DNA Binding and Sensor Specificity of FarR, a Novel TetR Family Regulator Required for Induction of the Fatty Acid Efflux Pump FarE in <i>Staphylococcus aureus</i>

Abstract

Divergent genes in <i>Staphylococcus aureus</i> 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 Δ<i>farER</i>, which exhibited a 2-fold reduction in MIC of linoleic acid. <i>farE</i> expressed from its native promoter on pLI<i>farE</i> conferred increased resistance to USA300 but not USA300 Δ<i>farER</i> Complementation of USA300 Δ<i>farER</i> with pLI<i>farR</i> also had no effect, whereas resistance was restored with pLI<i>farER</i> or through ectopic expression of <i>farE</i> 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 P <i>_farR</i> promoter, (ii) in palindrome PAL1 immediately in the 3' direction of P <i>_farR</i> , or (iii) within PAL2 upstream of the predicted P <i>_farE</i> promoter. FarR autorepressed its expression through cooperative binding to PAL1 and the adjacent TAGWTTA motif in P <i>_farR</i> Consistent with reports that <i>S. aureus</i> 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 <i>farE</i> 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 <i>farE</i> 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.<b>IMPORTANCE</b> Here, we describe the DNA binding and sensor specificity of FarR, a novel TetR family regulator (TFR) in <i>Staphylococcus aureus</i> 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 <i>farE</i> gene, encoding a resistance-nodulation-division (RND) family efflux pump that is induced in response to antimicrobial unsaturated fatty acids. Induction of <i>farE</i> 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.