U.S. patent application number 10/952915 was filed with the patent office on 2005-05-26 for 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.
This patent application is currently assigned to INSTITUT PASTEUR. Invention is credited to Arthur, Michel, Courvalin, Patrice, Dukta-Malen, Sylvie, Molinas, Catherine.
Application Number | 20050112641 10/952915 |
Document ID | / |
Family ID | 34714445 |
Filed Date | 2005-05-26 |
United States Patent
Application |
20050112641 |
Kind Code |
A1 |
Arthur, Michel ; et
al. |
May 26, 2005 |
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
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: 1
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.
Inventors: |
Arthur, Michel; (Paris,
FR) ; Dukta-Malen, Sylvie; (Fresnes, FR) ;
Molinas, Catherine; (Paris, FR) ; Courvalin,
Patrice; (Paris, FR) |
Correspondence
Address: |
OBLON, SPIVAK, MCCLELLAND, MAIER & NEUSTADT, P.C.
1940 DUKE STREET
ALEXANDRIA
VA
22314
US
|
Assignee: |
INSTITUT PASTEUR
Paris Cedex
FR
|
Family ID: |
34714445 |
Appl. No.: |
10/952915 |
Filed: |
September 30, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10952915 |
Sep 30, 2004 |
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09357375 |
Jul 20, 1999 |
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09357375 |
Jul 20, 1999 |
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08980357 |
Nov 28, 1997 |
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6013508 |
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08980357 |
Nov 28, 1997 |
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08286819 |
Aug 5, 1994 |
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5871910 |
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08286819 |
Aug 5, 1994 |
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08174682 |
Dec 28, 1993 |
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08174682 |
Dec 28, 1993 |
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07917146 |
Aug 10, 1992 |
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07917146 |
Aug 10, 1992 |
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PCT/FR91/00855 |
Oct 29, 1991 |
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Current U.S.
Class: |
435/6.16 ;
435/200; 435/221; 435/252.3; 435/320.1; 435/69.1; 536/23.2 |
Current CPC
Class: |
C12N 9/0004 20130101;
C12Q 1/689 20130101; C07K 16/1267 20130101; C12N 9/00 20130101;
C12N 15/65 20130101; C07K 14/315 20130101; C12N 15/52 20130101 |
Class at
Publication: |
435/006 ;
435/069.1; 435/221; 435/252.3; 435/320.1; 536/023.2; 435/200 |
International
Class: |
C12Q 001/68; C07H
021/04; C12N 009/54; C12N 015/74; C12N 001/21 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 31, 1990 |
FR |
9013579 |
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.degree. C. in a solution containing 0.1% SDS, 0.7% skim milk
6.times.SSC and washing at 65.degree. C. in 2.times.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:).
Description
[0001] 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.
[0002] 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.
[0003] 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.
[0004] 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.
[0005] 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).
[0006] 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.
[0007] 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.
[0008] 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 .mu.g/ml, respectively. The
MIC of vancomycin towards strains with low-level resistance are
included between 16 and 32 .mu.g/ml. These strains are apparently
sensitive to teicoplanin.
[0009] 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.
[0010] In principle, a gene sequence will be designated by "van . .
. and an amino acid sequence by "Van . . . ."
[0011] 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.
[0012] 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.
[0013] 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:
2 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
[0014] 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
[0015] 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
[0016] 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.
[0017] 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).
[0018] 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.
[0019] E. gallinarum is a constitutive isolate resistant to low
level of vancomycin (Dutka-Malen et al., Antimicrob. Agents
Chemother 34 (1990b) 1875-1879).
[0020] By the expression "polypeptides" is meant any sequence of
amino acids constituting proteins or being of a size less than that
of a protein.
[0021] 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:
[0022] for hybridization under conditions of high stringency:
[0023] a reaction temperature of 65.degree. C. overnight in a
solution containing 0.1% SDS, 0.7% skimmed milk powder, 6.times.SSC
(1.times.SSC 0.15 M NaCl and 0.015 M sodium citrate at pH=7.0)
[0024] washes at 65.degree. C. in 2.times.SSC-0.1% SDS;
[0025] for hybridization under slightly stringent conditions, the
hybridization temperature is 60.degree. C. overnight and the
temperature of the washings is 45.degree. C.
[0026] The expression of resistance to glycopeptides may be
expresses by the persistence of an infection due to microbes
usually sensitive to the glycopeptides.
[0027] 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.
[0028] Different levels of resistance to the glycopeptides exist in
the strains of Gram-positive cocci, in particular.
[0029] 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).
[0030] 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.
[0031] 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.
[0032] 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.
[0033] 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.
