Although Staphylococcus aureus is exposed to antimicrobial fatty acids on the skin, in nasal secretions, and in abscesses, a specific mechanism of inducible resistance to this important facet of innate immunity has not been identified. Here, we have sequenced the genome of S. aureus USA300 variants selected for their ability to grow at an elevated concentration of linoleic acid. The fatty acid-resistant clone FAR7 had a single nucleotide polymorphism resulting in an H 121 Y substitution in an uncharacterized transcriptional regulator belonging to the AcrR family, which was divergently transcribed from a gene encoding a member of the resistance-nodulation-division superfamily of multidrug efflux pumps. We named these genes farR and farE, for regulator and effector of fatty acid resistance, respectively. Several lines of evidence indicated that FarE promotes efflux of antimicrobial fatty acids and is regulated by FarR. First, expression of farE was strongly induced by arachidonic and linoleic acids in an farRdependent manner. Second, an H 121 Y substitution in FarR resulted in increased expression of farE and was alone sufficient to promote increased resistance of S. aureus to linoleic acid. Third, inactivation of farE resulted in a significant reduction in the inducible resistance of S. aureus to the bactericidal activity of 100 M linoleic acid, increased accumulation of [ 14 C]linoleic acid by growing cells, and severely impaired growth in the presence of nonbactericidal concentrations of linoleic acid. Cumulatively, these findings represent the first description of a specific mechanism of inducible resistance to antimicrobial fatty acids in a Gram-positive pathogen. IMPORTANCEStaphylococcus aureus colonizes approximately 25% of humans and is a leading cause of human infectious morbidity and mortality. To persist on human hosts, S. aureus must have intrinsic defense mechanisms to cope with antimicrobial fatty acids, which comprise an important component of human innate defense mechanisms. We have identified a novel pair of genes, farR and farE, that constitute a dedicated regulator and effector of S. aureus resistance to linoleic and arachidonic acids, which are major fatty acids in human membrane phospholipid. Expression of farE, which encodes an efflux pump, is induced in an farRdependent mechanism, in response to these antimicrobial fatty acids that would be encountered in a tissue abscess. Staphylococcus aureus has a dichotomous relation with human hosts, being able to establish an asymptomatic commensal relationship, but is also historically known as a leading cause of human infectious morbidity and mortality. Significantly, death attributed to S. aureus in the United States is now comparable to mortality rates for AIDS, tuberculosis, and viral hepatitis (1-3). Not surprisingly, therefore, S. aureus has been the subject of intensive research on mechanisms of pathogenesis and acquisition and transfer of antibiotic resistance and of efforts to identify potential vaccine antigens (4-6). Until the late ...

Divergent genes in Staphylococcus aureus USA300 encode the efflux pump FarE and TetR family regulator FarR, which confer resistance to antimicrobial unsaturated fatty acids. To study their regulation, we constructed USA300 ΔfarER, which exhibited a 2-fold reduction in MIC of linoleic acid. farE expressed from its native promoter on pLIfarE conferred increased resistance to USA300 but not USA300 ΔfarER. Complementation of USA300 ΔfarER with pLIfarR also had no effect, whereas resistance was restored with pLIfarER or through ectopic expression of farE. In electrophoretic mobility shift assays, FarR bound to three different oligonucleotide probes that each contained a TAGWTTA motif, occurring as (i) a singular motif overlapping the −10 element of the PfarR promoter, (ii) in palindrome PAL1 immediately in the 3′ direction of PfarR, or (iii) within PAL2 upstream of the predicted PfarE promoter. FarR autorepressed its expression through cooperative binding to PAL1 and the adjacent TAGWTTA motif in PfarR. Consistent with reports that S. aureus does not metabolize fatty acids through acyl coenzyme A (acyl-CoA) intermediates, DNA binding activity of FarR was not affected by linoleoyl-CoA. Conversely, induction of farE required fatty acid kinase FakA, which catalyzes the first metabolic step in the incorporation of unsaturated fatty acids into phospholipid. We conclude that FarR is needed to promote the expression of farE while strongly autorepressing its own expression, and our data are consistent with a model whereby FarR interacts with a FakA-dependent product of exogenous fatty acid metabolism to ensure that efflux only occurs when the metabolic capacity for incorporation of fatty acid into phospholipid is exceeded. IMPORTANCE Here, we describe the DNA binding and sensor specificity of FarR, a novel TetR family regulator (TFR) in Staphylococcus aureus. Unlike the majority of TFRs that have been characterized, which function to repress a divergently transcribed gene, we find that FarR is needed to promote expression of the divergently transcribed farE gene, encoding a resistance-nodulation-division (RND) family efflux pump that is induced in response to antimicrobial unsaturated fatty acids. Induction of farE was dependent on the function of the fatty acid kinase FakA, which catalyzes the first metabolic step in the incorporation of exogenous unsaturated fatty acids into phospholipid. This represents a novel example of TFR function.