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PLC结合蛋白的调控，PLC结合蛋白可与基因的编码区结合。从产气荚膜梭菌NCTC 8237（PLC高产菌株）中部分纯化PLC结合蛋白。一种多肽，其分子量估计为56 kDa的十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE），结合到PLC编码区内的片段。确定其N-末端氨基酸序列，并使用对应于该序列的简并引物产生PCR产物。将与PCR产物杂交的来自NCTC 8237染色体的6 kb EcoRI片段克隆到pUC 19中。对该片段进行核苷酸序列测定和氨基酸序列相似性分析，结果表明编码56 K多肽的基因为hemD-ctyGC，该片段中发现的其它开放阅读框架均与血红素生物合成酶有关。将该6-kb片段克隆到穿梭载体pJIR 418中，用该质粒转化产气荚膜梭菌菌株13，在大肠杆菌中表达了hemD-cysGC编码基因。然而，从大肠杆菌中提取的提取物对plc基因没有显示出阳性凝胶阻滞。SDS-PAGE分析表明，plc结合蛋白可能与56 K多肽共迁移，并对plc启动子上游DNA的弯曲作用进行了研究。它已被证明在PLC基因表达的调节中起作用，以响应温度的变化。

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.