Function of protein (s) which can bind to an alpha-toxin gene of Clostridium perfringens

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A study on a mechanism of regulation of an alpha-toxin gene (plc) in Clostridium perfringens has been made. We attempted to clone a gene encoding for a DNA binding protein which can bind to the plc gene. First, we cloned the plc gene from strain 13 into a plasmid pUC19. A region of chloramphenicol acetyl transferase gene (catP) starting from ribosome binding sequence to transcriptional terminator was inserted within a coding region of the plc gene. The resulting plasmid was introduced into C.perfringens strain 13. Thus obtained chloramphenicol-resistant strain was shown to have a chromosomal plc gene fused to a catP gene mediated by homologous recombination. This mutant strain formed a tiny colony on agar containing 100 mug/ml of chloramphenicol under the condition for transformation. After DNA library of type ANCTC8247 chromosomal DNA was constructed by using pJIR418 and the mutant strain, we tried to select transformants on agar containing 200 mug/ml of chloramphenicol based on the assumption that trans-acting factor bound to the plc gene can stimulate expression of the catP gene and thereby resistance of the strain increases resistance against the drug. However, we failed to obtain such clone. This could be either due to rearrangement of catP gene occurring in the presence of high concentration of the drug or due to possible toxicity displayd by a cloned gene into a high copy number of plasmid.We purified RNA plymerase from C.perfringens and also partially purified DNA binding proteins from Plc high producer, type A NCTC8237 of C.perfringens. Thus we established in vitro transcription system for examining the expression of plc gene in the presence or absence of DNA binding protein (s). Another important finding with respect to the plc gene expression is that static bent DNA present in the upstream DNA binding region is a cis-element stimulating the transcription from the gene and its stimulatory effect is prominent at low temperature.