Identification and Functional Analysis of Novel Virulence Genes in Clostridium perfringens.

利用差异显示技术鉴定了产气荚膜梭菌中受VirR/VirS双组分调控系统调控的新基因。从产气荚膜梭菌染色体DNA构建质粒文库，并将总共1，200个独立质粒与从野生型菌株13及其virR突变体衍生物TS 133的总RNA制备的cDNA探针杂交。三个克隆被新鉴定为受VirR/VirS调控，其中两个是正调控的，另一个是负调控的。通过将质粒DNA与cDNA探针杂交来鉴定VirR/VirS调节基因所在的片段，并确定每个片段的核苷酸序列。发现了几个开放阅读框架，其推导的氨基酸序列与蛋白酪氨酸磷酸酶、2 '，3'-环核苷酸2 '-磷酸二酯酶和半胱氨酸合酶具有显著的相似性。结果表明，全球调控系统VirR/VirS应调节各种基因，而不是毒素基因，积极和消极的，和新发现的基因被怀疑编码新的毒力因子产气荚膜梭菌。

Novel genes which are regulated in Clostridium perfringens by the two-component regulatory system, VirR/VirS, were identified using a differential display method. A plasmid library was constructed from C.perfringens chromosomal DNA and total of 1,200 independent plasmids was hybridized with cDNA probes prepared from total RNA of wild type strain 13 and its virR-mutant derivative TS133. Three clones were newly identified as being regulated by VirR/VirS, two of which were positively- and the other negatively-regulated. The fragment on which the VirR/VirS-regulated genes were located was identified by hybridizing the plasmid DNA with cDNA probes, and the nucleotide sequence of each fragment was determined. Several open reading frames were found and some of their deduced amino acid sequences showed significant similarities with protein-tyrosine phosphatase, 2', 3'-cyclic nucleotide 2'-phosphodiesterase, and cysteine synthase. The results suggested that the global regulatory system VirR/VirS should regulate various genes, other than toxin genes, both positively and negatively, and the newly identified genes were suspected to encode novel virulence factors in C.perfringens.