[0034] 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.
[0035] 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.
[0036] 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.
[0037] 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.
[0038] 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.
[0039] 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.sup.+-dependent oxidation
of 2-hydroxy carboxylic acids to form the corresponding
2-keto-carboxylic acids. In fact, the VanH protein might use
NADP.sup.+rather than NAD.sup.+. 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-.alpha.-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".
[0040] 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 .alpha.-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.
[0041] 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.
[0042] Also included in the framework of the invention are
nucleotide sequences coding for one of the amino acid sequences
described above.
[0043] 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.
[0044] 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.
[0045] 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.
[0046] 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:
[0047] HindIII, BglII, BglII, EcoRI, BamHI, XbaI, EcoRI.
[0048] 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,
[0049] 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,
[0050] 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.
[0051] The sequence SEQ ID NO 7 codes for the 3 resistance proteins
VanH, VanA and VanX.
[0052] The sequence SEQ ID NO 22 and the sequence SEQ ID NO 11
include a transposon shown in FIG. 7a; 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.
[0053] A specific sequence corresponding to the above definition is
one of the following sequences:
3 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
[0054] 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.
[0055] 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.
[0056] Variants include all of the fragments of the sequences
having the following properties.
[0057] These sequences code for the resistance proteins VanH, VanA
and VanX.
[0058] 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).
[0059] 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.
[0060] 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:
[0061] sequence upstream from the sequence coding for R: between
the bases 1 and 1476 of the sequence shown in FIG. 5,
[0062] 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,
[0063] sequence downstream from the sequence coding for ORF3:
between the bases 6168 and 7227 of the sequence shown in FIG.
5.
[0064] The sequence designated by SEQ ID NO 6 is also characterized
by the fragment bearing the restriction sites in the following
order:
[0065] BglIII-EcoRI-BamHI-EcoRI
[0066] The location of the regulatory proteins and the resistance
proteins is shown in FIG. 3.
[0067] 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.
[0068] 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.
[0069] 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.
[0070] Recombinant sequences characterized in that they comprise
one of the above nucleotide sequences also form part of the
invention.
[0071] 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.
[0072] 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.
[0073] 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.
[0074] In certain applications it is also possible to use yeasts,
fungi, insect or mammalian cells.
[0075] 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.
[0076] 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.
[0077] 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.
[0078] 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.
[0079] 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.
[0080] 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.
[0081] 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.
[0082] 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.
[0083] As examples, useful probes may be selected from the
intragenic part of the vanA gene.
[0084] 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:
4 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
[0085] 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.
[0086] 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.
[0087] 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.
[0088] 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.
[0089] 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.
[0090] 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.
[0091] 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.
[0092] 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 Kohler 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.
[0093] 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.
[0094] 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.
[0095] 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.
[0096] 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.
[0097] 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:
[0098] antibodies corresponding to the above definition, labelled
if necessary,
[0099] a reagent for the detection of an immunological reaction of
the antigen-antibody type,
[0100] if necessary, reagents to effect the lysis of the cells of
the sample to be tested.
[0101] 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.
[0102] 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).
[0103] The RPCR technique makes possible the amplification of the
NH.sub.2 and COOH terminal regions of the genes it is desired to
detect.
[0104] 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.
[0105] 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.
5 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
[0106] X represents one of the bases A, T, C or G or also
corresponds in all cases to inosine.
[0107] 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.
[0108] 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:
[0109] a nucleotide probe complying with the above specifications
and if necessary,
[0110] oligonucleoside triphosphates in an amount sufficient to
make possible the amplification of the desired sequence,
[0111] a hybridization buffer,
[0112] a DNA polymerization agent.
[0113] 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
[0114] 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,
[0115] b) the separation of the matrix from the elongation product
obtained, this latter then also being capable of behaving as a
matrix,
[0116] c) the repetition of step a) so as to produce a detectable
amount of the desired nucleotide sequences,
[0117] d) the detection of the product of amplification of the
nucleotide sequences.
[0118] 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.
[0119] Details relating to the implementation of the PCR procedures
may be obtained from the patent applications EP 0229701 and EP
0200362.
[0120] Other advantages and characteristics of the invention will
become apparent in the examples which follow and from the
figures.
FIGURES
[0121] 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 .mu.g/ml of vancomycin. The head of
the arrow indicates the position of the VanA protein.
[0122] FIG. 2:
[0123] 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.
[0124] 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.
[0125] FIG. 3: position of the sequences R, S, ORF1, ORF2,
ORF3.
[0126] FIG. 4: representation of SEQ ID NO 6.
[0127] FIG. 5: representation of SEQ ID NO 6 and the corresponding
protein.
