Payne P L, Straley S C
Department of Microbiology and Immunology, Albert B. Chandler Medical Center, University of Kentucky, Lexington, Kentucky 40536-0084, USA.
J Bacteriol. 1998 Aug;180(15):3882-90. doi: 10.1128/JB.180.15.3882-3890.1998.
The Yersinia pestis low-Ca2+ response stimulon is responsible for the temperature- and Ca2+-regulated expression and secretion of plasmid pCD1-encoded antihost proteins (V antigen and Yops). We have previously shown that lcrD, yscC, yscD, yscG, and yscR encode proteins that are essential for high-level expression and secretion of V antigen and Yops at 37 degreesC in the absence of Ca2+. In this study, we characterized yscO of the Yop secretion (ysc) operon that contains yscN through yscU by determining the localization of its gene product and the phenotype of an in-frame deletion. The yscO mutant grew and expressed the same levels of Yops as the parent at 37 degreesC in the presence of Ca2+. In the absence of Ca2+, the mutant grew independently of Ca2+, expressed only basal levels of V antigen and Yops, and failed to secrete these. These defects could be partially complemented by providing yscO in trans in the yscO mutant. Overexpression of YopM and V antigen in the mutant failed to restore the export of either protein, showing that the mutation had a direct effect on secretion. These results indicated that the yscO gene product is required for high-level expression and secretion of V antigen and Yops. YscO was found by immunoblot analysis in the soluble and membrane fractions of bacteria growing at 37 degreesC irrespective of the presence of Ca2+ and in the culture medium in the absence of Ca2+. YscO is the only mobile protein identified so far in the Yersinia species that is required for secretion of V antigen and Yops.
鼠疫耶尔森氏菌低钙应答刺激子负责质粒pCD1编码的抗宿主蛋白(V抗原和Yops)的温度和钙调节表达及分泌。我们先前已表明,lcrD、yscC、yscD、yscG和yscR编码的蛋白对于在37℃无钙条件下V抗原和Yops的高水平表达及分泌至关重要。在本研究中,我们通过确定其基因产物的定位和框内缺失的表型,对包含从yscN到yscU的Yop分泌(ysc)操纵子的yscO进行了表征。yscO突变体在37℃有钙存在时生长,且表达的Yops水平与亲本相同。在无钙条件下,该突变体的生长不依赖于钙,仅表达基础水平的V抗原和Yops,且无法分泌这些蛋白。通过在yscO突变体中反式提供yscO,这些缺陷可得到部分互补。在该突变体中过表达YopM和V抗原未能恢复这两种蛋白的输出,表明该突变对分泌有直接影响。这些结果表明,yscO基因产物是V抗原和Yops高水平表达及分泌所必需的。通过免疫印迹分析发现,无论有无钙存在,yscO在37℃生长的细菌的可溶性和膜部分以及无钙条件下的培养基中均存在。YscO是迄今为止在耶尔森氏菌属中鉴定出的唯一一种对于V抗原和Yops分泌必需的可移动蛋白。