Kang Yun, Lunin Vladimir V, Skarina Tatiana, Savchenko Alexei, Schurr Michael J, Hoang Tung T
Department of Microbiology, University of Hawaii at Manoa, Honolulu, Hawaii, USA.
Mol Microbiol. 2009 Jul;73(1):120-36. doi: 10.1111/j.1365-2958.2009.06757.x. Epub 2009 Jun 8.
The Pseudomonas aeruginosa PsrA autorepressor has dual roles as a repressor of the fadBA5beta-oxidation operon and an activator of the stationary-phase sigma factor rpoS and exsCEBA operon of the type III secretion system (TTSS). Previously, we demonstrated that the repression of the fadBA5 operon by PsrA is relieved by long-chain fatty acids (LCFAs). However, the signal affecting the activation of rpoS and exsC via PsrA is unknown. In this study, microarray and gene fusion data suggested that LCFA (e.g. oleate) affected the expression of rpoS and exsC. DNA binding studies confirmed that PsrA binds to the rpoS and exsC promoter regions. This binding was inhibited by LCFA, indicating that LCFA directly affects the activation of these two genes through PsrA. LCFA decreased rpoS and exsC expression, resulting in increased N-(butyryl)-l-homoserine-lactone quorum sensing signal and decreased ExoS/T production respectively. Based on the crystal structure of PsrA, site-directed mutagenesis of amino acid residues, within the hydrophobic channel thought to accommodate LCFA, created two LCFA-non-responsive PsrA mutants. The binding and activation of rpoS and exsC by these PsrA mutants was no longer inhibited by LCFA. These data support a mechanistic model where LCFAs influence PsrA regulation to control LCFA metabolism and some virulence genes in P. aeruginosa.
铜绿假单胞菌的PsrA自抑制因子具有双重作用，既是fadBA5β-氧化操纵子的阻遏物，又是III型分泌系统（TTSS）的稳定期σ因子rpoS和exsCEBA操纵子的激活剂。此前，我们证明长链脂肪酸（LCFAs）可解除PsrA对fadBA5操纵子的抑制作用。然而，通过PsrA影响rpoS和exsC激活的信号尚不清楚。在本研究中，微阵列和基因融合数据表明LCFA（如油酸）影响rpoS和exsC的表达。DNA结合研究证实PsrA与rpoS和exsC启动子区域结合。这种结合被LCFA抑制，表明LCFA通过PsrA直接影响这两个基因的激活。LCFA降低了rpoS和exsC的表达，分别导致N-（丁酰基）-L-高丝氨酸-L-内酯群体感应信号增加和ExoS/T产量降低。基于PsrA的晶体结构，对被认为可容纳LCFA的疏水通道内的氨基酸残基进行定点诱变，产生了两个对LCFA无反应的PsrA突变体。这些PsrA突变体对rpoS和exsC的结合及激活不再受LCFA抑制。这些数据支持了一种机制模型，即LCFAs通过影响PsrA的调控来控制铜绿假单胞菌中LCFA的代谢和一些毒力基因。
