT

TCA

AAA

CGA

TCC

AAA

AGA

CAT

TTA

1502

TYR

GLU

ASP

GLY

THR

ILE

ASN

ARG

HIS

TYR

GLU

MET

ALA

VAL

LEU

THR

GLU

TAC

GAG

GAT

GGT

ACA

ATT

AAT

CCT

CAT

TAC

TAT

GAA

ATG

GCT

GTT

TTA

ACA

CAA

1562

LEU

ARG

GLU

HIS

VAL

ARG

ALA

GLY

ASP

VAL

SER

ILE

VAL

GLY

SER

ARG

GLN

TYR

ARG

ASP

CTT

CGG

GAG

CAT

GTT

CGG

GCA

GGA

CAT

GTT

TCC

ATT

GTT

GGC

AGC

AGA

CAA

TAT

AGG

CAT

1622

PHE

GLU

TYR

LEU

PHE

SER

GLU

ASP

THR

TRP

ASN

GLN

SER

LYS

GLY

ASN

THR

ARG

LEU

TTT

GAG

GAA

TAT

TTG

TTT

TCG

GAA

GAT

ACA

TGG

AAT

CAA

TCG

AAG

GGG

AAT

ACG

AGA

TTA

1682

SER

VAL

SER

LEU

SER

PHE

GLN

ASP

TYR

ILE

THR

GLU

ARG

THR

SER

PHE

ASN

GLU

ARG

TCA

GTT

AGT

TTA

TCA

TTC

GAA

GAT

TAT

ATA

ACG

GAG

AGA

ACC

AGC

TTT

AAT

GAA

AGG

1742

LEU

LYS

TRP

LEU

ALA

ASN

SER

ASN

LYS

LEU

ASP

GLY

VAL

SER

LEU

GLU

LYS

GLY

LYS

TTA

AAG

TGG

TTA

GCT

GCC

AAT

TCC

AAT

AAG

TTA

GAT

GGG

GTT

TCT

CTT

GAA

AAA

GGA

AAG

1802

LEU

SER

LEU

ALA

ARG

LEU

GLN

LYS

ASP

VAL

PRO

GLU

ALA

LYS

PHE

SER

ALA

SER

CTA

TCA

CTT

GCA

CGC

TTA

GAA

AAA

GAT

GTT

CCA

GAA

GCA

AAA

TTT

AGT

GCA

AGC

1862

LEU

TYR

GLN

MET

LEU

PRO

ARG

ILE

LYS

LEU

THR

ASP

LEU

MET

ASP

VAL

ALA

HIS

ILE

CTT

TAT

CAG

ATG

CTA

CCA

AGA

ATA

AAA

TTA

ACT

GAT

TTA

CTC

ATG

GAT

GTG

GCC

CAT

ATA

1922

THR

GLY

PHE

HIS

GLU

GLN

PHE

THR

HIS

ALA

SER

ASN

ARG

LYS

PRO

ASP

LYS

GLU

ACA

GGA

TTT

CAT

GAG

CAA

TTC

ACT

CAT

GCT

TCC

AAT

CGA

AAA

CCA

GAT

AAG

GAA

1982

THR

ILE

MET

ALA

LEU

GLY

MET

GLY

MET

ASN

ILE

GLY

LEU

SER

LYS

MET

ACA

ATC

ATT

ATC

ATG

GCT

GCC

CTT

TTA

GGA

ATG

GGA

ATG

AAT

ATT

GGC

TTG

AGC

AAG

ATG

2042

ALA

GLU

ALA

THR

PRO

GLY

LEU

THR

TYR

LYS

GLN

LEU

ALA

ASN

VAL

SER

GLN

TRP

ARG

MET

GCC

GAA

GCC

ACA

CCC

GGA

CTT

ACA

TAT

AAG

CAA

CTA

GCC

AAT

GTA

TCT

CAA

TGG

CGC

ATG

2102

TYR

GLU

ASP

ALA

MET

ASN

LYS

ALA

GLN

ALA

ILE

LEU

VAL

ASN

PHE

HIS

LYS

LEU

GLN

TAT

GAA

GAT

GCC

ATG

AAT

AAA

GCC

CAA

GCC

ATA

TTA

GTA

AAC

TTT

CAT

AAA

TTA

CAA

2162

LEU

PRO

PHE

TYR

TRP

GLY

ASP

GLY

THR

SER

ASP

GLY

MET

ARG

MET

GLN

LEU

TTG

CCT

TTC

TAT

TGG

GGC

GAC

GGT

ACA

TCT

TCG

TCA

GAT

GGT

ATG

AGA

ATG

CAG

CTA

2222

GLY

VAL

SER

LEU

HIS

ALA

ASP

ALA

ASN

PRO

HIS

TYR

GLY

THR

GLY

LYS

GLY

ALA

THR

GGT

GTT

TCA

CTA

CAT

GCA

GAT

GCA

AAT

CCA

CAT

TAT

GGA

ACT