[0128] FIG. 6: sequence of the vanA gene and the corresponding
protein.
[0129] FIG. 7:
[0130] (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.
[0131] (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.
[0132] 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.
[0133] 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
[.varies.-.sup.35S] dATP (Amersham, England). The reaction products
were loaded onto 6% denaturing polyacrylamide gels.
[0134] 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.
[0135] 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.
[0136] (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 (*).
[0137] (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
Bougunec 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.
[0138] 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.sup.R methyl transferase.
[0139] (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.
[0140] (B): vanC region of the chromosomal DNA of BM4174.
[0141] (C): vanC region after integration of pAT217.
[0142] FIG. 13: Southern blot analysis of the integration of pAT217
into the vanC gene of BM4174.
[0143] (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 Schul, Germany) by using a Trans-Vac TE80 apparatus
(Hofer 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).
[0144] (right hand side): the probes were labelled with .sup.32P by
nick translation. The molecular masses (kb) are indicated.
[0145] 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.
[0146] 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
[0147] Materials and Methods for the Identification and
Characterization of the vanA Gene
[0148] Bacterial Strains and Plasmids
[0149] The origin of the plasmids used is given in the table
below.
6 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
[0150] Enterococcus faecium BM4147 was cultivated in 500 ml of
heart brain broth (BHI broth medium) until the optical density
(OD.sub.600) reaches 0.7. Induction was effected with 10 .mu.g/ml
of vancomycin (Eli Lilly Indianapolis Ind.). The subsequent steps
were performed at 4.degree. 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 .mu.m 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.
[0151] Preparation of the Minicells
[0152] 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 .mu.Ci of [.sup.35S]-L-methionine
(Amersham, Great Britain) according to the method of Rambach and
Hogness (1977, P.N.A.S. USA, 74; 5041-5045).
[0153] Preparation of the Membrane Fractions and the Cytoplasmic
Fractions of E. coli
[0154] E. coli JM83 and strains derived from it were placed in
culture in BHI medium until an optical density (OD.sub.600) 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.
[0155] Electrophoresis on SDS-polyacrylamide Gel (SDS-PAGE)
[0156] 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.
[0157] Purification of the Protein Band and Determination of the
N-Terminal Sequence
[0158] 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).
[0159] Construction of Plasmids
[0160] 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.
[0161] Subcloning in M13 and Nucleotide Sequence
[0162] 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
[.varies.-.sup.35S] dATP (Amersham, Great Britain) The reaction
products were revealed on 6% polyacrylamide gels containing a
denaturing buffer.
[0163] Data-Processing Analysis and Data on the Sequence
[0164] 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).
[0165] For each comparison 20 amino acid sequences were used to
calculate the mean values and the standard deviations of the random
results.
[0166] Genetic Complementation Tests
[0167] 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.degree. C. on plates
containing 100 .mu.g/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.degree. C. in BHI broth
medium containing ampicillin were placed on a BHI agar medium
containing both 100 .mu.g/ml of ampicillin and 50 .mu.M of
isopropyl-1-thio-.beta.-D-galacto-pyranoside (IPTG) and the plates
were incubated at a permissive temperature of 30.degree. C. and at
a non-permissive temperature of 42.degree. C. The complementation
test was considered to be positive if the colonies were present
after 18 hours of incubation at 42.degree. C.
Results
[0168] Identification of the VanA Protein and its N-Terminal
Sequence
[0169] 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.
[0170] Sub-Cloning of the vanA Gene
[0171] 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.
[0172] Nucleotide Sequence of the Insert in pAT214 and
Identification of the vanA Coding Sequence
[0173] 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.
[0174] Homology of the Amino Acid Sequences of VanA and the
D-ala-D-ala Ligase Enzymes
[0175] 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).
[0176] Genetic Complementation Test for the Activity of D-ala-D-ala
Ligase
[0177] 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.degree. C.) whereas E. coli ST640 (pAT214) grew
both at the permissive temperature and at the non-permissive
temperature (42.degree. C.).
[0178] 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.
[0179] II--VanS-VanR Two-Component Regulation System for the
Control of the Synthesis of Depsipeptides of the Precursor of
Peptidoglycans
[0180] Materials and Methods
[0181] Strains, Plasmids and Conditions of Culture
[0182] The restriction fragments of pIP816 (Tra.sup.-, Mob.sup.+,
Vm.sup.r) were cloned in derivatives of the vector pAT29 which
constitutes a shuttle vector between the Gram-positive and
Gram-negative bacteria (oriR pAM.beta.1, 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.sup.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.sup.R, Rif.sup.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.
