University of Western Ontario Department of Microbiology and Immunology, Schulich School of Medicine and Dentistry, London, Ontario, Canada.
University of Western Ontario Department of Microbiology and Immunology, Schulich School of Medicine and Dentistry, London, Ontario, Canada
J Bacteriol. 2019 Jan 11;201(3). doi: 10.1128/JB.00602-18. Print 2019 Feb 1.
Divergent genes in 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, which exhibited a 2-fold reduction in MIC of linoleic acid. expressed from its native promoter on pLI conferred increased resistance to USA300 but not USA300 Δ Complementation of USA300 Δ with pLI also had no effect, whereas resistance was restored with pLI or through ectopic expression of 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 promoter, (ii) in palindrome PAL1 immediately in the 3' direction of P , or (iii) within PAL2 upstream of the predicted P promoter. FarR autorepressed its expression through cooperative binding to PAL1 and the adjacent TAGWTTA motif in P Consistent with reports that 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 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 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. Here, we describe the DNA binding and sensor specificity of FarR, a novel TetR family regulator (TFR) in 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 gene, encoding a resistance-nodulation-division (RND) family efflux pump that is induced in response to antimicrobial unsaturated fatty acids. Induction of 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.
USA300 中的分化基因编码外排泵 FarE 和 TetR 家族调节剂 FarR，它们赋予了对抗抗菌非饱和脂肪酸的抗性。为了研究它们的调控机制，我们构建了 USA300Δ，其 linoleic acid 的 MIC 值降低了 2 倍。 从其天然启动子 pLI 表达赋予了对 USA300 的更高抗性，但对 USA300Δ没有影响。用 pLI 或通过 的异位表达进行 USA300Δ的互补也没有效果，而用 pLI 或通过 的异位表达恢复了抗性。在电泳迁移率变动分析中，FarR 结合到三个不同的寡核苷酸探针，每个探针都包含一个 TAGWTTA 基序，（i）一个单独的基序重叠 P 启动子的-10 元件，（ii）在 3'方向上直接在 P 中的回文 PAL1 中，或（iii）在预测的 P 启动子上游的 PAL2 内。FarR 通过协同结合 PAL1 和 P 中的相邻 TAGWTTA 基序来自我抑制其表达。与报道一致， 不通过酰基辅酶 A (acyl-CoA) 中间体代谢脂肪酸，FarR 的 DNA 结合活性不受 linoleoyl-CoA 的影响。相反， 的诱导需要脂肪酸激酶 FakA，它催化不饱和脂肪酸掺入磷脂的第一个代谢步骤。我们得出结论，FarR 是促进 表达所必需的，同时强烈自我抑制其自身表达，我们的数据与这样一种模型一致，即 FarR 与 FakA 依赖性的外源脂肪酸代谢产物相互作用，以确保只有在外源脂肪酸代谢产物的代谢能力超过将脂肪酸掺入磷脂的能力时，才会发生外排。在这里，我们描述了 FarR 的 DNA 结合和传感器特异性，它是 USA300 中的一种新型 TetR 家族调节剂 (TFR)。与大多数已经表征的 TFR 不同，后者的功能是抑制一个转录方向相反的基因，我们发现 FarR 是促进转录方向相反的 基因表达所必需的，该基因编码一种对抗菌非饱和脂肪酸有反应的抗性-结节-分裂 (RND) 家族外排泵。 的诱导依赖于脂肪酸激酶 FakA 的功能，该酶催化外源不饱和脂肪酸掺入磷脂的第一个代谢步骤。这代表了 TFR 功能的一个新例子。
