U.S. patent application number 10/646807 was filed with the patent office on 2004-04-22 for novel topoisomerase iv, corresponding nucleotide sequences and uses thereof.
This patent application is currently assigned to Aventis Pharma S.A.. Invention is credited to Blanche, Francis, Cameron, Beatrice, Crouzet, Joel, Famechon, Alain, Ferrero, Lucia.
Application Number | 20040077005 10/646807 |
Document ID | / |
Family ID | 29420864 |
Filed Date | 2004-04-22 |
United States Patent
Application |
20040077005 |
Kind Code |
A1 |
Blanche, Francis ; et
al. |
April 22, 2004 |
Novel topoisomerase IV, corresponding nucleotide sequences and uses
thereof
Abstract
The present invention generally relates to a novel topoisomerase
IV, the nucleotide sequences encoding this enzyme, their
corresponding vectors, and the use of this enzyme for screening
biologically active products.
Inventors: |
Blanche, Francis; (Paris,
FR) ; Cameron, Beatrice; (Paris, FR) ;
Crouzet, Joel; (Sceaux, FR) ; Famechon, Alain;
(Janville-sur-Juine, FR) ; Ferrero, Lucia; (Paris,
FR) |
Correspondence
Address: |
FINNEGAN, HENDERSON, FARABOW, GARRETT & DUNNER
LLP
1300 I STREET, NW
WASHINGTON
DC
20005
US
|
Assignee: |
Aventis Pharma S.A.
|
Family ID: |
29420864 |
Appl. No.: |
10/646807 |
Filed: |
August 25, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10646807 |
Aug 25, 2003 |
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09398184 |
Sep 17, 1999 |
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6649394 |
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09398184 |
Sep 17, 1999 |
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08776265 |
Jan 24, 1997 |
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6001631 |
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08776265 |
Jan 24, 1997 |
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PCT/FR95/01001 |
Jul 26, 1995 |
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Current U.S.
Class: |
435/6.12 |
Current CPC
Class: |
C12Q 1/533 20130101;
C12N 9/90 20130101 |
Class at
Publication: |
435/006 |
International
Class: |
C12Q 001/68 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 27, 1994 |
FR |
94/09288 |
Claims
1. Nucleotide sequence encoding a subunit of topoisomerase IV of
Staphylococcus aureus.
2. Nucleotide sequence characterized in that it is chosen from: (a)
all or part of the grlA (SEQ ID No. 2) or grlB (SEQ ID No. 3)
genes, (b) the sequences hybridizing with all or part of the (a)
genes and encoding a subunit of a topoisomerase IV, and (c) the
sequences derived from the (a) and (b) sequences because of the
degeneracy of the genetic code.
3. Nucleotide sequence according to claim 1 or 2, characterized in
that it is the grlA gene (SEQ ID No. 2).
4. Nucleotide sequence according to claim 1 or 2, characterized in
that it is the grlB gene (SEQ ID No. 3).
5. Nucleotide sequence according to claim 1 or 2, characterized in
that it is the grlA gene having a mutation leading to a resistance
towards molecules of the quinolone family.
6. Nucleotide sequence according to claim 5, characterized in that
it is the grlA gene having a base A as a substitution for a base C
at position 2270 of SEQ ID No. 2.
7. Recombinant DNA comprising a nucleotide sequence according to on
of claims 1 to 6.
8. Autonomously replicating and/or integrative expression vector
characterized in that it comprises a nucleotide sequence according
to one of claims 1 to 6.
9. Recombinant cell containing a nucleotide sequence according to
one of claims 1 to 6, a recombinant DNA according to claim 7 and/or
an expression vector according to claim 8.
10. Cell according to claim 8, characterized in that it is
preferably a bacterium.
11. Polypeptide resulting from the expression of at least one
sequence according to one of claims 1 to 6.
12. Polypeptide comprising all or part of the polypeptide GrlA (SEQ
ID No. 2), of the polypeptide GrlB (SEQ ID No. 3) or of a
derivative thereof.
13. Polypeptide according to claim 11 or 12, characterized in that
it is the polypeptide GrlA (SEQ ID No. 2).
14. Polypeptide according to claim 11 or 12, characterized in that
it is the polypeptide GrlB (SEQ ID No. 3).
15. Polypeptide according to claim 11 or 12, characterized in that
it is the polypeptide GrlA.sub.(Ser-80.fwdarw.Tyr).
16. Process for the production of a polypeptide according to one of
claims 11 to 15, charact riz d in that a r combinant cell according
to claim 9 or 10 is cultur d and th polypeptid produced is
recovered.
17. Isolated topoisomerase IV characterized in that it is capable
of being obtained from the expression of all or part of the grlA
gene (SEQ ID No. 2) and of all or part of the grlB gene (SEQ ID No.
3), or of their respective derivatives as defined in b) and c) of
claim 2.
18. Isolated topoisomerase IV according to claim 17, characterized
in that it is derived from the expression of all or part of the
grlA gene (SEQ ID No. 2) and of all or part of the grlB gene (SEQ
ID No. 3).
19. Isolated topoisomerase IV, characterized in that it has the
behaviour of a primary target towards the fluoroquinolones.
20. Isolated topoisomerase IV according to one of the preceding
claims, characterized in that it is topoisomerase IV of
Staphylococcus aureus.
21. Use of a topoisomerase IV according to one of claims 17 to 20
to target biologically active products.
22. Use of a topoisomerase IV according to one of claims 17 to 20
to search for products inhibiting th ATP-d p ndent DNA r laxing
reaction.
23. Use of a topoisomeras IV according to one of claims 17 to 20
for identifying products inhibiting the reaction of decatanation of
catenanes of DNA.
Description
[0001] The present invention relates to a novel topoisomerase IV,
the nucleotide sequences encoding this enzyme, their corresponding
vectors and the use of this enzyme for screening biologically
active products.
[0002] Topoisomerases are enzymes capable of modifying the
topological configuration of DNA rings, of making knots therein or
of interlacing separated rings. They are thus involved in the
replication, transcription and recombination of the entire genetic
information (Wang et al., 1990). The mechanism of all these
topological conversions is the same: the ring is opened so that a
segment of DNA passes through the gap before the ends are rejoined.
Two types of topoisomerase are involved in these conversions: type
I topoisomerases which cut a single DNA strand and type II
topoisomerases which cut both strands simultaneously.
[0003] Up until now, two type II bacterial topoisomerases have been
identified and studied more particularly: gyrase from Escherichia
coli (Gellert et al., 1976), and more recently, DNA topoisomerase
IV from E.coli (Kato et al., 1990).
[0004] Gyras is a .alpha..sub.2.beta..sub.2 tetramer whose a or
GyrA and .beta. or GyrB subunits are encoded by the gyrA and gyrB
genes respectively. Bacterial gyrases are the only known
topoisomerases capable of supercoiling relaxed DNA rings in the
presence of ATP.
[0005] As regards more particularly DNA topoisomerase IV from
E.coli, it relaxes supercoiled plasmid DNA, unknots T4 phage DNA
and unwinds (or decatenates) kinetoplast DNA (Kato et al., 1992;
Peng et al., 1993). The sequence of its corresponding genes, parC
and parE from E.coli, has made it possible to demonstrate regions
of high similarity between the subunits of gyrase and those of this
topoisomerase IV, ParC with GyrA (35.6% over the entire sequence)
and ParE with GyrB (40.1% over the entire sequence) respectively
(Kato et al., 1990).
[0006] E.coli gyrase has also been identified as being a primary
target of fluoroquinolones (Hooper et al., 1993). It has thus been
demonstrated that E.coli strains mutated at the level of the Ser83
residue in the GyrA subunit have a high resistance to
fluoroquinolones (Maxwell, 1992). Fluoroquinolones bind less to
DNA-mutated gyrase complexes than to DNA-wild-type gyrase
complexes. Indeed, other point mutations, mapped in the region
between residues 67 and 106 of GyrA, lead to strains resistant to
fluoroquinolones. This region is called QRDR (Yoshida et al., 1990;
Cullen et al., 1989). Similar results have been published with
strains of Staphylococcus aureus resistant to fluoroquinolones
(Goswitz et al., 1992; Sreedharan et al., 1990). Gyrase is
therefore nowadays recognized as being the primary target of
quinolones. However, a clinical strain of Staphylococcus aureus,
not containing any mutation in the QRDR region of GyrA, has also
been described as resistant to fluoroquinolones (Sreedharan et al.,
1991).
