Macrolide resistance gene mreA of Streptococcus agalactiae encodes a flavokinase

Abstract

The mreA gene from Streptococcus agalactiae COH31 gamma/delta, 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 gamma/delta. 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 gamma/delta; broad-spectrum resistance conferred by the gene in E. coli could involve multidrug efflux pumps by a mechanism that remains to be elucidated.

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.

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.

FIG. 3

Analysis of genomic DNA from S. agalactiae COH31 γ/δ (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).

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.