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Previous Article | Next Article 
Antimicrobial Agents and Chemotherapy, August 2001, p. 2280-2286, Vol. 45, No. 8
0066-4804/01/$04.00+0 DOI: 10.1128/AAC.45.8.2280-2286.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Macrolide Resistance Gene mreA of
Streptococcus agalactiae Encodes a
Flavokinase
Gervais
Clarebout,1
Corinne
Villers,2 and
Roland
Leclercq1,*
Service de Microbiologie, UPRESA 2128,
Hôpital Côte de Nacre, Université de Caen, 14033 Caen
Cedex,1 and Laboratoire de Biochimie,
UPRESA 2608 CNRS, Université de Caen, 14032 Caen
Cedex,2 France
Received 5 January 2001/Returned for modification 6 March
2001/Accepted 23 May 2001
 |
ABSTRACT |
The mreA gene from Streptococcus
agalactiae COH31 
/
, resistant to macrolides and
clindamycin by active efflux, has recently been cloned in
Escherichia coli, where it was reported to confer macrolide resistance (J. Clancy, F. Dib-Hajj, J. W. Petitpas, and
W. Yuan, Antimicrob. Agents Chemother. 41:2719-2723,
1997). Cumulative data suggested that the mreA gene was
located on the chromosome of S. agalactiae COH31 /.
Analysis of the deduced amino acid sequence of mreA
revealed significant homology with several bifunctional
flavokinases/(flavin adenine dinucleotide (FAD) synthetases, which
convert riboflavin to flavin mononucleotide (FMN) and FMN to FAD,
respectively. High-performance liquid chromatography experiments showed
that the mreA gene product had a monofunctional flavokinase
activity, similar to that of RibR from Bacillus subtilis. Sequences identical to those of the mreA gene and of a
121-bp upstream region containing a putative promoter were detected in strains of S. agalactiae UCN4, UCN5, and UCN6 susceptible
to macrolides. mreA and its allele from S. agalactiae UCN4 were cloned on the shuttle vector pAT28. Both
constructs were introduced into E. coli, where they
conferred a similar two- to fourfold increase in the MICs of
erythromycin, spiramycin, and clindamycin. The MICs of a variety of
other molecules, including crystal violet, acriflavin, sodium dodecyl
sulfate, and antibiotics, such as certain cephalosporins,
chloramphenicol, doxycycline, nalidixic acid, novobiocin, and rifampin,
were also increased. In contrast, resistance to these compounds was not
detected when the constructs were introduced into E. faecalis JH2-2. In conclusion, the mreA gene was
probably resident in S. agalactiae and may encode a
metabolic function. We could not provide any evidence that it was
responsible for macrolide resistance in S. agalactiae COH31
/; broad-spectrum resistance conferred by the gene in E. coli could involve multidrug efflux pumps by a mechanism that
remains to be elucidated.
| |
INTRODUCTION |
Streptococcus agalactiae
(group B streptococcus) is responsible for neonatal sepsis and
meningitis as well as serious invasive infections in adults, such as
postpartum endometritis (6). The first line of therapy for
these infections consists of administration of beta-lactam agents.
However, macrolides and related drugs are useful alternate therapies in
allergic patients.
Until recently, macrolide resistance in streptococci was considered to
result only from target modification by 23S rRNA methylases encoded by
erm genes, which conferred cross-resistance to macrolides, lincosamides, and streptogramin B components
(MLSB phenotype) (21). Another
phenotype, called M, related to efflux of only 14- and 15-member ring
macrolides, has been reported in various streptococcal species,
including Streptococcus pneumoniae, Streptococcus pyogenes, and S. agalactiae. The mechanism of
resistance relies on a proton-dependent efflux system encoded by
mef(A) class genes: (3, 15, 19). The
mef(A) genes belong to the major facilitator superfamily and are believed to encode a hydrophobic membrane protein
containing 12-membrane-spanning regions that pumps the antibiotic out of the cell. In addition, a novel efflux system distinct
from the Mef pump and encoded by mreA (for macrolide resistance efflux) was recently reported in a unique strain of S. agalactiae COH31 / by Clancy et al. (4). The
strain harboring this gene was resistant to 14-, 15-, and 16-member
macrolides and to clindamycin. The results of experiments with
radiolabeled erythromycin suggested the presence of a macrolide efflux
mechanism. The mreA gene was cloned from total DNA of
S. agalactiae COH31 / into Escherichia
coli, where it conferred macrolide resistance. The presence of the
gene in E. coli also resulted in a significant decrease in
erythromycin accumulation. Sequencing revealed that mreA
encodes a 310-amino-acid protein, with a predicted molecular mass of
35.4 kDa. This protein is hydrophilic with interspersed hydrophobic and
amphipathic sequences. The protein displayed homology with RibC, a
flavokinase/flavin adenine dinucleotide (FAD) synthetase from
Bacillus subtilis; however, its function has not been
studied (4). The present study demonstrates that the
product of the mreA gene displays a flavokinase activity and
is responsible for a broad-spectrum resistance to a variety of
compounds when cloned in E. coli, but not when expressed in
Enterococcus faecalis.