[0007] Nowadays, this phenomenon of resistance developed by
Staphylococcus aureus bacteria towards antibiotics and more
particularly towards fluoroquinolones is being increasingly
encountered at the therapeutic level. It would be particularly
important to be able to lift this resistance and this involves a
characterization of all the parameters which are associated with
it.
[0008] The main objective of the present invention is precisely the
identification, sequencing and characterization of nucleic
sequences encoding subunits of a novel topoisomerase, topoisomerase
IV of Staphylococcus aureus, composed of two subunits, GrlA and
GrlB.
[0009] Unexpectedly, the applicant has found that the primary
target of the fluoroquinolones in S. aureus is a topoisomerase IV
and not gyrase. It has thus demonstrated that clinical strains of
S. aureus, in which the QRDR region of the GyrA subunit of gyrase
is identical to th wild-type sequence, develop nevertheless a
resistance to fluoroquinolones because of a mutation which they
possess in the region of the GrlA subunit of topoisomerase IV,
homologous to the QRDR region.
[0010] The first subject of the present invention is a nucleotide
sequence encoding at least one subunit of topoisomerase IV of
Staphylococcus aureus.
[0011] The present invention describes in particular the isolation
and the characterization of the grlA and grlB genes. These genes
have been cloned, sequenced and expressed in E. coli, and their
enzymatic activity has been characterized. They were isolated from
a Staphylococcus aureus genomic DNA library. From the grlAB nucleic
sequence (SEQ ID No. 1), two open frames, corresponding to the grlB
and grlA genes respectively, have been identified. The grlA and
grlB genes have been sequenced in SEQ ID No. 2 and SEQ ID No. 3
respectively.
[0012] Preferably, the subject of the present invention is a
nucleotide sequence chosen from:
[0013] (a) all or part of the grlA (SEQ ID No. 2) or grlB (SEQ ID
No. 3) genes,
[0014] (b) the sequences hybridizing with all or part of the (a)
genes and encoding a subunit of a topoisomerase IV, and
[0015] (c) the sequences derived from the (a) and (b) sequences
because of the degeneracy of the genetic code.
[0016] It is clear that from the genes identified in the present
application, it is possible, by hybridization, to directly clone
other genes encoding a subunit of topoisomerase IV of bacteria
close to S. aureus such as for example Streptococci and
Enterococci. It is thus possible to clone this type of gene using,
as probe, the genes grlA, grlB or fragments thereof. Likewise, the
cloning of these genes may be carried out using degenerate
oligonucleotides derived from sequences of the grlA or grlB genes
or fragments thereof.
[0017] For the purposes of the present invention, derivative is
understood to mean any sequence obtained by one or more
modifications and encoding a product conserving at least one of the
biological properties of the original protein. Modification should
be understood to mean any mutation, substitution, deletion,
addition or modification of a genetic and/or chemical nature. These
modifications may be performed by techniques known to persons
skilled in the art.
[0018] Among the preferred derivatives, there may be mentioned more
particularly natural variants, molecules in which one or more
residues have been substituted, derivatives obtained by deletion(s)
of regions not or little involved in the interaction between the
binding sites considered or expressing an undesirable activity, and
derivatives having, compared with the native sequence, one or more
additional residues.
[0019] Still more preferably, the subject of the invention is the
nucleotide sequences represented by the grlA (SEQ ID No. 2) and
grlB (SEQ ID No. 3) genes.
[0020] It also relates to any grlA gene having a mutation leading
to a resistance to molecules of the quinolone and more particularly
of the fluoroquinolone family. As a representative of these mutated
genes, there may be mentioned more particularly the grlA gene
having a base change from C to A at position 2270 of SEQ ID No. 2.
The resulting gene is termed grlA.sub.(c-2270A). This mutation
leads to substitution of the Ser-80 residue with Tyr in the GrlA
protein. The resulting protein will be designated by
GrlA.sub.(Ser-80 Tyr).
[0021] Another subject of the present invention relates to a
recombinant DNA comprising at least one nucleotide sequence
encoding a subunit of topoisomerase IV of Staphylococcus aureus.
More preferably, it is a recombinant DNA comprising at least one
nucleotide sequence as defined above in (a), (b) and (c) and more
particularly the gene grlA (SEQ ID No. 2) grlA .sub.(C-2270A)
and/or the gene grlB (SEQ ID No. 3).
[0022] According to a preferred mode of the invention, the
nucleotide sequences defined above form part of an expression
vector which may be autonomously replicating or integrative.
[0023] Another subject of the invention relates to the polypeptides
resulting from the expression of the nucleotide sequences as
defined above. More particularly, the present invention relates to
the polypeptides comprising all or part of the polypeptides GrlA
(SEQ ID No. 2) or GrlB (SEQ ID No. 3) or of their derivatives. For
the purposes of the present invention, the term derivative
designates any molecule obtained by modification of the genetic
and/or chemical nature of the peptide sequence. Modification of the
genetic and/or chemical nature may be understood to mean any
mutation, substitution, deletion, addition and/or modification of
one or more residues. Such derivatives may be generated for
different purposes, such as especially that of increasing the
affinity of the peptide for its substrate(s), that of enhancing its
production levels, that of increasing its resistance to proteases,
that of increasing and/or of modifying its activity, or that of
conferring new biological properties on it. Among the derivatives
resulting from an addition, there may be mentioned, for example,
the chimeric polypeptides containing an additional heterologous
part attached to one end. The term derivative also comprises the
polypeptides homologous to the polypeptides described in the
present invention, derived from other cellular sources.
[0024] Preferably, they are the polypeptides GrlA (SEQ ID No. 2),
GrlB (SEQ ID No. 3) and GrlA.sub.(Ser-80Tyr). The subject of the
invention is also any recombinant cell containing a nucleotide
sequence, a recombinant DNA and/or a vector as defined above. The
recombinant cells according to the inv ntion may be both eukaryotic
and prokaryotic cells. Among the suitable eukaryotic cells, there
may be mentioned animal cells, yeasts, or fungi. In particular, as
regards yeasts, there may be mentioned yeasts of the genus
Saccharomyces, Kluyveromyces, Pichia, Schwanniomyces or Hansenula.
As regards animal cells, there may be mentioned COS, CHO and C127
cells, Xenopus eggs, and the like. Among the fungi, there may be
mentioned more particularly Micromonospora, Asperaillus ssp. or
Trichoderma ssp. Preferably, they are prokaryotic cells. In this
case, the following bacteria may be more particularly used:
Actinomycetes, Bacillus, and more preferably E. coli and
Staphylococcus. The recombinant cells of the invention may be
obtained by any method allowing the introduction of a foreign
nucleotide sequence into a cell. This may be especially
transformation, electroporation, conjugation, fusion of
protoplasts, or any other technique known to persons skilled in the
art.
[0025] The subject of the present invention is also a process for
the preparation of polypeptides as claimed from the culture of one
of these recombinant cells. The polypeptide(s) thus obtained are
recovered according to conventional methods after the culture.
[0026] The invention also relates to an isolated topoisomerase IV
capable of being obtained from the expression of all or part of the
grlA gene (SEQ ID No. 2) and of all or part of the grlB gene (SEQ
ID No. 3) or of their respective derivatives.
[0027] Derivative is understood to designate the sequences
hybridizing with all or part of the grlA or grlB gene and encoding
a subunit of a topoisomerase IV as well as all the sequences
derived from a degeneracy of the genetic code of these hybrid
sequences or of the sequences corresponding to all or part of the
grlA or grlB gene.
[0028] More preferably, it is an isolated topoisomerase IV derived
from the expression of all or part of the grlA gene (SEQ ID No. 2)
or of all or part of the grlB gene (SEQ ID No. 3).
[0029] The present invention relates more particularly to any
topoisomerase IV behaving as a primary target towards
fluoroquinolones.
[0030] According to a specific mode of the invention, it is
topoisomerase IV of Staphylococcus aureus.
[0031] The claimed topoisomerase IV according to the invention is
most particularly useful for targeting biologically active products
such as for example potential antibiotics and especially molecules
of the fluoroquinolone family. Advantageously, it may also be used
to assay and/or identify products inhibiting the ATP-dependent DNA
relaxing reaction and/or the products inhibiting the reaction of
decatenation of catenanes of DNA.
[0032] The applicant has thus developed an assay for enzymatic
activity which is specific for topoisomerase IV of S. aureus and
has shown that this activity is inhibited by antibiotic molecules
such as fluoroquinolones.