GGA

AAA

GGA

GCC

ACC

2282

ILE

TYR

ARG

PHE

THR

SER

ASP

GLN

PHE

SER

TYR

THR

LYS

ILE

HIS

THR

ASN

ATC

TAC

CGA

TTT

ACA

AGT

GAT

CAA

TTC

TCT

TAC

ACA

AAG

ATT

CAT

ACT

AAT

2342

SER

ARG

ASP

ALA

ILE

HIS

VAL

LEU

ASP

GLY

LEU

HIS

GLU

THR

ASP

LEU

ASN

ILE

TCA

AGA

GAT

GCG

ATT

CAT

GTT

TTG

GAT

GGT

TTG

TTA

CAT

GAG

ACG

GAT

CTA

AAC

ATA

2402

GLU

HIS

TYR

THR

ASP

THR

ALA

GLY

TYR

THR

ASP

GLN

ILE

PHE

GLY

LEU

THR

HIS

LEU

GAG

GAA

CAT

TAT

ACA

GAC

ACT

GCC

GGT

TAC

ACT

GAC

CAA

ATA

TTC

GGA

CTG

ACT

CAT

TTA

2462

LEU

GLY

PHE

LYS

PHE

ALA

PRO

ARG

ILE

ARG

ASP

LEU

SER

ASP

SER

LYS

LEU

PHE

THR

ILE

TTA

GGA

TTT

AAA

TTT

GCC

CCA

AGA

ATA

AGG

GAT

TTA

TCG

GAC

TCA

AAA

TTA

TTT

ACG

ATA

2522

ASP

LYS

ALA

SER

GLU

TYR

PRO

LYS

LEU

GLU

ALA

ILE

LEU

ARG

GLY

GLN

ILE

ASN

THR

LYS

GAT

AAA

GCA

AGT

GAG

TAT

CCA

AAA

CTA

GAA

GCC

ATT

TTA

CGT

GGA

CAA

ATA

AAT

ACA

AAG

2582

VAL

ILE

LYS

GLU

ASN

TYR

GLU

ASP

VAL

LEU

ARG

LEU

ALA

HIS

SER

ILE

ARG

GLU

GLY

THR

GTC

ATT

AAA

GAA

AAT

TAT

GAG

GAT

GTT

TTG

CGA

TTA

GCT

CAT

TCT

ATA

AGG

GAG

GGA

ACA

2642

AGT

TTC

AGC

ATC

CCT

TAT

GGG

GAA

GCT

AGG

TTC

CTA

TTC

AAG

ACA

AAA

CAG

CTT

AGC

VAL

SER

ALA

SER

LEU

ILE

MET

GLY

LYS

LEU

GLY

SER

TYR

SER

ARG

GLN

ASN

SER

LEU

ALA

GTT

TCA

GCA

TCC

CTT

ATT

ATG

GGG

AAG

CTA

GGT

TCC

TAT

TCA

AGA

CAA

AAC

AGC

TTA

GCT

2702

THR

ALA

LEU

ARG

GLU

MET

GLY

ARG

ILE

GLU

LYS

THR

ILE

PHE

ILE

LEU

ASN

TYR

ILE

SER

ACA

GCC

TTA

CGT

GAG

ATG

GGC

CGA

ATA

GAA

AAA

ACG

ATC

TTT

ATT

TTG

AAT

TAT

ATA

TCG

2762

ASP

GLU

SER

LEU

ARG

LYS

ILE

GLN

ARG

GLY

LEU

ASN

LYS

GLY

GLU

ALA

MET

ASN

GLY

GAT

GAA

TCA

TTA

AGA

AAA

ATA

CAA

AGA

GGA

TTG

AAT

AAA

GGA

GAA

GCC

ATG

AAT

GGA

2822

LEU

ALA

ARG

ALA

ILE

PHE

GLY

LYS

GLN

GLY

GLU

LEU

ARG

GLU

ARG

THR

ILE

GLN

HIS

TTG

GCA

AGA

GCT

ATT

TTC

GGA

AAA

CAA

GGT

GAG

CTT

AGA

GAA

CGC

ACC

ATA

CAG

CAT

2882

GLN

LEU

GLN

ARG

ALA

SER

ALA

LEU

ASN

ILE

ASN

ALA

ILE

SER

ILE

TRP

ASN

THR

CAA

TTG

CAA

AGA

GCC

AGT

GCT

TTA

AAC

ATA

ATT

ATC

AAT

GCT

ATA

AGT

ATT

TGG

AAT

ACT

2942

TCT

CCA

CCT

AAC

AGC

AGT

TGA

ATA

TAA

AAA

ACG

GAC

AGG

TAG

CTT

TAA

TGA

AGA

TTT

LEU

HIS

LEU

THR

ALA

VAL

GLU

TYR

LYS

ARG

THR

GLY

SER

PHE

ASN

GLN

ASP

LEU

CTC

CAC

CTA

ACA

GCA

GTT

GAA

TAT

AAA