| |
MATERIALS AND METHODS |
Bacterial strains, plasmids, and growth conditions.
The
bacterial strains and plasmids used in this study are listed in Table
1. Streptococcal strains were grown on
Trypticase soy (TS) agar (Bio-Rad, Marnes-la-Coquette, France)
supplemented with 5% horse blood. E. coli, Bacillus
subtilis, and Enterococcus faecalis strains were
cultured in TS broth or agar. All cultures were incubated at 37°C.
Susceptibility testing.
MICs of antibiotics were determined
by the agar dilution method with Mueller-Hinton medium (Bio-Rad)
supplemented with 5% horse blood inoculated with
104 CFU and incubated at 37°C under aerobic
conditions according to the recommendations of the Comité de
l'Antibiogramme de la Société Française de
Microbiologie (5). The antibiotics and molecules tested
were supplied by Sigma Chemical Co. (St. Louis, Mo.) or by their manufacturer.
PCR and cloning experiments.
DNA sequences specific for the
mreA gene were amplified by PCR with the primers mre3
(5'-ATA AAG AAA GTC AAT CAT G-3' [nucleotides 106 to 124])
and mre4 (5'-AT ACA AAA AAT TAA AGA G-3' [nucleotides 1064 to 1045]). The numbers in brackets refer to the numbers of the
mreA sequence in the GenBank database (accession no.
U92073). PCRs were performed with a GeneAmp PCR system 2400 cycler
(Perkin-Elmer Cetus, Norwalk, Conn.) with Taq DNA polymerase
(Eurobio, Les Ullis, France). The mreA gene preceded by a
121-bp sequence containing a putative promoter (4) was
amplified from the DNA of three macrolide-susceptible S. agalactiae strains, UCN4, UCN5, and UCN6, by PCR with
oligonucleotides mre5 (5'-CTT ATT AGA AAA TGA AGC AG-3'
[nucleotides 1 to 20]) and mre4. The various amplicons were
cloned into plasmid pCR2.1 (Invitrogen, Groningen, The Netherlands) in
the same orientation. The recombinant plasmids were introduced into
competent E. coli DH10B cells by
electrotransformation with a Gene Pulser (Bio-Rad) and
selected by using TS agar plates containing 50 µg of kanamycin per ml.
The fragments were then subcloned on the multicopy shuttle vector pAT28
(spectinomycin resistance) with the SacI and XbaI restriction sites (20). The plasmid constructs were made
with E. coli DH10B prior to transformation into
E. faecalis JH2-2, as described previously
(12), and were selected by using TS agar plates containing
60 and 150 µg of spectinomycin per ml, respectively.
The ribC and promoter sequences were amplified from B. subtilis Marburg 168 DNA by PCR with oligonucleotides ribc1
(5'-ATT GCC GTC TTT ACT GAA TCC G-3' [nucleotides 241 to
262]) and ribc2 (5'-AAA CTA TCA TAC TAA AAA TCG TGC C-3'
[nucleotides 1387 to 1363]). The numbers in brackets refer to
numbers of the ribC sequence in the GenBank database
(accession no. X95312). The amplicon was cloned into plasmid pCR2.1 and
introduced into competent E. coli DH10B cells.
Southern blot hybridization.