[0033] The present invention provides a new target for searching
for new antibiotics, as well as a screen specific for this target;
this screen is described in Example 7. This screen makes it
possible to demonstrate the products which inhibit DNA
topoisomerase IV of S. aureus. The following may be used in this
test: pure or mixed synthetic products, natural plant extracts,
bacterial cultures, fungi, yeasts or algae, pure or in the form of
a mixture. The test described in the present invention makes it
possible to detect both products which stabilize the cleavable
complex, a reaction intermediate of the reaction catalysed by the
enzyme, and also inhibitors acting through other mechanisms.
[0034] The examples and figures presented below by way of
nonlimiting illustration show other advantages and characteristics
of the present invention.
LEGEND TO THE FIGURES
[0035] FIG. 1: Restriction map of the 4565 bp fragment containing
the grlB and grlA genes of S. aureus.
[0036] FIG. 2: Construction of the plasmids for expression of grlA
and grlB. The constructs produced with grlA are schematically
represented in A and those of grlB are in B. The cloned S. aureus
DNA is represented by the shaded rectangles, the vectors derived
from M13 are in thick black line and the expression vectors are in
a fine black line, the SstI restriction site is in brackets because
it is a cloning site.
[0037] FIG. 3: PAGE-SDS electrophoresis gel stained with Coomasie
blue. Total cell extracts are deposited, lanes: 1 and 2, XL1-blue,
pXL2340; 3 and 4, XL1-blue, pRSETB; 5 and 6, XL1-blue, pXL2320. The
molecular weight markers (in hundreds) are indicated on the right
of the figure. The arrow shows the overproduced protein. The + or -
signs represent the induction with or without IPTG.
[0038] FIG. 4: ATP-dependent relaxation activity of the GrlAB
protein. The control experiments with purified DNA topoisomerase IV
of E. coli (Peng and Marians, 1993) and purified DNA gyrase of E.
coli (Hallet et al., 1990) are also described.
[0039] FIG. 5: Decatenation activity of the protein GrlAB. kDNA,
kinetoplast DNA; monomers, relaxed and decatenated DNA monomers.
TopoIV: purified DNA topoisomerase IV of E. coli (50 ng); Gyrase:
purified DNA gyrase of E. coli (50 ng); GrlA: GrlA protein extract
(2 .mu.g); GrlB: GrlB protein extract (2 .mu.g); GrlAB: GrlA
protein extract (2 .mu.g) mixed with the GrlB protein extract (2
.mu.g).
EXAMPLE 1
PCR amplification of DNA Fragments of Staphylococcus aureus Which
are Inside the grlA and grlB Genes
[0040] This example describes the production of DNA fragments of
Staphylococcus aureus which are inside the grlA and grlB genes.
These fragments were obtained after PCR amplification carried out
at 50.degree. C. with genomic DNA of the Staphylococcus aureus
strain RN4220 (Novick, 1990) and of the degenerate oligonucleotides
corresponding to the amino acids conserved in the N-terminal
regions of the subunits GyrA of E. coli and B. subtilis and ParC of
E. coli or of the subunits GyrB of E. coli and B. subtilis and ParE
of E. coli. More specifically, the sense oligonucleotides 2137 and
antisense oligonucleotides 2135 made it possible to amplify
fragments of 255 bp which can encode 85 amino acids which would
correspond to positions 39 to 124 on the E. coli GyrA sequence; the
sequence of the sense oligonucleotide 2137 is
5'-GCGCGAATTCGATGG(A,T)(C,T)T(A,T)AAACC(A,T)GT(A,T)CA-3' (SEQ ID
No. 4) and that of the antisense 2135 is
5'-CGCGAAGCTTTTC(T,A)GTATA(A,T)C(T,G)CA- T(A,T)GC(A,T)GC-3' (SEQ ID
No. 5). The oligonucleotides 2144 and 2138 led to the amplification
of 1 kb fragments which can encode 333 amino acids which would
correspond to positions 98 to 430 on the E. coli GyrB sequence; the
sequence of the sense oligonucleotide 2144 is
5'-GCGCGAATTCT(T,A)CATGC(A,T)GG(T,A)GG(T,A)AAATT-3' (SEQ ID No. 6),
and that of the antisense 2138 is
5'-CGCGAAGCTT(T,A)CC(T,A)CC(T,A)GC(T,A)GAAT- C(T,A)CCTTC-3' (SEQ ID
No. 7) . The fragments were cloned and a total of 40 clones were
analysed by sequencing their insert. The sequence of the
oligonucleotides used for the PCR was found for 31 clones out of
40; among the 31 clones, 20 have a sequence which is inside the
gyrA or gyrB gene of S. aureus; the other 11 clones contain either
a fragment A of 255 bp or a fragment B of 1 kb.
[0041] The amino acid sequence which the A fragment is thought to
encode has 59% identity with the GyrA subunit of S. aureus between
positions 44 to 125, the A fragment is therefore thought to be part
of an S. aureus grlA gene thus newly identified. Likewise, the
amino acid sequence which the B fragment is thought to encode has
51% identity with the GyrB subunit of S. aureus between positions
105 to 277, the B fragment is therefore thought to be part of an S.
aureus grlB gene thus newly identified.
EXAMPLE 2
Cloning and Sequencing of the grlA and grlB Genes of Staphylococcus
aureus
[0042] This example describes the molecular biology experiments
which have made it possible to clone and then sequence the grlA and
grlB genes of Staphylococcus aureus.
[0043] The A and B fragments described in Example 1 were used as
radioactive probe to identify, by hybridization, the grlA and grlB
genes in a genomic DNA library of S. aureus FDA 574 (CE ent.sup.+)
constructed in .lambda.gt11 by Clontech Laboratories (catalogue
XL1501b, batch 0721). Out of a total of 250,000 recombinant phages,
twelve phages hybridize with the A fragment or the B fragment but
do not hybridize with oligonucleotides specific for the gyrA or
gyrB genes. The size of the EcoRI inserts contained in these phages
varies between 0.7 and 3.5 kb and two phages, 16 and 111, whose
insert is of a larger size, were studied. The EcoR1 insert of 3.5
kb of the phage 16 was eluted and then digested with XbaI and the
two fragments of 1.5 and 2 kb were cloned into M13mp19 and M13mp18
(Boehringer Mannheim) in order to generate pXL2321 and pXL2322.
Likewise, the EcoRI insert of 3.6 kb of the phase 111 was eluted
and then digested with PstI and the 2 kb fragment was cloned into
M13mp19 in order to generate pXL2324.
[0044] The inserts contained in the recombinant phages pXL2321,
pXL2322 and pXL2324 were sequenced on both strands with the aid of
the universal primer or of internal oligonucleotides using the
Sanger method. The nucleic sequence grlAB (SEQ ID No. 1) of 4565 bp
was analysed with the programme by Staden et al., 1982 in order to
identify the coding sequences with the aid of a codon usage table
for S. aureus. Only two open frames ORF1 (positions 41 to 2029) and
ORF2 (positions 2032 to 4431) wer thus determined. On SEQ ID No. 1,
the coding strand is th 5'->3' top strand, the open frame ORF1
starts arbitrarily at ATG position 41 but it can also start at TTG
position 17 or 35, this codon being already described as initiation
codon in S. aureus; the stop codon of ORF1 overlaps with the
initiation codon GTG of ORF2, which is characteristic of a
translational coupling (Normark et al., 1983); such a coupling has,
for example, been described for the gyrA and gyrB genes of
Haloferax sp. (Holmes et al., 1991). These open frames have a
percentage of GC of 34.5% which is a value in agreement with the
values described for the S. aureus DNA in the literature (Novick,
1990). Moreover, the B fragment is identical to the sequence
described on SEQ ID No. 1 from position 333 to position 1348 in
ORF1 and the fragment A is identical to the sequence of SEQ ID No.
1 from position 2137 to position 2394 in ORF2. From the nucleotide
sequence, a restriction map is produced with enzymes which cut
least frequently, see FIG. 1.
[0045] This sequence analysis shows that ORF1 is the grlB gene and
ORF2 the grlA gene.
EXAMPLE 3
Primary Structure, Expression and Function of the GrlA and GrlB
Proteins Encoded by the grlA and grlB genes of Staphylococcus
aureus
[0046] This example describes the primary structure, the expression
in E. coli and the function of the GrlA and GrlB proteins of
Staphylococcus aureus. This function, which corresponds to a DNA
topoisom rase IV, is based, in this example, on sequence homology
and genetic complementation data.
