Gene Regulation by Staphylococcus Aureas rsbU and rsbV

备选sigma因子sigB是金黄色葡萄球菌中许多一般应激和能量消耗的主要调节因子。我们检测了rsbU和rsbV在sigB位点（rsbU-rsbV- rsbw -sigB）中的作用。我们采用同源重组法获得rsbV基因敲除突变体。我们将被CAT基因破坏的rsbV插入到框架中有tc耐药基因的pSPT181中，构建敲除质粒pEMVKO，然后电穿孔转化。42℃孵育后，我们在含有Cm5或Tc5的复制板上选择cm耐药和tc敏感菌株，它们应该是rsbv敲除突变体。然而，我们未能获得菌株。失效的原因可能是热依赖性复合。接下来，我们将完整的sigB位点（rsbU-sigB）补充到rsbU突变体RN6390 （rsbU缺失11bp）和sigB KO突变体EM1001中，并分别命名为RN1002和EM1002。当我们将这些菌株注射到无毛小鼠体内时，RN6390、EM1001和EM1002产生了与溃疡相似的皮下脓肿。另一方面，RN1002显示小结节性脓肿。RN6390的脓肿大小倾向于大于EM1001 (440mm^2 vs 370mm^2)，但差异无统计学意义。EM1002小于其他菌株（50mm^2）。当RN1002在m9最小溶液中孵育后暴露于加热时，其生长速度比其他菌株慢。我们的研究表明，包括rsbU和rsbV在内的sigB位点参与了金黄色葡萄球菌的应激反应和毒力。

The alternative sigma factor sigB is the master regulator of a lot of general stress and energy depletion in Staphylococcus aureus. We examined the role of rsbU and rsbV in the sigB locus (rsbU-rsbV-rsbW-sigB). To obtain rsbV knockout mutant, we used homologous recombination method. We constructed knockout plasmid, pEMVKO, by inserting rsbV, which was disrupted by CAT gene, into pSPT181 with TC-resistant gene in frame and then transformed by electroporation. After incubation at 42 C, we selected Cm-resistant and Tc-sensitive strains, which should be rsbV-knockout mutant, on replica plates containing Cm5 or Tc5. However, we failed to obtain the strain. The failure may attribute to the heat-dependent recombination. Next, we complemented intact sigB locus (rsbU-sigB) into RN6390, an rsbU mutant (11bp-depletion in rsbU), and EM1001, a sigB KO mutant, and named RN1002 and EM1002, respectively. When we injected these strains to the hairless mice, RN6390, EM1001, and EM1002 made subcutaneous abscess, which were similar to the ulcer. On the other hand, RN1002 showed the small nodular abscess. The abscess size of RN6390 tended to be larger than EM1001 (440mm^2 versus 370mm^2), but there is no statistical difference. EM1002 showed smaller than ant other strain (50mm^2). When exposed to heat after incubation inM9 minimal solution, RN1002 grew slower than any other strain. Our studies indicate that sigB locus including rsbU and rsbV contributes to the stress response and virulence of Staphylococcus aureus.