DNA from S. agalactiae COH31 / was digested with the restriction
endonuclease HindIII. DNA fragments were separated in a 0.7% agarose gel, denatured, and transferred onto a nylon membrane (Hybond-N; Amersham France, Les Ullis, France). The 738-bp
mreA-specific PCR product obtained with the primers mre1
(5'-AAT TTG AAA ATT GTC GTC TTA ACG T-3' [nucleotides 260 to 285]) and mre2 (5'-GTT GTT TTA CAA GAT CGT CAA TAC C-3'
[nucleotides 997 to 974]) was used as a probe. This product was
labeled with digoxigenin (Boehringer Mannheim France, Meylan, France),
and hybridization was detected by using an anti-digoxigenin-alkaline
phosphatase conjugate with a chromogenic enzyme substrate.
The chromosomal location of the mreA gene was determined by
restriction of total DNA of S. agalactiae COH31 / with
I-CeuI (New England Biolabs, Beverly, Mass.) an
intron-encoded endonuclease specific for rRNA genes, followed by pulsed
field gel electrophoresis as previously described (13).
DNA fragments were transferred onto a nylon membrane and successively
hybridized with 16S rRNA and mreA probes.
Inverse PCR.
In order to sequence the DNA regions located
upstream and downstream of mreA, DNA of S. agalactiae COH31 / was digested with HindIII
and ligated with T4 ligase. Inverse PCR was performed with
oligonucleotides mre1 and mre in2 (5'-CGC AAT CTT CTT TAG CTT GAA
TAT C-3' [nucleotides 176 to 152]) with Taq
polymerase (Eurobio). The reaction consisted of (i) an initial
step of 3 min at 94°C; (ii) 35 cycles of PCR, with 1 cycle consisting
of 30 s at 94°C, 30 s at 50°C, and 120 s at 72°C;
and (iii) a final step of 10 min at 72°C with 2 mM
MgCl2. A 3.5-kb amplified fragment was cloned in
pCR2.1 and sequenced with an automated ABI PRISM 377 system
(Perkin-Elmer Corp.). Nucleotide and amino acid sequences were analyzed
by using the software available online over the internet at the
National Center for Biotechnology Information web site
(http://www.ncbi.nlm.nih.gov/). The microbial databases used were those
of The Institute for Genomic Research (TIGR)
(http://www.tigr.org), the Doe Joint Genome Institute (JGI)
(http://www.jgi.doe.gov), and The University of Oklahoma
(http://www.genome.ou.edu).
Preparation of cell extracts, enzyme assay, and HPLC analysis of
flavins.
Cell extracts of E. coli DH10B containing
various constructs were prepared as follows. Cells of an overnight
culture (100 ml) were collected by centrifugation. The cell pellet was
washed with a mixture of 100 mM potassium phosphate (pH 7.5), 0.1 mM EDTA, and 1 mM dithiothreitol (buffer A). The cells were resuspended in buffer A and sonicated twice for 10 s. After centrifugation (18,000 × g for 30 min), an aliquot of the
supernatant was directly used in the flavokinase assay. All procedures
were carried out at 4°C. Protein concentrations were determined by
the method of Bradford, with reagents from the Bio-Rad protein assay
and with bovine serum albumin as a standard (2).
The 
-lactamase activity encoded by the plasmid pCR2.1 was measured
to standardize the extract. Assays were performed by UV spectrophotometry with freshly prepared penicillin G solutions in 100 mM phosphate buffer (pH 7.0). The assays were run at 37°C and
monitored at 235 nm (22).
Flavokinase activity was measured in a final volume of 1 ml of
potassium phosphate (pH 7.5) containing 50 µM riboflavin, 3 mM ATP,
15 mM MgCl2, and 10 mM
Na2SO3. A similar FAD
synthetase assay containing 50 µM flavin mononucleotide (FMN) instead
of riboflavin was performed to measure the formation of FAD from FMN
and ATP (14). The mixture was preincubated for 5 min at 37°C; the reaction was started by addition of the cell extract and
stopped by boiling after 5 or 30 min of incubation. A centrifugation eliminated the denatured proteins.