[0047] 3.1--Primary Structure and Sequence Analysis of the GrlA and
GrlB Proteins
[0048] This example describes computer analysis of the sequence of
the grlA and grlB genes of Staphylococcus aureus carried out using
the sequence data presented in Example 2. The grlB gene encodes a
GrlB protein of 663 amino acids (molecular weight 74,318), and the
grlA gene encodes a GrlA protein of 800 amino acids (molecular
weight 91,040). The coding parts of the grlB and grlA genes, the
sequences of the GrlB and GrlA proteins are presented in SEQ ID No.
3 and SEQ ID No. 2 respectively and the properties of each of these
proteins (amino acid composition, isoelectric point, polarity
index) are presented in Tables 1 and 2 below.
1 TABLE 1 Protein: GrlA: First residue = 1 and last residue = 800
Molecular mass (monoisotopic) = 91040.8438 Molecular mass (average)
= 91097.2578 Polarity ind x (%) = 52.00 Iso lectric point = 6.77 OD
260 (1 mg/ml) = 0.298 OD 280 (1 mg/ml) = 0.487 NUMBER % NOMB WEIGHT
% WEIGHT 1 Phe F 22 2.75 3235.51 3.55 2 Leu L 74 9.25 8368.22 9.19
3 Ile I 77 9.63 8707.47 9.56 4 Met M 19 2.38 2489.77 2.73 5 Val V
59 7.38 5845.04 6.42 6 Ser S 51 6.38 4438.63 4.88 7 Pro P 22 2.75
2135.16 2.35 8 Thr T 43 5.38 4345.05 4.77 9 Ala A 37 4.63 2628.37
2.89 10 Tyr Y 28 3.50 4565.77 5.02 12 His H 20 2.50 2741.18 3.01 13
Gln Q 26 3.25 3329.52 3.66 14 sn N 45 5.63 5131.93 5.64 15 Lys K 66
8.25 8454.27 9.29 16 Asp D 54 6.75 6211.45 6.82 17 Glu E 67 8.38
8645.85 9.50 18 Cys C 0 0.00 0.00 0.00 19 Trp W 2 0.25 372.16 0.41
20 Arg R 44 5.50 6868.45 7.54 21 Gly G 44 5.50 2508.94 2.76
[0049]
2 TABLE 2 GrlB protein: First residue = 1 and last residue = 663
Molecular mass (monoisotropic) = 74318.3516 Molecular mass
(average) = 74363.9219 Polarity ind x (%) = 53.70 Iso lectric point
= 7.21 OD 260 (1 mg/ml) = 0.404 OD 280 (1 mg/ml) = 0.603 NUMBER %
NOMB WEIGHT % WEIGHT 1 Phe F 26 3.92 3823.78 5.15 2 Leu L 55 8.30
6219.62 8.37 3 Ile I 36 5.43 4071.03 5.48 4 Met M 10 1.51 1310.40
1.76 5 Val V 50 7.54 4953.42 6.67 6 Ser S 41 6.18 3568.31 4.80 7
Pro P 15 2.26 1455.79 1.96 8 Thr T 41 6.18 4142.95 5.57 9 Ala A 33
4.98 2344.22 3.15 10 Tyr Y 19 2.87 3098.20 4.17 12 His H 14 2.11
1918.82 2.58 13 Gln Q 26 3.92 3329.52 4.48 14 Asn N 36 5.43 4105.55
5.52 15 Lys K 63 9.50 8069.98 10.86 16 Asp D 40 6.03 4601.08 6.19
17 Glu E 61 9.20 7871.60 10.59 18 Cys C 0 0.00 0.00 0.00 19 Trp W 4
0.60 744.32 1.00 20 Arg R 34 5.13 5307.44 7.14 21 Gly G 59 8.90
3364.27 4.53
[0050] The Kanehisa programme, described in 1984, was used to align
the GrlB and GrlA proteins with the following type II bacterial DNA
topoisomerases, the E. coli, B. subtilis or S. aureus gyrases or
the E. coli topoisom rase IV. The degrees of identity, a Table 3,
are high and are between 32 and 55%. More specifically, GrlB
exhibits a greater degree of identity with the GyrB subunits of E.
coli (49%) and of S. aureus (52%) than with ParE of E. coli (38%),
whereas GrlA exhibits comparable degrees of identity with the GyrA
subunits of E. coli (32%) and of S. aureus (39%) than with ParE of
E. coli (33%).
[0051] The GyrB subunits of Staphylococcus aureus (Margerrison et
al., 1992), Bacillus subtilis (Moriya et al., 1985), and
Escherichia coli (Adachi et al., 1987) are called SAGYRB, BSGYRB
and ECGYRB respectively, GrlB is called SAGRLB and ECPARE
corresponds to ParE of E. coli (Kato et al., 1990). A similar
nomenclature is used for the GyrA, GrlA and ParC subunits. The
numbers under the name of the proteins are the numbers of amino
acids in them.
3TABLE 3 B or B-like SAGYRB SAGRLB BSGYRB ECGYRB subunits 644 663
638 804 SAGRLB 52% BSGYRB 68% 55% ECGYRB 55% 49% 57% ECPARE 40% 38%
40% 40% A or A-like SAGYRA SAGRLA BSGYRA ECGYRA subunits 887 800
821 875 SAGRLA 39% BSGYRA 65% 40% ECGYRA 39% 32% 41% ECPARC 38% 33%
36% 32%
[0052] Multiple alignments between the type II bacterial topoisom
rases, performed with the CLUSTAL programme of Higgins t al., 1988,
show numerous conserved regions between the sequences of the
various B, GrlB and ParE subunits and in the N-terminal part of the
sequence of the A, GrlA and ParC subunits. The residues conserved
in the N-terminal region of the B subunits of these proteins are in
fact the residues involved in the binding of ATP and identified
from X-ray crystallization data with the E. coli GyrB (Wigley et
al., 1991). The residues conserved in the N-terminal region of the
A subunits of these proteins are either the residues AAMRYTE (SEQ
ID No. 8) close to the active site of gyrase Tyr-122, identified on
the E. coli GyrA (Horowitz et al., 1987), or the residues YEPHGDS
(SEQ ID No. 9) modified in the strains resistant to
fluoroquinolones (Hooper et al., 1993).
[0053] 3.2--Expression of the grlA and grlB genes in E. coli.
[0054] This example describes the constructs produced in order to
express, in E. coli, the grlA or grlB genes under the control of
the pT7 promoter (Studier et al., 1990). The expression plasmid
pXL2320, see FIG. 2, containing the grlB gene in the vector pRSETB
(Studier et al., 1990; Invitrogen) was constructed by cloning 1)
the 1 kb EcoRI-XbaI insert of pXL2321 into pXL2322 at the XbaI and
EcoRI sites in order to gene rat pXL2323; 2) th 1.9 kb KpnI-EcoRI
insert of pXL2323 at th KpnI and EcoRI sites of the vector pRSETB
in order to gene rat pXL2319; the 0.5 kb NdeI-KpnI insert of
pXL2325 at th NdeI and KpnI sites of pXL2319 in order to obtain
pXL2320. (pXL2325 contains the first 500 bas s of the gene where a
CAT sequence has been introduced by mutagenesis, just upstream of
the ATG initiation codon, in order to create an NdeI site). The
grlB gene expression cassette contained in pXL2320 was cloned at
the BalI and EcoRI sites of pKT230 (Bagdasarian et al., 1981) in
order to obtain pXL2439. The expression plasmid pXL2340, see FIG.
2, containing the grlA gene in the vector pRSETB was constructed by
cloning 1) the 1.7 kb Ndel-EcoRI insert of pXL2324 at the NdeI and
EcoRI sites of the vector pRSETB in order to generate pXL2338; the
0.75 kb NdeI insert of pXL2337 at the NdeI sites of pXL2338 in
order to obtain pXL2340. (pXL2337 contains the first 750 bases of
the gene where a CATATG sequence has been introduced by
mutagenesis, in place of the GTG initiation codon in order to
create an NdeI site).
[0055] The plasmids pXL2320, or pXL2340 were introduced into the E.
coli XL-1-Blue strain (Stratagen) and the expression of the genes
was induced when the T7 phage RNA polymerase was produced after
induction of the gene, encoding the T7 phage RNA polymerase, cloned
into the helper phage M13/T7 (Studier et al., 1990, Invitrogen).
The cellular extracts were analysed by electrophoresis on a
PAGE-SDS gel stained with Coomasie blue as has already been
described (Denfle et al., 1987). In FIG. 3 is represented the
production of a protein with a i) molecular weight of 79,000, when
the grlB gene is induced in the E. coli strain XL1-blue, pXL2320;
and ii) molecular weight of 90,000, when the grlA gene is induced
in the E. coli strain XL1-Blue, pXL2340. The measured molecular
weights are in agreement with the molecular weights deduced from
the sequence.