[0183] The integrative plasmid pAT113 (Mob.sup.+, Em.sup.R,
Km.sup.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).
[0184] The cultures were grown in brain-heart broth (BHI--Brain
Heart Infusion Broth) or on agar at 37.degree. 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.
[0185] Recombinant DNA Procedures
[0186] 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.).
[0187] Construction of Plasmids
[0188] The origin of the vectors and the inserts used for the
recombinant plasmids constructed here is the following:
[0189] (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).
[0190] (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.
[0191] (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.
[0192] (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.
[0193] Sub-Cloning in M13 and Sequencing
[0194] 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 [.varies.-.sup.35S] dATP (Amersham). The reaction products were
resolved on gradient gels of polyacrylamide in a 6% buffer.
[0195] Enzymatic Test
[0196] The JH2-2 derivatives of E. faecalis were grown to an
optical density OD.sub.600 of 0.7 in a BHI broth supplemented with
spectinomycin (300 .mu.g/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.degree.
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
[0197] 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.
[0198] Nucleotide Sequence of the vanR and vanS Genes
[0199] 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.
[0200] 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).
[0201] 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.
[0202] 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.
[0203] 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).
[0204] Inactivation of the van Genes by Insertion
[0205] 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.
[0206] 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.
[0207] 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.
[0208] Trans-Activation of the Transcription of the van Genes
[0209] 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.
[0210] 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.
[0211] III--Characterization of the Sequence of the vanC Gene of
Enterococcus gallinarum BM4174
[0212] 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
[0213] 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.
[0214] 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.
[0215] Amplification, Cloning and Sequencing of the vanC Gene
[0216] 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.
[0217] 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.
[0218] Inactivation of the vanC Gene by Insertion
[0219] 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.
[0220] 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.
[0221] 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
[0222] (contained in the sequences I (Ia, Ib), II presented below
or in the sequence shown in FIG. 5).
[0223] Amino Acid Sequences
[0224] 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.
[0225] 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.
[0226] 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.
[0227] 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.
[0228] 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.
[0229] SEQ ID NO 16: sequence of the transposase corresponding to
the amino acids included between the nucleotides 150 and 3112 of
the sequence Ib.
[0230] 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.
[0231] SEQ ID NO 18: VanY sequence comprising the amino acids
situated between the positions of the nucleotides 9046 and 9960 of
the sequence Ia.
[0232] SEQ ID NO 19: VanZ sequence comprising the amino acids
situated between the positions of the nucleotides 10116 and 10598
of the sequence Ia.
[0233] SEQ ID NO 20: VanC amino acid sequence shown in list II.
[0234] Nucleotide Sequences
[0235] SEQ ID NO 6: nucleotide sequence containing the sequence
coding for the 5 proteins as well as the flanking sequences, shown
in FIG. 5.
[0236] 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.
[0237] 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.
[0238] 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.
[0239] 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.
[0240] 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.
[0241] SEQ ID NO 12: sequence coding for the transposase, starting
at the base 150 and terminating at the base 3112 of the sequence
Ib.
[0242] SEQ ID NO 13: sequence coding for the resolvase, starting at
the base 3187 and terminating at the base 3759 of the sequence
Ia.
[0243] SEQ ID NO 14 sequence coding for VanY, starting at the base
9046 and terminating at the base 9960 of the sequence Ia.
[0244] SEQ ID NO 15: sequence coding for VanZ, starting at the base
10116 and terminating at the base 10598 of the sequence Ia.
[0245] SEQ ID NO 21: sequence coding for VanC, shown in the list II
in relation to the protein VanC.
[0246] SEQ ID NO 22 complete sequence Ia of the transposon of E.
faecium, starting at the base 1 and terminating at the base
10851.
[0247] 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.
[0248] 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.
