Paredes-Sabja Daniel, Setlow Peter, Sarker Mahfuzur R
Department of Biomedical Sciences, College of Veterinary Medicine, Oregon State University, Corvallis, OR 97331, USA.
Department of Molecular, Microbial and Structural Biology, University of Connecticut Health Center, Farmington, CT 06030, USA.
Microbiology (Reading). 2009 Oct;155(Pt 10):3464-3472. doi: 10.1099/mic.0.030965-0. Epub 2009 Jul 23.
The genome of the Clostridium perfringens food poisoning isolate SM101 encodes a subtilisin-like protease, CspB, upstream of the sleC gene encoding the enzyme essential for degradation of the peptidoglycan cortex during spore germination. SleC is an inactive pro-SleC in dormant spores that is converted to active SleC during spore germination and Csp proteases convert pro-SleC to the active enzyme in vitro. In this work, the germination and viability of spores of a cspB deletion mutant of strain SM101, as well as cspB expression, were studied. The cspB gene was expressed only during sporulation, and only in the mother cell compartment. cspB spores were unable to germinate significantly with either a rich nutrient medium, KCl, or a 1 : 1 chelate of Ca(2+) and dipicolinic acid (DPA); the viability of these spores was approximately 10(4)-fold lower than that of wild-type spores, although cspB and wild-type spores had similar viability on plates containing lysozyme, and cspB spores could not process inactive pro-SleC into active SleC during spore germination. Germination of cspB spores was blocked prior to DPA release and cortex hydrolysis, and germination and viability defects in these spores were complemented by an ectopic cspB. These results indicate that Csp proteases are essential to generate active SleC and allow cortex hydrolysis early in C. perfringens spore germination. However, Csp proteases likely play another role in spore germination, since cspB spores did not release DPA upon exposure to germinants, while sleC spores have been shown previously to release DPA, albeit slowly, upon exposure to germinants.
产气荚膜梭菌食物中毒分离株SM101的基因组在编码芽孢萌发过程中肽聚糖皮层降解所需酶的sleC基因上游编码一种枯草杆菌蛋白酶样蛋白酶CspB。SleC在休眠芽孢中是无活性的前体SleC，在芽孢萌发过程中转化为活性SleC，并且Csp蛋白酶在体外将前体SleC转化为活性酶。在这项研究中，对SM101菌株的cspB缺失突变体芽孢的萌发和活力以及cspB表达进行了研究。cspB基因仅在芽孢形成期间表达，且仅在母细胞区室中表达。cspB芽孢在富含营养的培养基、KCl或Ca(2+)与吡啶二羧酸（DPA）的1:1螯合物中均不能显著萌发；这些芽孢的活力比野生型芽孢低约10(4)倍，尽管cspB芽孢和野生型芽孢在含有溶菌酶的平板上具有相似的活力，并且cspB芽孢在芽孢萌发过程中不能将无活性的前体SleC加工成活性SleC。cspB芽孢的萌发在DPA释放和皮层水解之前被阻断，并且这些芽孢的萌发和活力缺陷可被异位cspB互补。这些结果表明，Csp蛋白酶对于产生活性SleC以及在产气荚膜梭菌芽孢萌发早期允许皮层水解至关重要。然而，Csp蛋白酶可能在芽孢萌发中还发挥另一种作用，因为cspB芽孢在接触萌发剂时不释放DPA，而之前已表明sleC芽孢在接触萌发剂时会释放DPA，尽管释放缓慢。