The high-performance liquid chromatography (HPLC) analysis required a
C8 Satisfaction column (4.6 by 250 mm) (CIL,
Cluzeau, France) and fluorescence detector (excitation, 470 nm;
emission, 530 nm) (ThermoQuest, Les Ullis, France). The solvent system
was composed of 40% methanol in 100 mM potassium phosphate (pH 4) and
used at a flow rate of 1 ml/min. Flavokinase activity was expressed as
micrograms of FMN formed from riboflavin and ATP per minute and per
milligram of total protein. FAD synthetase activity was expressed as
micrograms of FAD formed from FMN and ATP per minute and per milligram
of total protein.
| |
RESULTS |
Detection of mreA in erythromycin-susceptible
Streptococcus agalactiae strains.
A 960-bp DNA
fragment internal to mreA was amplified from DNA of S. agalactiae COH31 / and, surprisingly, that of three clinical
erythromycin-susceptible S. agalactiae isolates with oligonucleotides mre3 and mre4. To assess if mutations could explain the differences in erythromycin susceptibility of the strains, we have
amplified and sequenced a 1,065-bp DNA fragment including the entire
mreA gene and a 121-bp upstream region containing a putative
promoter (4) and the allelic sequences from the three erythromycin-susceptible strains. Previous cloning of this 1,065-bp fragment in the two opposite orientations by Clancy et al. has shown
that it contained the sequences required to confer a two- to
fourfold decrease in macrolide susceptibility in E. coli (4). Sequencing revealed complete identity
between all of the DNA fragments. In a recent study, the
mreA gene was found by PCR in all strains of a collection of
88 clinical isolates of S. agalactiae resistant to
macrolides and containing erm or mef genes,
whereas it was not found in two strains of group G streptococcus
strains (E. Bingen, personal communication). We did not detect by
PCR mreA sequences in group A streptococci (10 strains) and
pneumococci (10 strains).
mreA confers a broad-spectrum drug resistance when
cloned in E. coli
mreA with
its putative promoter and the allele mreAS from
S. agalactiae UCN4, susceptible to erythromycin, were
cloned on plasmid pCR2.1 to generate plasmids pUV6 and pUV7,
respectively. The inserts were subsequently subcloned on the shuttle
plasmid pAT28 to generate plasmids pUV8 and pUV9, respectively. The
recombinant plasmids were introduced into E. coli
DH10B. In this host, all constructs conferred the same levels of
resistance to erythromycin (MIC = 128 µg/ml), spiramycin (a
16-member macrolide) (MIC = 1,024 µg/ml), and clindamycin
(MIC = 128 µg/ml). These MICs corresponded to an increase of
a factor 2 or 4, as reported previously by Clancy et al.
(4). This increase was repeatedly found in several experiments.
In addition, MreA and its allele conferred in E. coli a
similar four- to eightfold increase in MICs of acriflavin, as reported previously (4), as well as an increase in MICs of a
variety of other compounds, including cationic dyes, such as crystal
violet; detergents, such as sodium dodecyl sulfate; various
antibiotics, including cefoxitin, cefepime, and ceftazidime; lipophilic
compounds, such as rifampin (zwitterionic) and doxycycline; and
hydrophobic agents, such as novobiocin and nalidixic acid, as well as
chloramphenicol, an uncharged antibiotic. Except for the
cross-resistance to macrolides (MIC of erythromycin = 4 µg/ml) and clindamycin (MIC = 0.5 µg/ml) in S. agalactiae COH31 /, no other differences in the MICs of the
tested compounds for the four tested S. agalactiae strains were found.
The pUV8 and pUV9 constructs were introduced into E. faecalis JH2-2. The stability of the pAT28 derivatives in
E. faecalis was verified by confirming the
expression of spectinomycin resistance at crucial steps of the
experiments. The presence of mreA was also verified
by PCR. HPLC experiments showed that flavokinase was expressed in
E. faecalis, although at a slightly lower level than in
E. coli. Retransformation of E. coli
with the recombinant plasmid extracted from E. faecalis
led to increased MICs of erythromycin and of the other compounds at the
expected level. In contrast, in the E. faecalis
background, pUV8 and pUV9 did not confer any increase in the MICs of
the compounds tested, including erythromycin.
