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

用差异显示方法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。以产气荚膜梭菌染色体DNA为模板构建了质粒库，将1200个独立的质粒与野生型13菌株及其病毒突变体TS133的总RNA制备的cDNADNA探针杂交。新发现的三个克隆受Virr/VIRS的调控，其中两个是正调控的，另一个是负调控的。将载体DNA与cDNA探针杂交，确定病毒/VIRS调控基因所在的片段，并测定各片段的核苷酸序列。发现了几个开放阅读框，其中一些推导的氨基酸序列与蛋白质酪氨酸磷酸酶、2‘，3’-环核苷酸2‘-磷酸二酯酶和半胱氨酸合成酶有显著的相似性。结果表明，除毒素基因外，全球调控系统VirR/VIRS应对多种基因进行正向和负向调控，新发现的基因可能编码产气荚膜梭菌新的毒力因子。

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.