CGG

ACA

GGT

AGC

TTT

AAT

GAA

GAT

TTG

3002

LEU

HIS

MET

SER

PRO

LEU

GLY

TRP

GLU

HIS

ILE

ASN

LEU

GLY

GLU

TYR

HIS

PHE

TTA

CAC

CAT

ATG

TCG

CCC

TTA

GGT

TGG

GAA

CAT

ATT

AAT

TTA

CTA

GGA

GAA

TAC

CAT

TTT

3062

ASN

SER

GLU

LYS

VAL

SER

LEU

ASN

SER

LEU

ARG

PRO

LEU

LYS

LEU

SER

AAC

TCA

GAG

AAA

GTA

GTC

TCA

TTA

AAT

TCT

TTA

AGA

CCA

CTA

AAA

CTT

TCT

TAA

CGT

TG

3121

TTA

AAA

ACG

AGG

GAT

TCG

TCA

GGA

AAA

TAG

GCT

TAG

CGT

TGT

AAA

TCC

GCA

TTT

TCC

TGA

3181

CGC

TAC

CCC



LIST OF SEQUENCES: ii

SacI

GAGCTCTTCCTTCAACGCACTTCTGTACCAAGAGTTGTTGTC	42

CATTTGATCACTAACAATAGCTTCCCCTGCTTTCTTCAAGCCCTTTGTCATAAAATCGTTAGATTTTCA	111

TCATAAAAATACGAGAAAGACAACAGGAAGACCGCAAATTTTCTTTTCTTTTCCTAGGTACACTGAATG	180
RBS M K K I A V L F G G

TAACCTTAAAAGAAAAAAGGAAAGGAAGAAAATGATGAAAAAAATTGCCGTTTTATTTGGAGGG	244
N S B E Y S V S L T S A A S V I Q A I D

AATTCTCCAGAATACTCAGTGTCACTAACCTCAGCAGCAAGTGTGATCCAAGCTATTGAC	304
P L K Y E V M T I G I A P T M D W Y W Y

CCGCTGAAATATGAAGTAATGACCATTGGCATCGCACCAACAATGGATTGGTATTGGTAT	364
Q G N L A N V R N D T W L E D H K N C H

CAAGGAAACCTCGCGAATGTTCGCAATGATACTTGGCTAGAAGATCACAAAAACTGTCAC	424
Q L T F S S Q G F I L G E K R I V P D V

CAGCTGACTTTTTCTAGCCAAGGATTTATATTAGGAGAAAAACGAATCGTCCCTGATGTC	484
L F P V L H G K Y G E D G C I Q G L L E

CTCTTTCCAGTCTTGCATGGGAAGTATGGCGAGGATGGCTGTATCCAAGGACTGCTTGAA	544
L M N L P Y V G C H V A A S A L C M N K

CTAATGAACCTGCCTTATGTTGGTTGCCATGTCGCTGCCTCCGCATTATGTATGAACAAA	604
W L L H Q L A D T M G I A S A P T L L L

TGGCTCTTGCATCAACTTGCTGATACCATGGGAATCGCTAGTGCTCCCACTTTGCTTTTA	664
S R Y E N D P A T I D R F I Q D H G F P

TCCCGCTATGAAAACGATCCTGCCACAATCGATCGTTTTATTCAAGACCATGGATTCCCG	724
I F I K P N E A G S S K G I T K V T D K

ATCTTTATCAAGCCGAATGAAGCCGGTTCTTCAAAAGGGATCACAAAAGTAACTGACAAA	784
T A L Q S A L T T A F A Y G S T V L I Q

ACAGCGCTCCAATCTGCATTAACGACTGCTTTTGCTTACGGTTCTACTGTGTTGATCCAA	844
K A I A G I E I G C G I L G N E Q L T I

AAGGCGATAGCGGGTATTGAAATTGGCTGCGGCATCTTAGGAAATGAGCAATTGACGATT	904
G A C D A I S L V D G F F D F E E K Y Q

GGTGCTTGTGATGCGATTTCTCTTGTCGACGGTTTTTTTGATTTTGAAGAGAAATACCAA	964
L I S A T I T V P A P L P L A L E S Q I

TTAATCAGCGCCACGATCACTGTCCCAGCACCATTGCCTCTCGCGCTTGAATCACAGATC	1024
K E Q A Q L L Y R N L G L T G L A R I D

AAGGAGCAGGCACAGCTGCTTTATCGAAACTTGGGATTGACGGGTCTGGCTCGAATCGAT	1084
F F V T N Q G A I Y L N E I N T M P G F

TTTTTCGTCACCAATCAAGGAGCGATTTATTTAAACGAAATCAACACCATGCCGGGATTT	1144
T G H S R Y P A M M A E V G L S Y E I L

ACTGGGCACTCCCGCTACCCAGCTATGATGGCGGAAGTCGGGTTATCCTACGAAATATTA	1204