[0056] 3.3--Complementation of the parCts and parEts Mutants of
Salmonella typhimurium by the grlA and grlB Genes of Staphylococcus
aureus
[0057] This example describes the heterologous complementation of
the S. typhimurium parCts and parEts mutants by the S. aureus grlA
and grlB genes. The plasmids pXL2320, pXL2340, pXL2439 or the
vector pRSETB were introduced into the S. typhimurium strains
SE7784 (parC281(Ts) zge-2393::Tn10 leu485) or SE8041 (parE206(Ts)
zge-2393::Tn10 leu485) (Luttinger et al., 1991). No plasmid
complements the heat-sensitive phenotype; on the other hand, when
the plasmids pXL2340 and pXL2439 are introduced simultaneously into
the SE7784 strain or into the SE8041 strain, the heat-sensitive
phenotype of both strains is complemented. Consequently, the
coexpression of the grlA and grlB genes of S. aureus allows the
complementation of the ParC Ts or ParE Ts phenotype of the S.
typhimurium mutants.
EXAMPLE 4
The DNA Topoisomerase IV of S. aureus is the Primary Target of the
Fluoroquinolones
[0058] This example describes the presence of a point mutation
Ser-80 in the GrlA subunit with all the analysed clinical strains
of S. aureus resistant to the fluoroquinolones whereas a mutation
in the QRDR region (Quinolone Determining Region) (equivalent to
the Ser-80 region of GrlA) in the GyrA subunit does not exist with
the clinical strains of S. aureus weakly resistant to the
fluoroquinolones. Consequently, the GrlA subunit is shown to be the
primary target of the fluoroquinolones in S. aureus.
[0059] The genomic DNA of eight clinical strains of S. aureus and
of a laboratory strain was prepared and used to amplify at
42.degree. C. by PCR: i) the first 500 base pairs of gyrA using the
sense oligonucleotide 5'-GGCGGATCCCATATGGCTGAATTACCTCA-3' (SEQ ID
No. 10) and the antisense oligonucleotide 5'-GGCGGAAT
TCGACGGCTCTCTTTCATTAC-3' (SEQ ID No. 11); ii) and the first 800
base pairs of grlA using the sense oligonucleotide
5'-GGCCGGATCCCATATGAGTGAAATAATTCAAGATT-3' (SEQ ID No. 12) and the
antisense oligonucleotide 5'-GGCCGAATTCTAATAATTAACTGTTTACGTCC-3'
(SEQ ID No. 13). Each amplified fragment was cloned into the phage
M13mp18 and the sequence of the first 300 base pairs of ach of the
genes was read n 2 clones. The gyrA sequence is identical to that
published by Magerrison and that of grlA to that described in SEQ
ID No. 1, with the exception of the mutations presented in Table 4.
The mutations in gyrA exist with the strains highly resistant to
fluoroquinolones (SA4, SA5, SA6, SA35, SA42R and SA47; MIC for
ciprofloxacin >16 mg/l); these mutations are a base change which
leads to changes in the amino acids Ser-84 or Ser-85 or Glu-88. A
mutation in grlA exists with all the strains resistant to
fluoroquinolones and corresponds to the changing of the residue
Ser-80 to Phe or Tyr.
4TABLE 4 MIC mg/l Mutation in gyrA Mutation in grlA Strain
Ciprofloxacin Base Codon Base Codon RN4220* 1 no no no no SA42* 0.5
no no no no SAH** 2 no no 2281 .sup.84Glu->Lys G->A SA1* 2 no
no 2270 .sup.80Ser->Phe C->T SAA** 4 no no 2281
.sup.84Glu->-Lys G->A SA3** 4 no no 2270 .sup.80Ser->Phe
C->T SA2** 16 no no 2270 .sup.80Ser->Tyr C->A SA47* 16
2533 .sup.84Ser->Leu 2270 .sup.80Ser->Tyr C->T* C->A
SA4** 32 2544 .sup.88Glu->Lys 2270 .sup.80Ser->Phe G->A
C->T SA5** 32 2533 .sup.84Ser->Leu 2270 .sup.80Ser->Phe
C->T C->T SA6** 32 2533 .sup.84Ser->Leu 2270
.sup.80Ser->Phe C->T C->T SA35* 64 2535 .sup.85Ser->Pro
2270 .sup.80Ser->Tyr T->C C->A SA42R* >128 2533
.sup.84Ser->Leu 2270 .sup.80Ser->Tyr C->T* C->A
*already published by Sreedharan et al. (1990) **strains obtained
from French state hospitals.
EXAMPLE 5
PCR (Polymerase Chain Reaction) Amplification of the S. aureus DNA
Fragment Which is Inside grlA Containing a Point Mutation Which
Leads to a Substitution in GrlA from Ser-80 to Tyr
(Ser-80.fwdarw.Tyr)
[0060] This example describes the production of the DNA fragment
which is inside grlA of an S. aureus strain, SA2, resistant to
fluoroquinolones. The grlA fragment contains a base change from C
to A at position 2270 of the wild-type gene (FIG. 1). This mutation
leads to a substitution of the residue S r-80 to Tyr in the GrlA
protein. It has been shown that a substitution of the residue
Ser-80 to Phe or Tyr exists with all the strains weakly resistant
to fluoroquinolones (Example 4). The fragment which is inside grlA
was obtained after PCR amplification carried out at 50.degree. C.
with the genomic DNA of the SA2 strain and of the oligonucleotides
3358 and 3357 which correspond to position 2036 and 3435
respectively on the sequence of grlA. More specifically, the sense
oligonucleotide 3358 (SEQ ID No. 12) (Example 4) and the antisense
oligonucleotide 3357 made it possible to amplify a fragment of 1399
base pairs; the sequence of the antisense oligonucleotide 3357 is
5'-GGCCGAGCTCCAATTCTTCTTTTATGACATTC-3' (SEQ ID No. 14). The
oligonucleotide 3358 was also used to introduce, by mutagenesis, a
sequence CATATG, in place of the GTG initiation codon in order to
create an NdeI site. The amplified grlA fragment was cloned into
the BamHI/SstI cloning sites of pUC18 (Boehringer Mannheim), and 6
clones containing this plasmid, pXL2692, were analysed after
sequencing their insert. In all cases, a sequence CATATG was
introduced in place of the CTG initiation codon, and the point
mutation at position 2270 of grlA (C.fwdarw.A) was again found.
EXAMPLE 6
Expression in E. coli of the grlA Gene Containing a Base Change
Corresponding to the Change of the Residue Ser-80 to Tyr
[0061] This example describes the construct prepared in order to
express, in E. coli, the mutated grlA gene under the control of the
T7 promoter (Studier et al., 1990). The expression plasmid pXL2742,
containing the mutated grlA gene, was constructed by cloning the
0.75 kb insert of pXL2692 into the NdeI site of pXL2338 (Example
3.2). The plasmid pXL2742 was introduced into the E. coli XL1-Blue
strain and the expression of the grlA gene was carried out as
described in Example 3.2. The production of a protein having a
molecular weight of 90,000 was obtained with the plasmid pXL2742
containing the grlA gene. The molecular weight measured is in
agreement with the molecular weight deduced from the sequence of
the grlA gene, and that already obtained for the wild-type GrlA
protein (Example 3.2).
EXAMPLE 7
DNA Topoisomerase IV Activity of the GrlAB Protein of S. aureus
[0062] This example illustrates how an acellular extract containing
the GrlAB protein can be prepared and how the enzymatic activity of
the GrlAB protein present in this extract can be detected and
measured.
[0063] 7.1--Preparation of the Cell Extracts
[0064] An acellular extract of the E. coli strain XL1-blue pXL2340
expressing the GrlA protein is prepared for example in the
following manner:
[0065] The E. coli strain XL1-blue pXL2340 is cultured as follows:
250 ml of LB medium containing ampicillin at 50 mg/l are inoculated
at {fraction (1/100)} with a culture of E. coli XL1-blue pXL2340,
and incubated at 30.degree. C.; when the optical density at 600 nm
is 0.3, 1 mM IPTG is added; after incubating for 30 min at
37.degree. C., the strain is infected with the helper phage M13/T7
with a multiplicity of infection of 5 pfu per cell for 4 hours.
