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

Abstract

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.

Novel Functions and Signaling Specificity for the GraS Sensor Kinase of Staphylococcus aureus in Response to Acidic pH

“…It is now evident that these commonly encountered environmental signals confer enhanced resistance to antimicrobial uFFA, with different genetic requirements depending on acidic or nonacidic growth conditions and exposure to calcium or subinhibitory antimicrobial peptides. We previously found that the RND family efflux pump FarE was required for S. aureus resistance to antimicrobial uFFA (5,11), and in our present work, this requirement was maintained under nonacidic growth conditions for enhanced resistance to linoleic acid in unbuffered TSB supplemented with calcium. However, at acidic pH, enhanced resistance to both C 18:2 and C 16:1 uFFA was dependent on GraS and GraS-dependent expression of MprF.…”

“…Strain and plasmid construction. Genetic manipulation of S. aureus was conducted according to established guidelines and as described in previous work (4,11,65). All recombinant plasmids were initially constructed in E. coli DH5␣.…”

“…GraS and the GraS-regulated gene mprF are required for resistance to antimicrobial uFFA at acidic pH. We recently established that efflux pump FarE is induced by and required for resistance to antimicrobial uFFA through a mechanism that is dependent on the fatty acid kinase encoded by fakA (5,11), which is also required for metabolic incorporation of uFFA into phospholipids (7,44), while GraS responds to acidic pH in macrophage phagosomes (7). We therefore queried the role of these genes in enhanced resistance to antimicrobial uFFA that is manifested at acidic pH.…”

“…The anterior nares of the nose are exposed to secretions of the upper respiratory tract, including antimicrobial unsaturated free fatty acids (uFFA), of which, linoleic acid (C 18:2 ) is the most abundant in human nasal secretions (8), while the major antimicrobial uFFA on skin is sapienic acid (C 16:1 ), an isomer of palmitoleic acid that is uniquely produced by human sebaceous glands (9,10). We found that these antimicrobial uFFA induce the expression of secreted proteases and also an RND family efflux pump that contributes to resistance (4,5,11). Other environmental signals relevant to innate immunity on skin and the anterior nares include acidic pH (12)(13)(14) and antimicrobial peptides (15)(16)(17)(18).…”

Microbial fatty acid transport proteins and their biotechnological potential

“…However, the environments where some pathogenic bacteria must thrive in, such as the skin or the mouth, contain unsaturated fatty acids and therefore they need more advanced protection mechanisms such as fatty acid export systems (Choi et al, 2013; Parsons et al, 2012). In the Gram‐positive bacterium Staphylococcus aureus , an opportunistic pathogen found on the skin, a fatty acid export system was found when screening for strains resistant to linoleic acid (Alnaseri et al, 2019). The strain S. aureus FAR7 showed a mutation in the transcription factor farR , which led to the upregulation of the farE gene, encoding an efflux pump from the RND superfamily.…”