V E Q L E A L A E E D K R *

GTAGAGCAATTGATTGCACTGGCAGAGGAGGACAAACGATGAACACATTACAATTGATCAATA	1267

AAAACCATCCATTGAAAAAAAATCAAGAGCCCCCGCACTTAGTGCTAGCTCCTTTTAGCGATCACGATG	1336

TTTACCTGCAG	1347
PstI

Claims

1-28. (canceled)

29. A composition comprising at least one isolated protein which is encoded by a nucleic acid hybridizing to a sequence selected from the group consisting of SEQ ID NO:1 (vanH), SEQ ID NO:5 (vanX), SEQ ID NO:7 (vanC), SEQ ID NO:11 (vanR), SEQ ID NO:13 (vanS), SEQ ID NO:18 (transposase), SEQ ID NO:20 (resolvase), SEQ ID NO:22 (vanY) SEQ ID NO:24 (vanZ), SEQ ID NO:9 (V1) and SEQ ID NO:10 (V2) under high stringency conditions comprising hybridization overnight at 65° C. in a solution containing 0.1% SDS, 0.7% skim milk 6×SSC and washing at 65° C. in 2×SSC and 0.1% SDS, wherein said at least one isolated protein being necessary for conferring resistance to glycopeptides in gram-positive bacteria.

30. The composition according to claim 29, wherein the at least one isolated protein selected from the group consisting of VanH (SEQ ID NO:2), VanX (SEQ ID NO:6), VanC (SEQ ID NO:8), VanR (SEQ ID NO:12), VanS (SEQ ID NO:14), transposase (SEQ ID NO:19), resolvase (SEQ ID NO:21), VanY (SEQ ID NO:23), VanZ (SEQ ID NO:25) or a combination thereof.

31. The composition according to claim 1 comprising:

(a) an isolated protein encoded by a nucleotide sequence hybridizing to VanR (SEQ ID NO:11) under high stringency conditions, wherein said protein when combined with (b) is capable of increasing the level of said resistance to glycopeptides in gram-positive bacteria; and

(b) an isolated protein encoded by a nucleotide sequence hybridizing to VanS (SEQ ID NO:13) under high stringency conditions, wherein said protein when combined with (a) is capable of increasing the level of said resistance to glycopeptides in gram-positive bacteria.

32. A composition according to claim 31, comprising VanR (SEQ ID NO:12) and VanS (SEQ ID NO:14).

33. The composition according to claim 32, further comprising at least one isolated protein encoded by a nucleotide sequence hybridizing to SEQ ID NO:17 under high stringency conditions.

34. The composition according to claim 33, further comprising at least one isolated protein encoded by a nucleotide sequence hybridizing to SEQ ID NO:1 (vanH), SEQ ID NO:5 (vanX) or SEQ ID NO:3 (vana) under high stringency conditions.

35. The composition according to claim 35, further comprising VanH (SEQ ID NO:2), VanX (SEQ ID NO:) and VanA (SEQ ID NO:).