After centrifugation (5000.times.g; 20 min), the cells obtained
using 1.5 litres of culture are resuspended in 20 ml of 50 mM
Tris/HCl buffer pH 7.8 containing 10 mM EDTA, 150 mM NaCl, 1 mM
DTT, 0.12% Brij 58 and 0.75 mg/ml of lysozyme. After 30 min at
4.degree. C., the mixture is centrifuged for 1 h at 50,000.times.g
and the supernatant containing the GrlA protein is recovered. A
change of buffer is carried out on this sample by chromatographing
the extract through a column filled with Sephadex G625 (Pharmacia)
equilibrated and eluted with the 50 mM Tris/HCl buffer pH 7.5
containing 1 mM EDTA, 5 mM DTT, 100 mM NaCl and 10% sucrose.
[0066] An acellular extract containing the GrlB protein is prepared
in a similar manner using the E. coli strain XL1-blue pXL2320.
[0067] 7.2--Purification of the DKN Topoisom rase IV of S.
aureus.
[0068] This example illustrates how an S. aureus enzyme catalysing
the segregation of the daughter chromosomes during the final phase
of replication (topoisomerase IV) can be purified.
[0069] The purification of the two GrlA and GrlB subunits of
topoisomerase IV is carried out as described below, using the
decatenation activity assay described in Example 7.3 to detect the
presence of the GrlA and GrlB proteins during the purification, as
is commonly used by persons skilled in the art. During the assay of
this enzymatic activity, complamentation of the fractions
containing the GrlA protein is obtained with 1 .mu.g of proteins of
an extract of the E. coli strain XL1-blue pXL2320 expressing the
GrlB subunit, and complementation of the fraction containing the
GrlB protein is obtained with 1 .mu.g of proteins of an extract of
the E. coli strain XL1-blue pXL2340 expressing the GrlA subunit. A
preferred mode of preparation of the enzymatic extracts is
described in Example 7.1. Between each stage, the fractions
containing the desired protein are frozen and stored at -70.degree.
C. The purification of the A subunit may be carried out by
chromatography, for example, according to the following
procedure:
[0070] an acellular extract prepared as described in Example 7.1
using about 5 g of cells of E. coli XL1-blue pXL2340 is
chromatographed on a MonoQ HR 10/10 column (Pharmacia) at a flow
rate of 3 ml/min with a linear gradient of NaCl (0.1M to 0.6M over
60 min) in a 10 mM Tris/HCl buffer pH 8.0 containing 1 mM EDTA, 1
mM DTT and 10% glycerol (w/v). The active fractions are combined
and the sample is chromatographed on a Superdex 200 HiLoad 26/60
column (Pharmacia) equilibrated and eluted with 50 mM Tris/HCl
buffer pH 7.5 containing 1 mM EDTA, 5 M DTT and 0.25 M NaCl. The
GrlA protein, which exists in the form of a symmetrical peak, is
coeluted with the desired activity. After this stage, the
preparation shows a single visible band in SDS-PAGE after
developing with silver nitrate, and this band migrates with an
apparent molecular weight of about 90,000.
[0071] The purification of the B subunit may be carried out by
chromatography, for example, according to the following
procedure:
[0072] an acellular extract prepared as described in Example 5
using about 5 g of cells of E. coli XL1-blue pXL2320 is injected
onto a Novobiocin-Sepharose CL-6B column (6 ml of gel prepared
according to the procedure described by Staudenbauer et al., 1981,
Nucleic Acids Research) equilibrated in 50 mM Tris/HCl buffer pH
7.5 containing 1 mM EDTA, 5 mM DTT and 0.3 M NaCl. After washing
the column with the same buffer, the GrlB protein is eluted with 50
mM Tris/HCl buffer pH 7.5 containing 1 mM EDTA, 5 mM DTT and 2 M
NaCl and 5 M urea. This fraction is then chromatographed on a
Superdex 200 HiLoad 26/60 gel permeation column (Pharmacia)
equilibrated and eluted with 50 mM Tris/HCl buffer pH 7.5
containing 1 mM EDTA, 5 mM DTT and 0.25 M NaCl. The GrlB protein,
which exists in the form of a symmetrical peak, is coeluted with
the desired activity. After this stage, the preparation has a
single visible band in SDS-PAGE after developing with silver
nitrate, and this band migrates with an apparent molecular weight
of about 80,000.
[0073] 7.3--Detection of the Enzymatic Activities of the GrlAB
Protein.
[0074] The various enzymatic activities of the GrlAB protein are
detected by incubating, in the same reaction mixture, equal
quantities of the two types of extracts prepared by the process
described above or by any other process which makes it possible to
recover the intracellular enzymatic proteins of the microorganism
while preserving their activity, such as for example the procedures
involving the use of presses (such as the French Press, the
X-Press), or the use of ultrasound.
[0075] The ATP-dependent supercoiled DNA relaxing activity can be
detected by carrying out the procedure, for example, in the
following manner:
[0076] a mixture of an extract of the E. coli strain XL1-blue
pXL2320 (1 .mu.g of proteins) and of an extract of the E. coli
strain XL1-blue pXL2340 (1 .mu.g of proteins) is incubated for 1 h
at 37.degree. C. in 30 .mu.l of 50 mM Tris/HCl buffer pH 7.7
containing 4 mM ATP, 6 mM MgCl.sub.2, 5 mM DTT, 1 mM spermidine, 20
mM KCl, 50 .mu.g/ml of bovine serum albumin and 500 ng of
supercoiled plasmid pBR322. The reaction is stopped by adding 7
.mu.l of a 5% SDS and 2.5 mg/ml proteinase K mixture and the
samples are incubated for a second period of 30 min at 37.degree.
C. and then analysed by electrophoresis on 1% agarose gel in 0.1M
Tris/borate buffer pH 8.3 containing 2 mM EDTA at 6V/cm for 3 h.
The separation of the relaxed and nicked (open circular) DNAs is
carried out by performing an additional 2 h electrophoretic run
after addition of ethidium bromide (1 .mu.g/ml) to the running
buffer. The DNA is then quantified by scanning the negatives of
photographs of the gels (Polaroid type 665 film) with the aid of a
Bioimage 50S apparatus (Millipore).
[0077] FIG. 4 shows that the acellular extracts of the strains E.
coli XL1-blue pXL2320 and E. coli XL1-blue pXL2340 exhibit in a
mixture an intense DNA relaxing activity whereas each of the
extracts is inactive when it is incubated alone. The reaction is
ATP-dependent. Furthermore, these two extracts, alone or in the
form of a mixture, exhibit no DNA supercoiling activity, an
activity typical of gyrase.
[0078] Th ATP-dependent activity of decatenation of intertwined
circular DNA molecules (catenanes) can be detected by carrying out
the procedure, for example, in the following manner: a mixture of
an extract of the E. coli strain XL1-blue pXL2320 (2.5 .mu.g of
proteins) and of an extract of the E. coli strain XL1-blue pXL2340
(2.5 .mu.g of proteins) is incubated for 1 h at 37.degree. C. in 40
.mu.l of 50 mM Tris/HCl buffer pH 7.7 containing 1 mM ATP, 6 mM
MgCl2, 200 mM glutamate, 10 mM DTT, 10 mM NaCl, 50 .mu.g/ml of
bovine serum albumin and 800 ng of kinetoplast DNA [consisting of a
network of intertwined DNA molecules (catenanes) obtained from
Crithidia fasciculata; TopoGene]. The reaction is stopped by adding
7 .mu.l of a 250 mM EDTA solution (incubation 5 min at 37.degree.
C.), 5 .mu.l of a 5% SDS and 2.5 mg/ml proteinase K mixture
(incubation 30 min at 37.degree. C.). The mixture is then analysed
by electrophoresis on a 1% agarose gel in a 0.1M Tris/borate buffer
pH 8.3 containing 2 mM EDTA at 6V/cm for 2 h 30 min. After staining
the DNA with ethidium bromide (1 .mu.g/ml), the DNA is quantified
by scanning the negatives of photographs of the gels (Polaroid type
665 film) with the aid of a Bioimage 50S apparatus (Millipore). By
working, for example, under the conditions described above, the
extracts of the two strains E. coli XL1-blue pXL2320 and E. coli
XL1-blue pXL2340 exhibit, in the form of a mixture, an activity of
complete decatenation of the starting kinetoplast DNA. This
activity is detected by the appearance of a DNA band with a size of
about 2.5 kb and by the disappearance of the band of catenated DNA
of very large size which penetrates very little into the gel during
the electrophoretic run (FIG. 5). The E. coli gyrase introduced as
a control into this assay exhibits no decatehation activity
contrary to DNA topoisomerase IV of E. coli which completely
decatenates the kinetoplast DNA (FIG. 5).