7 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 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 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 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 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
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 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 TTT GCT TCT 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 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 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 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 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 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 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 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 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 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 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 GLN LEU ASN GLU ILE GLY MET ASP
ILE ILE TYR GLU GLU LYS VAL SER GLY CAA TTT CAG 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 ASP LEU GLN GLU ASP 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 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 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 LYS TYR HIS LYS ASN HIS ALA
GLY MET ASN TYR ALA VAL AAG TTT AAA GGT CGA TTA AAG 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 ASN AGG GCT
TCA TTA TAC AGG AAA TTA TCA GAA GTG AAT AAT TAG CCA TTC TGT ATT CCC
CTA 3781 ATG GGC AAT ATT TTT AAA GAA GAA AAG GAA ACT ATA AAA TAT
TAA CAG CCT CCT AGC GAT 3841 GCC GAA AAG CCC TTT GAT AAA AAA AGA
ATC ATC ATC TTA AGA AAT TCT TAG TCA TTT ATT 3901 ATG TAA ATG CTT
ATA AAT TCG GCC CTA TAA TCT GAT AAA TTA TTA AGG GCA AAC TTA TGT
3961 VanR MET SER ASP LYS ILE LEU ILE VAL ASP ASP GLU HIS GLU ILE
ALA GAA AGG GTG ATA ACT ATG AGC GAT AAA ATA CTT ATT GTG GAT GAT GAA
CAT GAA ATT GCC 4021 ASP LEU VAL GLU LEU TYR LEU LYS ASN GLU ASN
TYR THR VAL PHE LYS TYR 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 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 ASP TYR ILE THR LYS PRO PHE ARG PRO LEU GLU
LEU ILE ALA ARG VAL LYS ALA GCG GAT GAT TAT ATA ACG AAG CCC TTT CGC
CCA CTG GAG TTA ATT GCT CGG GTA AAG GCC 4321 GLN LEU ARG ARG TYR
LYS LYS PHE SER GLY VAL LYS GLU GLN ASN GLU ASN VAL ILE VAL CAG TTG
CGC CGA TAC AAA 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 SER GLU LEU 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 AAA AAC
GAC VanS LEUVALILELYSLEULYSASN LYS LYS ASN ASP 4682 TYR SER LYS LEU
GLU ARG LYS LEU TYR MET TYR ILE VAL ALA ILE VAL VAL 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 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 VAL MET TYR LEU ALA HIS ASP ILE
LYS THR PRO LEU THR GAA CAA AGA AAA AAT GAC GTT GTT ATG TAC TTG GCG
CAC GAT ATT AAA ACG CCC CTT ACA 5162 SER ILE ILE GLY TYR LEU SER
LEU 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 PHE GLU ILE THR ARG TYR
ASN LEU GLN THR ILE THR LEU THR LYS THR HIS ILE ASP TTT TTT GAG ATT
ACA CGG TAT AAC CTA CAA ACG ATA ACG CTA ACA AAA ACG CAC ATA GAC
5342 LEU TYR 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 ASN ILE LEU LYS ASN ALA ALA ALA TYR SER
GLU ASP ASN SER ILE GCG AGA GTC TTT AAC 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 VAL SER ILE GLU PHE LYS ASP THR GLY ATT GAC ATT ACC
GCG GGC CTC TCC GGG GAT GTG GTG TCA ATC GAA TTC AAG AAC ACT GGA
5582 SER ILE PRO LYS ASP LYS LEU ALA 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 SER ASP THR GLY GLY
ALA GLY LEU GLY LEU ALA ILE ALA LYS GLU ILE ILE VAL CGT TCT TCC GAT
ACG GGT GGC GCG GGA CTT GGA TTG GCG ATT GCA AAA GAA ATT ATT GTT
5702 GLN HIS GLY GLY GLN ILE TYR ALA GLU SER ASN ASP ASN TYR THR
THR PHE ARG VAL GLU CAG CAT GGA GGG CAG ATT TAC GCG GAA AGC AAT GAT
AAC TAT ACG ACG TTT AGG GTA GAG 5762 LEU PRO ALA MET PRO ASP LEU
VAL ASP LYS ARG ARG SER CTT CCA GCG ATG CCA GAC TTG GTT GAT AAA AGG
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
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 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 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 LEU ALA LEU LYS ARG ALA GLY VAL LYS TYR ILE AAA TCA
GAG ATT TCC GCC TCT ATT CTT CTT GCG CTG AAG AGA GCC GGT GTG AAA TAT
ATT 6243 SER THR ARG SER ILE GLY CYS ASN HIS ILE ASP THR THR ALA
ALA LYS ARG MET GLY ILE TCT ACC CGA AGC ATC GGC TGC AAT CAT ATA GAT
ACA ACT GCT GCT AAG AGA ATG GGC ATC 6303 THR VAL ASP ASN VAL ALA
TYR SER PRO ASP SER VAL ALA ASP TYR THR MET MET LEU ILE ACT GTC GAC