MreA is a flavokinase.
MreA shared a significant degree of
similarity with several members of an enzyme family possessing a
bifunctional flavokinase/FAD synthetase activity. Flavokinases (EC
2.7.1.26) catalyze the conversion of riboflavin to FMN, whereas FAD
synthetases (EC 2.7.7.2) convert FMN to FAD; these two reactions
require ATP as a cofactor (1). The optimal aligment of the
amino acid sequence of MreA revealed a 37% identity with RibC, a
bifunctional flavokinase/FAD synthetase from B. subtilis
(316 amino acids), and 30.4 and 32.7% identity with flavokinases/FAD
synthetases RibF from E. coli (313 amino acids) and
Haemophilus influenzae (312 amino acids), respectively (Fig.
1) (7, 8; K. Kitatsuji,
S. Ishino, S. Teshiba, and M. Arimoto, 1993, European patent
application 0 542 240 A2). Two conserved motifs were found in the
C-terminus ends of MreA and FAD synthetases (Fig. 1). Furthermore, the
hydrophobic cluster analysis showed that MreA and FAD synthetases
proteins shared similar presumed secondary structures (data not shown).
These results suggested that mreA could encode a
bifunctional flavokinase/FAD-synthetase.
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FIG. 1.
Alignment of the N-terminus amino acid sequence of RibR
from B. subtilis with the C termini of the
mreA gene product (MreA), and the bifunctional
flavokinase/FAD-synthetase from B. subtilis,
E. coli (E.c), and H.
influenzae (H.i) with the CLUSTALW program. Dashes indicate
gaps introduced to increase the number of matches. Homologous and
similar amino acids are represented by double and single dots,
respectively. Asterisks represent amino acid residues identical among
the five sequences. The boxed blocks of amino acids correspond to
motifs of riboflavin kinase/FAD synthetase according to Block Searcher
results.
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To elucidate the function of MreA, flavokinase and FAD synthetase
activities were measured in cell extracts of E. coli
containing either pUV6 (mreA cloned in pCR2.1) or pUV7
(mreAS cloned in pCR2.1). The flavokinase and FAD synthetase
enzymatic activities were investigated by HPLC with riboflavin and FMN
as substrate donors, respectively. The preliminary calibration of HPLC
methodology showed that peaks of FAD, FMN, and riboflavin were resolved
at 4.3, 5.9, and 7.9 min, respectively, similar to the retention times
reported by Mack et al. (14). After 30 min of incubation
at 37°C of the cell extracts containing MreA (Fig.
2) or MreAS (data not shown) with
riboflavin, there was a decrease in the size of the riboflavin peak
concomitant with an increase in FMN production, which demonstrated that
both cell extracts displayed a flavokinase activity. Despite the high
level of flavokinase activity, no significant FAD peak was detected
after 30 min of incubation of cell extracts in the presence of FMN as a
donor, indicating the absence of FAD synthetase activity (Fig. 2B).
Flavokinase activities measured in cell extracts of E. coli containing mreA or mreAS were similar
and corresponded respectively to 0.087 and 0.083 µg of FMN produced
per min per mg of protein. Control experiments with extracts from
E. coli containing pCR2.1 did not reveal any
flavokinase or FAD synthetase activity (data not shown). Probably the
level of FMN or FAD produced by the flavokinase of E. coli encoded by a gene present as a single copy in the chromosome
was too low to be detected by the HPLC technique. The RibC control
showed a flavokinase activity (0.062 µg of FMN produced per min per
mg of protein) and a FAD synthetase activity (0.049 µg of FAD
produced per min per mg of protein) when riboflavin and FMN,
respectively, were used as substrate donors (Fig. 2).
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FIG. 2.
HPLC chromatograms of the products of flavokinase/FAD
synthetase assays. Flavokinase (A, B, E, and F) and FAD synthetase (C,
D, G, and H) activities were evaluated by fluorescence detection in the
presence of 50 µM riboflavin (RB) and 50 µM FMN, respectively. Cell
extracts of E. coli DH10B/pUV6 containing
mreA (A, B, C, and D) or E. coli
DH10B/pUV10 containing ribC (E, F, G, and H) were added
to the reaction mixture, and the activity assays were incubated for 5 min (A, C, E, and G) or 30 min (B, D, F, and H) and stopped by boiling.
