Identification and characterization of farR and farE as a regulator and effector of fatty acid resistance in Staphylococcus aureus
Although Staphylococcus aureus is exposed to antimicrobial fatty acids on the skin, in nasal secretions and in abscesses, specific mechanisms for regulating gene expression and intrinsic resistance in response to these fatty acids have not been reported. Through in vitro selection for increased resistance of S. aureus to linoleic acid, I identified fatty acid resistant clone FAR7, where a single nucleotide polymorphism caused a His121Tyr substitution in an uncharacterized member of the TetR family of transcriptional regulators, which is divergently transcribed from a gene encoding a member of the resistance-nodulation-division superfamily of multi-drug efflux pumps. I named these genes farE and farR, for regulator and effector of fatty acid resistance, respectively. S. aureusDfarER exhibited loss of inducible resistance to linoleic acid, and although FarR is a TetR family regulator which typically repress expression of a divergent gene, I found that FarR is needed to induce farE. Compared to wild type S. aureus, FAR7 exhibited increased expression of farR and farE under non-inducing conditions, and a significantly higher induced level of farE. Electrophoretic mobility shift assays revealed a FarR binding site in the farER intergenic segment, that overlaps with the +1 transcription start site of farR as determined by 5'-RACE. The variant FarR7 produced by S. aureus FAR7 failed to bind to this operator site, and nucleotide substitutions within the operator abolished binding of native FarR. Conversely, FarR and FarR7 bound equally well to a second operator site upstream of the predicted farE promoter. Therefore, like other TetR regulators, FarR represses its own expression, and a His121Tyr substitution in FarR causes a loss of auto-repression and increases expression of both farR and farE. My data reports the first description of a specific mechanism of inducible resistance to antimicrobial fatty acids in a Gram-positive pathogen and defines a new paradigm for regulation of a divergently transcribed gene by a TetR family regulator.
Antimicrobial fatty acids, efflux pumps, TetR family regulator, inducible resistance, Staphylococcus aureus
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