AAT GTG GCG TAC TCG CCG GAT AGC GTT GCC GAT TAT ACT ATG 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 VAL GLY THR GLY TTG GAC AGC GAC
CGT GGC AAG GTA CTC AGC GAC ATG ACA GTT GGT GTG 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 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 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
GLY ALA 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 GLU GLU
PHE 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 LEU LYS LEU GLN ARG MET PRO ASN VAL ILE 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 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 GLU HIS ASP VAL SER VAL
LYS SER ALA ILE GLU ILE ALA ALA ASN ILE ASN LYS GLU GAG 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 LYS CCT TGC GCG GAA
TGG GAA AAC GAC AAT TGC TAT TCA GCT GTA CTC TCG CCG GAT AAA AAA
7201 MET HIS GLY LEU LEU VAL LYS 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 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 ASP ARG PRO VAL ALA ALA THR PHE THR TYR PRO VAL PHE
VAL LYS PRO ALA ARG AAA GAT GAT AGG CCG GTG GCA GCT ACG TTT ACC TAT
CCT GTT TTT GTT AAG CCG GCG CGT 7501 SER GLY SER SER PHE GLY VAL
LYS LYS VAL ASN SER ALA ASP GLU LEU ASP TYR ALA ILE TCA GGC TCA TCC
TTC GGT GTG AAA 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 ALA LEU VAL VAL GLY GLU VAL ASP GLN ILE GTC GGT TGT GCG
GTA TTG GGA AAC AGT GCC GCG TTA GTT 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 GLU ARG GLY ARG ILE GLN GLU AAC GCA GTT ATA
ACC GTT CCC GCA GAC CTT TCA GCA GAG GAG CGA GGA CGG ATA CAG GAA
7801 THR ALA LYS LYS ILE TYR LYS ALA LEU GLY CYS ARG GLY LEU ALA
ARG VAL ASP MET PHE ACG GCA AAA 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 MET ALA ALA ALA GLY ILE ALA LEU
PRO GLU LEU ILE ASP TAC AGT CGT TAT CCC CGT ATG 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 LEU LYS ALA LYS GLN LEU ALA ALA THR GLN GLY TYR GLY LEU
LEU LEU TRP ASP GLY CTT TTG AAG GCA AAA GAA CTG GCT 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 ALA GLN PRO GLU ASN 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 TYR PRO ASN ILE ASP ARG THR GLU MET
ILE SER LYS GLY TYR CTG ACA AAG GAA AGT TAT TAT CCC AAT ATT GAC CGA
ACT GAG ATG ATT TCA AAA GGA TAC 8343 VAL ALA SER LYS SER 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 ALA ASN GLY ILE SER
CYS ASN GLU ALA GLN ASN ARG ARG 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 TRP HIS
TYR VAL LEU ARG ASP ATG GAA AAC AGT GGG TTT GAA GCA TAT AGC CTC GAA
TGG 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 ATA AGA CTG
ATC GCA TAG AGG GGT 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 LYS TTC TAG AAA TAT TTC ATA
CTT CCA ACT ATA TAG TTA AGG AGG AGA CTG AAA ATG AAG AAG 9061 LEU
PHE PHE LEU LEU LEU LEU LEU PHE LEU ILE TYR LEU GLY TYR ASP TYR VAL
ASN GLU TTG TTT 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 GLU GLN
VAL TTA GAA AAT AGT GGG ACT TCT GAA AAT ACC CAA GAG AAA ACA ATT ACA
GAA GAA CAG GTT 9241 TYR GLN GLY ASN LEU LEU 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 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 GLY
VAL SER HIS PHE ILE ILE ASN SER GLY TYR ARG ASP PHE ASP GLU GLN GTA
AAG GGT GGC GTT AGT CAT TTT ATT 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 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 GLU ASN ALA TRP LYS TYR GLY PHE ILE
LEU ARG TYR PRO GLU ASP GGA AAG TGG ATA GAA 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 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 GLU LYS THR ILE SER VAL
SER VAL ASN GLY GLU LYS TYR GLU ILE PHE TYR TYR PRO GAA GAA AAA ACC
ATT TCT GTT AGT GTA AAT GGG GAA AAA TAT GAG ATC TTT TAT TAT CCT
9841 VAL THR LYS ASN THR THR ILE HIS VAL PRO THR ASN LEU ARG TYR
GLU ILE SER GLY ASN GTT ACT AAA AAT ACC 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 ARG AAT ATA GAC GGT
GTA ATT GTG ACA GTG TTT CCC GGA TCA ACA CAT ACT AAT TCA AGG 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 ILE PHE ILE PRO PHE GLY LEU LEU 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 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 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 LYS LEU ASP ARG
VAL ILE ILE PHE VAL GLY ILE LEU LEU LEU VAL LEU LEU AAT CAA AAA AAA
TTA GAC AGA GTT ATT 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 GGA ATT GGT CTC 10801
TTT AAT AAA TTT CCT TAA CGT TGT AAA TCC GCA TTT TCC TGA CGG TAC
CCC
[0249] Ib (-) Strand
[0250] (corresponds to the sequence of the strand complementary to
the (+) strand from 1 to 3189.