EXAMPLE 8
DNA Topoisomerase IV Activity of the GrlAB Protein of S. aureus
whose GrlA Subunit Exhibits a Substitution of the Residue Ser-80 to
Tyr (Ser-80.fwdarw.Tyr)
[0079] 8.1--Preparation of a Cell Extract Containing the GrlAB
protein of S. aureus whose GrlA Subunit Exhibits a substitution of
the residue Ser-80 to Tyr (Ser-80.fwdarw.Tyr).
[0080] This example illustrates how an acellular extract containing
the protein GrlA.sub.(Ser-80.fwdarw.Tyr)B can be prepared, and how
the enzymatic activity of the protein GrlA.sub.(Ser-80.fwdarw.Tyr)B
can be detected and measured.
[0081] An acellular extract of the E. coli strain XL1-Blue pXL2742
expressing the protein GrlA.sub.(Ser-80.fwdarw.Tyr) is prepared,
for example, as described in Example 7 for the wild-type GrlA
protein.
[0082] 8.2--Purification of a DNK Topoisomerase IV of S. aureus
Exhibiting an Ser-80-Tyr mutation in the GrlA Subunit.
[0083] This example illustrates how a topoisomerase IV of S. aureus
exhibiting an Ser-80.fwdarw.Tyr mutation in the GrlA subunit can be
purified. The GrlA subunit of topoisomerase IV having an
Ser-80.fwdarw.Tyr mutation is purified according to a procedure
identical to that described in Example 7.2 using a culture of the
E. coli strain XL1-blue pXL2742 constructed as described in Example
6.
[0084] 8.3--Detection of the Enzymatic Activities.
[0085] The ATP-dependent activities of supercoiled DNA relaxation,
on the one hand, and of decatenation of intertwined circular DNA
molecules, on the other hand, are detected in this extract as
described in Example 7, by incubating, in the same reaction
mixture, an acellular extract of the E. coli strain XL1-Blue
pXL2742 containing the protein GrlA.sub.(Ser-80.fwdarw.Tyr) and an
extract of the E. coli strain XL1-Blue pXL2320 containing the GrlB
protein.
EXAMPLE 9
Inhibition by Fluoroquinolones, of the DNA Topoisomerase IV
Activity of the Wild-type GrlAB Protein of S. aureus and Resistance
to Fluoroquinolones of the Protein Containing an Ser-80.fwdarw.Tyr
Transition in the GrlA subunit
[0086] The two methods described in Example 7 for the assay of DNA
topoisomerase IV activities can be used to detect novel molecules
which act as inhibitors of topoisom rase IV of S. aureus or to
characterize the behaviour of topoisomerase IV of S. aureus towards
molecules already identified as inhibitors of other topoisomerases
(for example the fluoroquinolones).
[0087] In the test of relaxation of supercoiled DNA for example,
the disappearance or the decrease in the relaxed DNA band during
analysis of the reaction mixture after incubation of the GrlAB
protein of S. aureus in the presence of a molecule or of a mixture
of several molecules indicates that this molecule (or these
molecules), inhibit the relaxation activity of GrlAB, and is
therefore potentially antibacterial. However, since the studies
carried out up until now (described in Example 7) have demonstrated
that the GrlAB protein is a topoisomerase IV, and since it is
nowadays established that the major function of the topoisomerases
IV is decatenation (or disentanglement) of the intertwined daughter
chromosomes during the final stages of replication, it seems more
judicious to search for the inhibitors of the GrlAB protein using a
test of decatenation of DNA using, for example, the test described
in Example 7.3. To carry out the experiments described in the
examples which follow, the incubations are carried out with the
purified wild-type GrlAB proteins as described in Example 7, and
with the mutant protein GrlA.sub.(Ser-80.fwdarw.Tr)B as described
in Example 8. The two wild-type and mutant GrlAB proteins are
reconstituted by mixing equimolar quantities of their two GrlA and
GrlB subunits.
[0088] In the decatenation test, if the disappearance or the
decrease in the intensity of the decatenated DNA band is observed
during analysis of the reaction mixture after incubation of the
GrlAB protein in the presence of a molecule or of a mixture of
several molecules, this indicates that this molecule (or these
molecules) inhibits the decatenation activity of the GrlAB protein,
and is therefore potentially antibacterial. Since it has been
demonstrated in the present invention that the GrlAB protein is the
primary target for the molecules of the fluoroquinolone family, it
appears that the fluoroquinolones must act as inhibitors in the
decatenation test described in Example 7. Indeed, when the purified
GrlAB protein is incubated in the presence of increasing quantities
of a fluoroquinolone, for example ciprofloxacin, it appears that
above a concentration of 10 .mu.g/ml, ciprofloxacin completely
inhibits the activity of decatenation of the kinetoplast DNA.
Ciprofloxacin inhibits 50% of the activity of decatenation of
kinetoplast DNA at a concentration of 4.0 .mu.g/ml.
[0089] Likewise, sparfloxacin which is another fluoroquinolone
inhibits 50% of the activity of decatenation of kinetoplast DNA at
a concentration of 6.0 .mu.g/ml. Likewise, since it has been
demonstrated in the present invention (Example 4) that the presence
of an Ser-80.fwdarw.Tyr mutation on the GrlA subunit of the mutant
GrlAB protein confers on the strain a certain level of resistance
to fluoroquinolones, for example ciprofloxacin, it appears that the
fluoroquinolones must act on this mutant DNA topoisomeras IV as
inhibitors which are less efficient in the decatenation test
described in Example 7.
[0090] Indeed, when the mutant protein
GrlA.sub.(Ser-80.fwdarw.Tyr)B is incubated in the presence of
increasing quantities of a fluoroquinolone, for example
ciprofloxacin, it appears that ciprofloxacin inhibits 50% of the
activity of decatenation of kinetoplast DNA at a concentration of
60 .mu.g/ml, that is to say a concentration 15 times as high as
that necessary to obtain the same effect with the wild-type
enzyme.
[0091] Likewise, in the presence of the mutant enzyme
GrlA.sub.(Ser-80.fwdarw.Tyr)B, sparfloxacin inhibits 50% of the
activity of decatenation of kinetoplast DNA at a concentration of
500 .mu.g/ml, that is to say a concentration 80 times as high as
that necessary to obtain the same effect with the wild-type
enzyme.
[0092] Norfloxacin inhibits 50% of the activity of decatenation of
kinetoplast DNA at a concentration of 12 .mu.g/ml with the
wild-type GrlAB enzyme and exhibits the same inhibitory activity at
a concentration of 125 .mu.g/ml with the enzyme
GrlA.sub.(Ser-80.fwdarw.Tyr)B. Ofloxacin inhibits 50% of the
activity of decatenation of kin toplast DNA at a concentration of
10 .mu.g/ml with the wild-typ GrlAB nzyme and has the sam
inhibitory activity at a concentration of 250 .mu.g/ml with the
enzyme GrlA.sub.(Ser-80.fwdarw.Tyr)B.
[0093] Novobiocin, whos mechanism of action is different from that
of the fluoroquinolones, should therefor in principle have the same
inhibitory activity on both the wild-type GrlAB enzyme and on the
mutant GrlA.sub.(Ser-80.fwdarw.Tyr)B enzyme in the decatenation
test described in Example 7. Indeed, novobiocin inhibits 50%, of
the activity of decatenation of kinetoplast DNA at a concentration
of about 30 .mu.g/ml whatever the enzyme used (wild-type GrlAB or
mutant GrlA.sub.(Ser-80.fwdarw.Tyr)).
ABBREVIATIONS
[0094] DNA: deoxyribonucleic acid
[0095] RNA: ribonucleic acid
[0096] MIC: minimum inhibitory concentration
[0097] IPTG: isopropylthio-.beta.-D-galactoside
[0098] LB: Luria-Bertani medium
[0099] PAGE: electrophoresis gel containing acrylamide and
N,N'-methylenebisacrylamide
[0100] PCR: polymerase chain reaction
[0101] pfu: plaque forming unit
[0102] QRDR: region of the GyrA subunit where the point mutations
leading to resistance to fluoroquinolones are mapped
[0103] SDS: sodium dodecyl sulphate
[0104] Tris: tris(hydroxymethyl)aminomethane
REFERENCES
[0105] Adachi, T., Mizuuchi, M., Robinson, E. A., Apella, E., O'D
a, M. H., Gell rt, M., and Mizuuchi K. (1987) DNA sequence of the
E. coli gyrB gene: application of a new sequencing strategy. Nucl
Acid Res 15: 771-784.
