Transport processes are used by all organisms to obtain essential nutrients and to expel wastes and other potentially harmful substances from cells. Such processes are important means by which resistance to selected antimicrobial agents in bacteria is achieved. The recently described Staphylococcus aureus norA gene encodes a membrane-associated protein that mediates active efflux of fluoroquinolones from cells. SA-1199B is a fluoroquinolone-resistant strain of S. aureus from which we cloned an allele of norA (norA1199). Similar to that of norA, the protein product of norA1199 preferentially mediates efflux of hydrophilic fluoroquinolones in both S. aureus and an Escherichia coli host, a process driven by the proton motive force. Determination of the nucleotide sequence of norA1199 revealed an encoded 388-amino-acid hydrophobic polypeptide 95% homologous with the norA-encoded protein. Significant homology with other proteins involved in transport processes also exists, but especially with tetracycline efflux proteins and with the Bacillus subtilis Bmr protein that mediates active efflux of structurally unrelated compounds, including fluoroquinolones. In S. aureus, the norA1199-encoded protein also appears to function as a multidrug efflux transporter. Southern hybridization studies indicated that norA1199 (or an allele of it) is a naturally occurring S. aureus gene and that related sequences are present in the S. epidermidis genome. The nucleotide sequence of the wild-type allele of norA1199, cloned from the fluoroquinolone-susceptible parent strain of SA-1199B, did not differ from that of norA1199 throughout the coding region. Northern (RNA) and Southern hybridization studies showed that increased transcription, and not gene amplification, of norA1199 is the basis for fluoroquinolone resistance in SA-1199B.

The gene of the Staphylococcus aureus fluoroquinolone efflux transporter protein NorA confers resistance to a number of structurally dissimilar drugs, not just to fluoroquinolones, when it is expressed in Bacillus subtilis. NorA provides B. subtilis with resistance to the same drugs and to a similar extent as the B. subtilis multidrug transporter protein Bmr does. NorA and Bmr share 44% sequence similarity. Both the NorA- and Bmr-conferred resistances can be completely reversed by reserpine.

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The NorA protein of Staphylococcus aureus mediates the active efflux of hydrophilic fluoroquinolones from the cell, conferring low-level resistance upon the organism. This protein also is capable of transporting additional structurally diverse compounds, indicating that it has a broad substrate specificity. Increased transcription of the norA gene, leading to a greater quantity of the NorA protein within the cytoplasmic membrane, is felt to be the mechanism by which strains possessing such changes resist fluoroquinolones. S. aureus SA-1199 and its in vivo-selected derivative SA-1199B are fluoroquinolone-susceptible and -resistant isolates, respectively; SA-1199B resists hydrophilic fluoroquinolones via a NorA-mediated mechanism in a constitutive manner. SA-1199-3 is an in vitro-produced derivative of SA-1199 in which NorA-mediated multidrug resistance is expressed inducibly. Compared with organisms exposed to subinhibitory concentrations of a NorA substrate for the first time, preexposure of SA-1199-3 to such a compound followed by growth in the presence of that substrate results in the elimination of a 2-to 6-h period of organism killing that occurs prior to the onset of logarithmic growth. The uptake of radiolabeled fluoroquinolone is markedly reduced by preexposure of SA-1199-3 to NorA substrates; such prior exposure also results in a dramatic increase in RNA transcripts that hybridize with a norA probe. Preexposure of SA-1199 and SA-1199B to such substrates results in small increases or no increases in these transcripts. No sequence differences between SA-1199 and SA-1199-3 within the norA gene or flanking DNA were found. It appears likely that the regulation of norA in SA-1199-3, which may be effected by one or more genetic loci outside the norA region of the chromosome, differs from that of SA-1199 and SA-1199B.

Efflux is an important mechanism of multidrug resistance (MDR) in bacteria. The multidrug and toxin extrusion (MATE) family is the most recently described group of MDR efflux proteins, none of which have previously been identified in Staphylococcus aureus. Two independently derived S. aureus mutants having efflux-related MDR phenotypes were studied using microarray technology and a marked overexpression of an open reading frame (ORF; mepA) encoding a protein homologous with MATE family proteins was observed in both. There was concomitant overexpression of ORFs in close proximity to mepA (ϳ100 bp) encoding a MarR-type regulator (mepR, upstream of mepA) and a protein of unknown function (mepB, downstream). Experiments in which mepA was overexpressed or disrupted revealed that the encoded protein has a broad substrate profile that includes several monovalent and divalent biocides and the fluoroquinolone antimicrobial agents norfloxacin and ciprofloxacin. The function of MepB is obscure, it does not contribute to the MDR phenotype conferred by MepA. MepR overexpression reversed the MDR phenotypes of both mutants by repressing mepA transcription. All three ORFs are preferentially transcribed as a single mepRAB unit, suggesting that the three genes form an operon.