8 1 CAA AAT ATC ACC TCA TTT TTG AGA CAA GTC TTA TGA GAC GCT CTT AAC
TAT GAT TTT ATC 61 AGT CTA 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 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 ARG ARG
GLU GLU ASN ARG TTC ACT TTT TCC AAA CGG GAT TTA GAA ATA GTT AAT AAG
CGA AGG AGG GAA 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 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 ILE LEU
PRO ALA ILE THR 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 LYS LEU PHE ASN THR VAL SER LYS SER LEU THR
ASN GLU GCA AGG GCA ATG GCT GAA AAG AAG CTA TTT AAT ACG GTT AGT AAA
TCT CTA ACA AAT GAG 722 GLN LYS GLU LYS LEU GLU GLY ILE ILE THR SER
GLN HIS PRO SER GLU SER ASN LYS THR CAA AAA GAA AAG CTT GAA GGG ATT
ATT ACC TCG CAG CAT CCA TCC GAA TCC AAT AAA ACG 782 ILE LEU GLY TRP
LEU LYS GLU PRO 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 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 LEU
GLN LEU THR CGT GAC TTT CAA GAA AAT AAA CGT TAT TCG ATA TTA ACC ATC
TAT TTA TTA CAA CTT ACT 1022 GLN GLU LEU THR ASP LYS ALA PHE GLU
ILE HIS ASP ARG GLN ILE LEU SER LEU 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 GLU ILE GLN LYS GLN ASN GLY LYS 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 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 SER VAL GLU
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 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 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 LEU GLN ALA VAL GLU ILE 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 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 ASP GLY THR THR ILE ASN ARG HIS TYR TYR
GLU MET ALA VAL LEU THR GLU TAC GAG GAT GAT GGT ACA 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 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 SER PHE ASN GLU ARG TCA GTT AGT
TTA TCA TTC GAA GAT TAT ATA ACG GAG AGA ACC AGC AGC TTT AAT GAA AGG
1742 LEU LYS TRP LEU ALA 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 GLU ALA LYS LYS PHE SER ALA SER CTA TCA CTT GCA
CGC TTA GAA AAA GAT GTT CCA GAA GAA GCA AAA AAA TTT AGT GCA AGC
1862 LEU TYR GLN MET LEU PRO ARG ILE LYS LEU THR ASP LEU 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 ASN ARG LYS PRO ASP LYS GLU GLU ACA GGA TTT CAT
GAG CAA TTC ACT CAT GCT TCC AAT AAT CGA AAA CCA GAT AAG GAA GAA
1982 THR ILE ILE ILE MET ALA ALA LEU 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 HIS LYS LEU GLN TAT GAA GAT GCC ATG AAT AAA GCC CAA GCC ATA TTA
GTA AAC TTT CAT CAT AAA TTA CAA 2162 LEU PRO PHE TYR TRP GLY ASP
GLY THR THR SER SER SER ASP GLY MET ARG MET GLN LEU TTG CCT TTC TAT
TGG GGC GAC GGT ACA ACA TCT TCG TCA GAT GGT ATG AGA ATG CAG CTA
2222 GLY VAL SER SER LEU HIS ALA ASP ALA ASN PRO HIS TYR GLY THR
GLY LYS GLY ALA THR GGT GTT TCA 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 SER TYR TYR THR LYS ILE ILE HIS THR ASN ATC TAC CGA TTT
ACA AGT GAT CAA TTC TCT TCT TAC TAC ACA AAG ATT ATT CAT ACT AAT
2342 SER ARG ASP ALA ILE HIS VAL LEU ASP GLY LEU LEU HIS HIS GLU
THR ASP LEU ASN ILE TCA AGA GAT GCG ATT CAT GTT TTG GAT GGT TTG TTA