Aliquots were removed and separated on an HPLC column. The
chromatograms show three clearly resolved peaks of riboflavin (7.9 min), FMN (5.9 min), and FAD (4.3 min). Peak intensity is given in
arbitrary fluorescence units.
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Location of the mreA gene.
The mreA
and the rrs probes hybridized with the same
I-CeuI-generated fragment of S. agalactiae COH31
/ DNA (Fig. 3). This observation
was strongly in favor of a chromosomal location of the mreA
gene. Southern blot experiments with a probe specific for
mreA confirmed that only one chromosomal copy of the gene could be detected in S. agalactiae COH31 / (data
not shown). DNA regions located upstream and downstream of the
mreA gene were amplified by inverse PCR. A 3.5-kb amplified
fragment was cloned in pCR2.1, introduced in E.coli
DH10B, and sequenced. Upstream of mreA, sequence analysis
identified an open reading frame (ORF) that could encode a
214-amino-acid protein that displayed 48% identity with B. subtilis TruB, a 309-amino-acid protein (17). TruB is
a tRNA pseudouridine 55 (psi 55) synthase, an enzyme specific for the
conversion of U55 to pseudouridine in the CG loop of most tRNAs
(10). Nucleotide sequence analysis showed the presence of
an additional 414-bp ORF downstream of mreA, 43 bp after the termination codon of the gene. This ORF could encode a protein of 137 amino acids, which shared 28% identity with arsenate reductase (ArsC) of B. subtilis, a protein of 118 amino acids.
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FIG. 3.
Analysis of genomic DNA from S.
agalactiae COH31 / (lane 1) and UCN4 (lane 2), digested
with I-CeuI by pulsed-field gel electrophoresis (left)
and hybridization (middle and right). The digested fragments were
transferred to a nylon sheet and hybridized to an in vitro
digoxigenin-labeled 16S probe (middle). After dehybridization, the
filter was hybridized to a digoxigenin-labeled mreA
probe (right).
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The homologs of truB, ribC, and arsC
were identified on the chromosome of gram-positive microorganisms,
including S. pyogenes, S. pneumoniae, Enterococcus
faecium, and B. subtilis, according to the BLAST
program for unfinished and finished bacterial genomes (http://www.tigr.org./tdb/mdb/mdbcomplete.html). The organization of the genes was similar within these organisms, consistent with a
likely chromosomal location of the mreA gene in S. agalactiae (Fig. 4).
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FIG. 4.
Genetic organization of mreA and
ribC homologs in different Streptococcus
species, in E. faecium, and in B.
subtilis. These results were obtained after analysis with Blast
2 program of sequences of B. subtilis from Kunst et al.
(11), S. pneumoniae M1 from Oklahoma
University (http://www.genome.ou.edu), S. pneumoniae
type 4 from the TIGR database (http://www.tigr.org), and E.
faecium from the JGI database (http://www.jgi.doe.gov). The
percentages of identity relative to the B. subtilis
genes, obtained with the ALIGN program, are indicated within the
arrows. rpsO, ribosomal protein S15 gene;
pnpA, polynucleotide phosphorylase gene;
truB, tRNA pseudouridine 55 synthase gene;
arsC, arsenate reductase gene. Numbers refer to genomic
position.
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DISCUSSION |
Analysis of the deduced amino acid sequence of MreA revealed that
this protein displayed homology with various bifunctional flavokinases/FAD synthetases. We have shown that cell extracts of
E. coli expressing this protein possessed only a
monofunctional riboflavin kinase activity and were devoid of FAD
synthetase activity. By using site-specific mutagenesis on the
flavokinase/FAD synthetase gene from E. coli, it was
shown that the flavokinase activity of the enzyme was associated with
the C-terminal region, and the FAD synthetase activity was associated
with the N-terminal region (Kitatsuji et al., patent application).
