Study on function of phospholipase C gene binding protein from Clostridium perfringens

磷脂酶C(PLC)是产气荚膜梭菌的主要毒力因子之一。PLC编码基因(Plc)的表达受位于plc启动子上游的弯曲DNA和可与基因编码区结合的plc结合蛋白的调控。从产PLC的产气荚膜梭菌NCTC8237菌株中分离纯化了部分plc结合蛋白。经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳法(SDS-PAGE)测定，一种多肽与plc编码区的片段结合，其分子质量为56 kDa。对其N端氨基酸序列进行测定，并用与之对应的简并引物扩增产物。从NCTC 8237染色体上扩增出一段6kb的EcoRI片段，与扩增产物杂交，克隆到pUC19中。对该片段进行核苷酸序列分析和预测氨基酸序列的相似性搜索表明，56K多肽的编码基因为hemd-ctyGC，该片段中发现的其他开放阅读框都与血红素生物合成的酶有关。将该6kb片段克隆到穿梭载体pJIR418中，转化产气荚膜梭菌13，可在转化子中表达编码Hemd-cysGC的基因。然而，从转化子中提取的产物对plc基因没有明显的凝胶抑制作用。并对位于plc启动子上游的弯曲DNA的作用进行了研究。已经证明，它在调节plc基因表达以响应温度变化方面发挥作用。

Phospholipase C (PLC) is one of the major virulence factors of Clostridium perfringens. The expression of a PLC-encoding gene (plc) is regulated by bent DNA locating upstream of a plc promoter and also by plc-binding protein, which can bind to the coding region of the gene. The plc-binding protein was partially purified from C.perfringens NCTC 8237, a PLC high-producing strain. One polypeptide, of which molecular mass was estimated to be 56 kDa by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) , bound to a fragment within the plc coding region. Its N-terminal amino acid sequence was determined and PCR product was generated using degenerate primers corresponding to the sequence. A 6-kb EcoRI fragment from the NCTC 8237 chromosome, which hybridized with the PCR product, was cloned into pUC19. Nucleotide sequencing of this fragment and similarity search of the predicted amino acid sequence revealed that the gene encoding for the 56 K polypeptide is hemD-ctyGC and other open reading frames found in the fragment are all related to the enzymes for heme biosynthesis. When the 6-kb fragment was cloned into a shuttle-vector pJIR418 and C.perfringens strain 13 was transformed with the plasmid, othe hemD-cysGC-encoding gene was expressed in the transformant. However, extract from the transformant did not show a positive gel retardation to the plc gene. It may be possible that the plc-binding protein comigrates with the 56 K polypeptide on SDS-PAGE.Study was also conducted on a role of the bent DNA locating upstream of the plc promoter. It has been shown to play a role in the regulation of the plc gene expression in response to alteration of temperature.