CAT CAT GAG ACG GAT CTA AAC ATA 2402 GLU 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 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
ARG LYS ILE GLN ARG GLY LEU ASN LYS GLY GLU ALA MET ASN GLY GAT GAA
TCA TTA AGA AGA AAA ATA CAA AGA GGA TTG AAT AAA GGA GAA GCC ATG AAT
GGA 2822 LEU ALA ARG ALA ILE PHE PHE GLY LYS GLN GLY GLU LEU ARG
GLU ARG THR ILE GLN HIS TTG GCA AGA GCT ATT TTC 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 ILE 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 AAC AGC AGT TGA ATA TAA AAA ACG GAC AGG TAG
CTT TAA TGA AGA TTT LEU HIS LEU THR THR ALA VAL GLU TYR LYS LYS ARG
THR GLY SER PHE ASN GLN ASP LEU CTC CAC CTA ACA ACA GCA GTT GAA TAT
AAA AAA CGG ACA GGT AGC TTT AAT GAA GAT TTG 3002 LEU HIS HIS MET
SER PRO LEU GLY TRP GLU HIS ILE ASN LEU 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 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
[0251]
9 LIST OF SEQUENCES: ii SacI
GAGCTCTTCCTTCAACGCACTTCTGTACCAAGAGTTGTTGTC 42
CATTTGATCACTAACAATAGCTTCCCCTGCTTTCTTCAAGCCCTTTGTCATAAAATCGTTAGATTTTCA
111 TCATAAAAATACGAGAAAGACAACAGGAAGACCGCAAATTTTCTTTTCTTTTCCTAG-
GTACACTGAATG 180 RBS M K K I A V L F G G
TAACCTTAAAAGAAAAAAGGAAAGGAAGAAAATGATGAAAAAA- ATTGCCGTTTTATTTGGAGGG
244 N S B E Y S V S L T S A A S V I Q A I D
AATTCTCCAGAATACTCAGTGTCACTAACCTCAGCAGCA- AGTGTGATCCAAGCTATTGAC 304
P L K Y E V M T I G I A P T M D W Y W Y
CCGCTGAAATATGAAGTAATGACCATTGGCATCGCACCA- ACAATGGATTGGTATTGGTAT 364
Q G N L A N V R N D T W L E D H K N C H
CAAGGAAACCTCGCGAATGTTCGCAATGATACTTGGCTA- GAAGATCACAAAAACTGTCAC 424
Q L T F S S Q G F I L G E K R I V P D V
CAGCTGACTTTTTCTAGCCAAGGATTTATATTAGGAGAA- AAACGAATCGTCCCTGATGTC 484
L F P V L H G K Y G E D G C I Q G L L E
CTCTTTCCAGTCTTGCATGGGAAGTATGGCGAGGATGGC- TGTATCCAAGGACTGCTTGAA 544
L M N L P Y V G C H V A A S A L C M N K
CTAATGAACCTGCCTTATGTTGGTTGCCATGTCGCTGCC- TCCGCATTATGTATGAACAAA 604
W L L H Q L A D T M G I A S A P T L L L
TGGCTCTTGCATCAACTTGCTGATACCATGGGAATCGCT- AGTGCTCCCACTTTGCTTTTA 664
S R Y E N D P A T I D R F I Q D H G F P
TCCCGCTATGAAAACGATCCTGCCACAATCGATCGTTTT- ATTCAAGACCATGGATTCCCG 724
I F I K P N E A G S S K G I T K V T D K
ATCTTTATCAAGCCGAATGAAGCCGGTTCTTCAAAAGGG- ATCACAAAAGTAACTGACAAA 784
T A L Q S A L T T A F A Y G S T V L I Q
ACAGCGCTCCAATCTGCATTAACGACTGCTTTTGCTTAC- GGTTCTACTGTGTTGATCCAA 844
K A I A G I E I G C G I L G N E Q L T I
AAGGCGATAGCGGGTATTGAAATTGGCTGCGGCATCTTA- GGAAATGAGCAATTGACGATT 904
G A C D A I S L V D G F F D F E E K Y Q
GGTGCTTGTGATGCGATTTCTCTTGTCGACGGTTTTTTT- GATTTTGAAGAGAAATACCAA 964
L I S A T I T V P A P L P L A L E S Q I
TTAATCAGCGCCACGATCACTGTCCCAGCACCATTGCCT- CTCGCGCTTGAATCACAGATC 1024
K E Q A Q L L Y R N L G L T G L A R I D
AAGGAGCAGGCACAGCTGCTTTATCGAAACTTGGGAT- TGACGGGTCTGGCTCGAATCGAT 1084
F F V T N Q G A I Y L N E I N T M P G F
TTTTTCGTCACCAATCAAGGAGCGATTTATTTAAAC- GAAATCAACACCATGCCGGGATTT 1144
T G H S R Y P A M M A E V G L S Y E I L
ACTGGGCACTCCCGCTACCCAGCTATGATGGCGG- AAGTCGGGTTATCCTACGAAATATTA 1204
V E Q L E A L A E E D K R *
GTAGAGCAATTGATTGCACTGGCAGAGGAGGACAAACGATGAACACATTAC- AATTGATCAATA
1267 AAAACCATCCATTGAAAAAAAATCAAGAGCCCCCGCACTT-
AGTGCTAGCTCCTTTTAGCGATCACGATG 1336 TTTACCTGCAG 1347 PstI
[0252]
Sequence CWU 1
1
* * * * *