Wood Lynn F, Ohman Dennis E
McGuire Veterans Affairs Medical Center, Richmond, VA 23249, USA.
Department of Microbiology and Immunology, Virginia Commonwealth University Medical Center, Richmond, VA 23298, USA.
Microbiology (Reading). 2015 Jan;161(Pt 1):30-40. doi: 10.1099/mic.0.081182-0. Epub 2014 Oct 21.
The ECF (extracytoplasmic function) alternative sigma factor, σ(22) (AlgT/U), is required for expression of the algD promoter of the operon for alginate biosynthesis in Pseudomonas aeruginosa. Alginate production promotes chronic pulmonary infections by this opportunistic pathogen in patients with cystic fibrosis and chronic obstructive pulmonary disease. σ(22) is normally sequestered, but its deregulation and activation occur either by mutation in mucA (encoding an anti-sigma factor) or in response to envelope stress, such as inhibition of peptidoglycan synthesis. The σ(22) stress response system includes many genes in addition to those for alginate. In the present study, we characterized an intergenic region between ORFs PA2559 and PA2560 in PAO1 for a σ(22)-dependent, stress-responsive transcript, described here as PA2559.1. Northern analysis and transcript end-mapping indicated the PA2559.1 transcript was ~310 nt in length. Examination of the DNA sequence upstream of +1 revealed a σ(22) core promoter motif, GAATTT-N16-TCTGT, and site-directed mutagenesis confirmed this to be a σ(22)-dependent promoter that was highly activated during cell wall stress. PA2559.1 also contained an ORF that demonstrated increased translational activity upon cell wall stress. As determined by mutant analysis, the protein encoded by PA2559.1 was shown to play a positive role in the σ(22)-dependent activation of the algD promoter under stress in both sessile (i.e. biofilm) and planktonic conditions. Thus, it appeared to act as a stress response facilitator and so was named SrfA. The sequence of SrfA was found to be novel in nature and extremely well conserved only in P. aeruginosa, suggesting that it is of high evolutionary importance in this species.
胞外功能（ECF）替代西格玛因子σ(22)（AlgT/U）是铜绿假单胞菌藻酸盐生物合成操纵子中algD启动子表达所必需的。藻酸盐的产生会促使这种机会致病菌在囊性纤维化和慢性阻塞性肺疾病患者中引发慢性肺部感染。σ(22)通常被隔离，但它的失调和激活要么通过mucA（编码一种抗西格玛因子）的突变发生，要么是对包膜应激（如肽聚糖合成抑制）的反应。σ(22)应激反应系统除了藻酸盐相关基因外还包括许多基因。在本研究中，我们对PAO1中ORF PA2559和PA2560之间的一个基因间区域进行了表征，该区域存在一个依赖于σ(22)的应激反应转录本，在此称为PA2559.1。Northern分析和转录本末端定位表明PA2559.1转录本长度约为310 nt。对转录起始位点上游DNA序列的检查揭示了一个σ(22)核心启动子基序GAATTT - N16 - TCTGT，定点诱变证实这是一个依赖于σ(22)的启动子，在细胞壁应激期间被高度激活。PA2559.1还包含一个ORF，该ORF在细胞壁应激时表现出翻译活性增加。通过突变分析确定，PA2559.1编码的蛋白质在固着（即生物膜）和浮游条件下的应激状态下，对algD启动子的σ(22)依赖性激活发挥积极作用。因此，它似乎起到了应激反应促进因子的作用，所以被命名为SrfA。发现SrfA的序列在本质上是新颖的，并且仅在铜绿假单胞菌中极其保守，这表明它在该物种中具有高度的进化重要性。
