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

点击翻译按钮获取中文摘要

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.