[0106] Bagdasarian, M., Lurz, R., Ruckert, B., Franklin, F. C.,
Bagdasarian, M. M., Frey, J., and Timmis, K. (1981)
Specific-purpose plasmid cloning vectors. II. Broad host range,
high copy number, RSF1010-derived vectors, and a host-vector system
for gene cloning in Pseudomonas. Gene 16: 237-247.
[0107] Colman, S. D., Hu, P. C., and Bott, K. F. (1990) Mycoplasma
pneumoniae DNA gyrase genes. Mol Microbiol 4: 1129-11134.
[0108] Cullen, M. E., Wyke, A. W., Kuroda, R., and Fisher, L. M.
(1989) Cloning and characterization of a DNA gyrase gene from
Escherichia coli that confers clinical resistance to 4-quinolones
Antimicrob Agents Chemother 33: 886-894.
[0109] Denefle, P., Kovarik, S., Guiton, J. D., Cartwright, T., and
Mayaux, J.-F. (1987) Chemical synthesis of a gene coding for human
angiogenin, its expression in Escherichia coli and conversion of
the product into its active form. Gene 56: 61-70.
[0110] Gellert, M., Mizuuchi, K., O'Dea, M. E. and Nash, H. A.
(1976) DNA gyrase: an enzym that introduces superhelical turns into
DNA Proc Natl Acad Sci USA 73: 3872-3876.
[0111] Goswitz, J. J., Willard, K. E., Fasching, C. E., and Pet
rson, L. R. (1992) Detection of gyrA g ne mutations associated with
ciprofloxacin resistance in methicillin-resistant Staphylococcus
aureus: analysis by polymerase chain reaction and automated direct
DNA sequencing. Antimicrob .mu.gents Chemother 36: 1166-1169.
[0112] Higgins, D. G., and Sharp, P. M. (1988) Clustal: a package
for performing multiple sequence alignment on a microcomputer. Gene
73: 237-244.
[0113] Holmes, M. L., and Dyall-Smith, M. (1991) Mutations in the
DNA gyrase result in novobiocin resistance in halophilic
archaebacteria. J Bacteriol 173: 642-648.
[0114] Hooper, D. C., Wolfson, J. S. (1993) Mechanisms of quinolone
action and bacterial killing. In Quinolone Antimicrobial .mu.gents.
Hooper, D. C., Wolfson, J. S. (eds) Washington: American Society of
Microbiology, pp. 53-75.
[0115] Horowitz, D. S., and Wang, J. C. (1987) Mapping of the
active site tyrosine of Escherichia coli DNA gyrase. J Biol Chem
262: 5339-5344.
[0116] Huang, W. M. (1992) Multiple DNA gyrase-like genes in
Eubacteria. In Molecular Biology of DNA Topoisomerases and its
Application to Chemotherapy. Andoh, T., Ikeda, H., and Oguro, M.
(eds). London: CRC Press, pp. 39-48.
[0117] Kanehisa, M. (1984) Use of statistical criteria for
screening potential homologies in nucleic acids sequences. Nucl
Acids Res 12: 203-215.
[0118] Kato, J., Suzuki, H., and Ikeda, H. (1992) Purification and
characterization of DNA topoisomerase-IV in Escherichia coli. J
Biol Chem 267: 25676-25684.
[0119] Kato, J., Nishimura, Y., Imamura, R., Niki, H., Higara, S.,
and Suzuki, H. (1990) New topoisomerase essential for chromosome
segregation in E. coli. Cell 63: 393-404.
[0120] Luttinger, A. L., Springer, A. L., and Schmid, M. B. (1991)
A cluster of genes that affects nucleoid segregation in Salmonella
typhimurium. New Biol 3: 687-697.
[0121] Margerrison, E. E. C., Hopewell, R. and L. M. Fisher. (1992)
Nucleotide sequence of the Staphylococcus aureus gyrB-gyrA locus
encoding the DNA gyrase A and B proteins. J Bacteriol 174:
1596-1603.
[0122] Maxwell, A. (1992) The molecular basis of quinolone action.
J. Antimicrob. Chemother. 330: 409-416.
[0123] Moriya, S., Ogasawara, N. and Yoshida, H. (1985) Structure
and function of the region of the replication origin of Bacillus
subtilis chromosome. III. Nucleotide sequence of some 10,000 base
pairs in the origin region. Nucl Acid Res 13: 2251-2265.
[0124] Normark, S., Bergtrom, S., Edlund, T., Grundstrom, T.,
Jaurin, B., Lindberg, F., and Olsson, O. (1983) Overlapping genes.
Ann Rev Gen t 17: 499-525.
[0125] Novick, R. P. (1990) Th staphylococcus as a molecular
genetic system. In Molecular Biology of the Staphylococci. Novick,
R. P. (ed). New York: VCH Publishers, pp. 1-37.
[0126] Parales, R. E., and Harwood, C. S. (1990) Nucleotide
sequence of the gyrB gene of Pseudomonas putida. Nucl Acid Res 18:
5880-5880.
[0127] Peng, H., Marians, K. J. (1993 (a)) Escherichia coli
topoisomerase IV Purification, charcterization, subunit structure,
and subunit interactions. J Biol Chem 268: 24481-24490.
[0128] Peng, H., Marians, K. J. (1993 (b)) Decatenation activity of
topoisomerase IV during oriC and pBR322 DNA replication in vitro.
Proc Natl Acad Sci USA 90: 8571-8575.
[0129] Sambrook J., Fritsch, E. F., and Maniatis, T. (1989)
Molecular Cloning: a Laboratory Manual 2nd edn. Cold Spring Harbor,
New York: Cold Spring Harbor Laboratory Press.
[0130] Sreedharan, S., Peterson, L., and Fisher, L. M. (1991)
Ciprofloxacin-resistance in coagulase-positive and -negative
Staphylococci: role of mutations at serine 84 in the DNA gyrase A
protein of Staphylococcus aureus: and Staphylococcus epidermidis
Antimicrob Agents Chemother 35: 2151-2154.
[0131] Sreedharan, S., Oram, M., Jensen, B., Peterson, L., and Fish
r, L. M. (1990) DNA gyrase gyrA mutations in
ciprofloxacin-resistant strains of Staphylococcus aureus: close
similarity with quinolone resistance mutations in Escherichia coli.
J Bacteriol 172: 7260-7262.
[0132] Staden, R., and McLachlan, A. D. (1982) Codon preference and
its use in identifying protein coding regions in long DNA
sequences. Nucl Acid Res 10: 141-156.
[0133] Staudenbauer, W. L., and Orr, E. (1981) DNA gyrase: affinity
chromatography on novobiocin-Sepharose and catalytic properties
Nucleic Acid Research 9: 3589-3603
[0134] Stein, D. C., Danaher, R. J., and Cook, T. M. (1991)
Characterization of a gyrB mutation responsible for low-level
nalidixic acid resistance in Neisseria gonorrhoeae. Antimicrob
Agents Chemother 35: 622-626.
[0135] Studier, W. F., Rosenberg, A. H., Dunn, J. J., and
Duberndorff, J. W. (1990) Use of T7 RNA polymerase to direct
expression of cloned genes. Methods Enzymol. 185: 89-60.
[0136] Swamberg, S. L. and Wang, J. C. (1987) Cloning and
sequencing of the Escherichia coli DAN gyrA gene coding for the A
subunit of DNA gyrase. J Mol Biol 197: 729-736.
[0137] Thiara, A. M., and Cundliffe, E. (1993) Expression and
analysis of two gyrB genes from the novobiocin producer,
Streptomyces sphaeroides. Mol Microbiol 8: 495-506.
[0138] Wang, J. C., and Liu, L. F. (1990) DNA replication:
topological aspect and the roles of DNA topoisomerases. In DNA
Topology and its Biological Effects. Cozzarelli, N. R., and Wang,
J. C. (eds). New York: Cold Spring Harbor Laboratory Press, pp.
321-340.
[0139] Wang, Y., Huang, W. M. and Taylor, D. E. (1993) Cloning and
nucleotide sequence of the Campylobacter jejuni gyrA gene and
characterization of quinolone resistance mutations. Antimicrob
Agents and Chemother 37: 457-463.
[0140] Wigley, D. B., Davies, G. J., Dodson, E. J., Maxwell, A.,
and Dodson, G. (1991) Crystal structure of an NH.sub.2-terminal
fragment of the DNA gyrase B protein. Nature 351: 624-629.
[0141] Yoshida, H., Bogaki, M., Nakamura, M., and Nakamura, S.
(1990) Quinolone resistance determining region in the DNA gyrase
gene of Escherichia coli. Antimicrobe Agents Chemother 34:
1271-1272.
Sequence CWU 1
1
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