Opdyke Jason A, Scott June R, Moran Charles P
Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, Georgia 30322, USA.
J Bacteriol. 2003 Aug;185(15):4291-7. doi: 10.1128/JB.185.15.4291-4297.2003.
Secondary RNA polymerase sigma factors in many bacteria are responsible for regulating a vast range of processes including virulence. A protein (sigma(X)) in the gram-positive human pathogen Streptococcus pyogenes (the group A Streptococcus or GAS) was recently shown to function in vitro as a secondary sigma factor. We report here the isolation of a mutant in which both sigX genes are inactivated, show that sigma(X) functions in GAS cells, and show that the amount of sigma(X) is controlled at two levels. Primer extension analysis indicates that sigX transcription is low in GAS cells grown in Todd-Hewitt yeast broth, and immunoblot assays with a sigma(X)-specific polyclonal antibody demonstrate that the protein does not accumulate in these cells. To increase the level of sigX transcription in GAS, we constructed a strain that constitutively expresses the sigX gene from a heterologous promoter. Expression of sigX from this promoter led to transcription of the sigma(X)-dependent cinA promoter in GAS cells. We found that expression of the sigX gene in a clpP mutant strain resulted in greater accumulation of sigma(X) protein, which resulted in higher levels of transcription from the sigma(X)-dependent promoters cinA, smf, and cglA. In addition, a clpP mutant containing sigX only at its wild-type loci on the chromosome generated more transcription from the sigma(X)-dependent cinA promoter than did the wild-type parental strain. Therefore, sigma(X) activity in GAS is limited by low-level transcription of the sigX structural genes and by clpP, which appears to negatively regulate sigma(X) accumulation.
许多细菌中的二级RNA聚合酶σ因子负责调控包括毒力在内的大量过程。革兰氏阳性人类病原体化脓性链球菌（A组链球菌或GAS）中的一种蛋白质（σ(X)）最近被证明在体外作为二级σ因子发挥作用。我们在此报告分离出一个突变体，其中两个sigX基因均失活，表明σ(X)在GAS细胞中发挥作用，并表明σ(X)的量在两个水平上受到控制。引物延伸分析表明，在托德-休伊特酵母肉汤中生长的GAS细胞中，sigX转录水平较低，用σ(X)特异性多克隆抗体进行的免疫印迹分析表明该蛋白质在这些细胞中不积累。为了提高GAS中sigX的转录水平，我们构建了一个从异源启动子组成型表达sigX基因的菌株。从该启动子表达sigX导致GAS细胞中依赖于σ(X)的cinA启动子转录。我们发现，在clpP突变体菌株中表达sigX基因导致σ(X)蛋白积累更多，从而导致依赖于σ(X)的启动子cinA、smf和cglA的转录水平更高。此外，仅在其染色体上野生型位点含有sigX的clpP突变体比野生型亲本菌株从依赖于σ(X)的cinA启动子产生更多转录。因此，GAS中σ(X)的活性受到sigX结构基因的低水平转录和clpP的限制，clpP似乎对σ(X)的积累起负调控作用。