Recently, a monofunctional flavokinase, RibR, has been reported in
B. subtilis (18). This 230-amino-acid protein
is about 100 amino acids shorter than the bifunctional FAD synthetases,
including RibC from B. subtilis. Similarly to the C terminus
of MreA, RibR showed significant homology with the C-terminus regions
of RibC and RibF of E. coli and H. influenzae, starting near residue 200. In particular, a conserved motif of 10 amino acid residues, GR(K/T)(L/I)GFPTAN,
between residues 15 and 24 (numbering with respect to the
ribR gene product) was found.
Cumulative and convergent data strongly suggested that the
mreA gene was chromosomal in S. agalactiae COH31
/. Sequencing of the flanking regions revealed that it was
surrounded by truB and arsC homologs. Analysis of
the data banks showed that a similar genetic linkage between homologs
of truB and arsC and flavokinase genes could be
found in the chromosome of other gram-positive organisms, including
several Streptococcus species and B. subtilis. The gene organization appeared different in the E. coli
chromosome, where ribF was located between infB
and rpsO (16).
Taken together, these observations suggested that the mreA
gene was resident in S. agalactiae and could putatively
encode a metabolic function. Several of our results questioned the role of the mreA gene in conferring resistance to
macrolides in S. agalactiae COH31 /. First,
sequences identical to mreA were found in
macrolide-susceptible strains of S. agalactiae
conferring similar levels of macrolide resistance after
cloning in E. coli. No difference in the sequences
upstream of the mreA gene, which included a putative
promoter, could be detected, and the gene was present in one copy on
the chromosome of S. agalactiae COH31 /. However, we
did not compare the transcription of the mreA gene in the
erythromycin-susceptible or erythromycin-resistant strains. We could
not characterize the macrolide resistance phenotype after disruption of
the mreA gene, since we were unable to introduce plasmid DNA
by electrotransformation in S. agalactiae COH31
/. In addition, the phenotype of isolated resistance to
macrolides and clindamycin displayed by S. agalactiae COH31
/ could not be reproduced after cloning of the gene either in
E. coli or in a gram-positive host. In E. coli, it conferred broad-spectrum resistance, while in
E. faecalis, no expression of resistance could be
detected. The mechanism by which the flavokinase MreA conferred
macrolide resistance in E. coli remains unclear.
The enzyme did not inactivate erythromycin (4).
Intriguingly, Clancy et al. have shown in studies of
[14C]erythromycin accumulation that an
energy-dependent macrolide efflux mechanism was associated with the
presence of mreA in E. coli
(4). This erythromycin efflux was inhibited by uncouplers of oxidative phosphorylation, such as CCCP (carbonyl
cyanide-m-chlorophenylhydrazone) and arsenate, proving that
efflux was an energy-dependent process. Interestingly, the spectrum of
resistance conferred by mreA extends to numerous other
compounds, including other flavins, such as acriflavin, and various
antibiotics. This effect did not appear to be specifically related to
the presence of the mreA gene, but rather to flavokinase
activity, since similar MIC increases were observed when
ribC was introduced into E. coli (data not
shown). Both the pattern of broad-spectrum antibiotic resistance and
the energy-dependent efflux of erythromycin suggested that multidrug efflux pumps, possibly belonging to the resistance nodulation division
(RND) family, could intervene to confer resistance. This requirement
for the presence of a functional gram-negative pump for expression of
resistance would be consistent with the lack of expression of
mreA in gram-positive hosts. This hypothesis is currently
under investigation.
| |
ACKNOWLEDGMENTS |
G. Clarebout was the recipient of a FEDER fellowship from the
Conseil Régional de Basse-Normandie.
We are grateful to A. Coquerel and D. Debruyne from the
Department of Pharmacology for help with the HPLC experiments and J. Clancy for the gift of the S. agalactiae
COH31 / strain.
| |
FOOTNOTES |
*
Corresponding author, CHU de Caen, Service de
Microbiologie, Avenue Côte de Nacre, 14033 Caen Cedex, France.
Phone: (33) 02 31 06 45 72. Fax: (33) 02 31 06 45 73. E-mail:
leclercq-r{at}chu-caen.fr.
| |
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Antimicrobial Agents and Chemotherapy, August 2001, p. 2280-2286, Vol. 45, No. 8
0066-4804/01/$04.00+0 DOI: 10.1128/AAC.45.8.2280-2286.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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Top
Abstract
Introduction
