MgrA is a multiple regulator of two new efflux pumps in Staphylococcus aureus

Abstract

In an analysis of the resistance mechanisms of an mgrA mutant, we identified two genes encoding previously undescribed transporters, NorB and Tet38. norB was 1,392 bp and encoded a predicted 49-kDa protein. When overexpressed, NorB led to an increase in resistance to hydrophilic quinolones, ethidium bromide, and cetrimide and also to sparfloxacin, moxifloxacin, and tetracycline, a resistance phenotype of the mgrA mutant. NorA and NorB shared 30% similarity, and NorB shared 30 and 41% similarities with the Bmr and Blt transporters of Bacillus subtilis, respectively. The second efflux pump was a more selective transporter that we have called Tet38, which had 46% similarity with the plasmid-encoded TetK efflux transporter of S. aureus. tet38 was 1,353 bp and encoded a predicted 49-kDa protein. Overexpression of tet38 produced resistance to tetracycline but not to minocycline and other drugs. norB and tet38 transcription was negatively regulated by MgrA. Limited binding of MgrA to the promoter regions of norB and tet38 was demonstrated by gel shift assays, suggesting that MgrA was an indirect regulator of norB and tet38 expression. The mgrA norB double mutant was reproducibly twofold more susceptible to the tested quinolones than the mgrA mutant. The mgrA tet38 double mutant became more susceptible to tetracycline than the wild-type parent strain. These data demonstrate that overexpression of NorB and Tet38 contribute, respectively, to the hydrophobic quinolone resistance and the tetracycline resistance of the mgrA mutant and that MgrA regulates expression of norB and tet38 in addition to its role in regulation of norA expression.

FIG. 1.

Amino acid sequence alignment of TetL of B. subtilis (GenBank accession number YTBSRT), TetK of S. aureus (GenBank accession number P02983), and Tet38 of S. aureus (GenBank accession number AY825285). *, identical residues; :, conserved residues. Solid highlights represent the TMS of the correspondent transporter: blue for TetL, red for TetK, and yellow for Tet38.

FIG. 2.

Amino acid sequence alignment of NorB and NorA proteins of S. aureus and Bmr and Blt proteins of B. subtilis. GenBank accession numbers for NorA, Bmr, and Blt are BAA14147, M33768, and L32599, respectively. Identical residues are represented in red, and conserved residues are represented in blue. Green represents identical residues between two or three proteins.

FIG. 3.

Northern blot analysis of RNAs isolated from various S. aureus strains. The same amount of RNA (10 μg) was loaded in each lane and was verified by ethidium bromide staining before RNAs were transferred onto a nylon membrane. (A) RNAs isolated from S. aureus ISP794 (wild type), QT1 (mgrA::cat), and ISP94(pQT10) (tet38 overexpressor) were hybridized with a tet38-specific probe. (B) RNAs isolated from S. aureus ISP794, QT1, and ISP794(pQT8) (norB overexpressor) were hybridized with a norB-specific probe. (C) Ethidium bromide staining of the RNA after electrophoresis. The gel was appropriately divided for the Northern analysis.

FIG. 4.

Gel mobility shift analysis of the interaction of the MgrA-His₆ protein with the norA (150 bp), norB (150 bp), and tet38 (200 bp) upstream putative promoter fragments. The labeled DNA fragments were incubated with increasing amounts of purified proteins. MgrA binds to the tested DNA and retards its mobility, creating different gel shift patterns for different promoter fragments. MgrA binding to the norA promoter was previously demonstrated to be specific (35). The binding of MgrA to the norB promoter is specific, proven by a deplacement of the shifted DNA by unlabeled norB DNA, while nonspecific DNA had no effect on MgrA-norB promoter binding.

FIG. 5.

Schematic representation of the orientation of the norB and tet38 genes and their neighboring ORFs on the S. aureus N315 published genome. The arrows indicates the gene orientations.

FIG. 6.

Phylogenetic tree of NorB, Tet38, and related proteins. Each protein has the accession number indicated. Bmr (NP_390281), Blt (NP_390536), and TetL(YTBSRT) are from B. subtilis. NorA (A37838), QacA (CAA39963), QacB(AAQ10697), MdeA(NP_372939), and SepA (BAB83937) are from S. aureus. EmrA (P27303) and EmrB (P27304) are from E. coli. The tree was created and analyzed with the web program available at http://tcdb.ucsd.edu/progs/msaTMS.php (40).