A study on a mechanism for the transcriptional regulation of an epsilon-toxin gene by a novel type of bent DNA

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An epsilon-toxin gene of Clostridium perfringens has bent DNA in a promoter region. It has been shown to possess another weakly-bent DNA in the coding region, which regulates epsilon-toxin gene expression along with the upstream bent DNA. When a DNA fragment coveting the two bent DNA regions were PCR amplified and cloned into an E coli plasmid, A-tract consisting of 8 adenine residues located at the downstream bent DNA lost the 8th adenine. This suggests that cloning of the fragment causes plasmid instability. We constructed plasmids expressing C. perfringens LrpC with and without His' by using our plasmid vector pFF. Unfortunately, transformats of C. perfringens strains 13 and SM101 carrying these plasmids failed to produce large amounts of recombinant LrpC proteins. Therefore, we failed to purify LrpC from these cultures. One likely reason for the difficulty of purification is possible proteolytic breakdown of the recombinant product during purification. We constructed a clostripain- … More like protease-deficient mutant, since this is the most potent thiol-protease among proteases produced by the organism and probably decreases the yield of LrpC products. We are currently attempting to purify LrpC by using this mutant To assess whether or not his-tagged LrpC functions normally, we examined biological properties of both transformants carrying plasmids containing LrpC and LrpC-his genes. The result indicates LrpC and LrpC-his are involved in the onset of spore-formation, as demonstrated for Bacillus subtilis, proving that LrpC-his is fractional in C perfringens. We prepared cell lysate from Strain 13 with and without a plasmid containing the LrpC gene and also PCR-products corresponding to the fragment containing the two bent DNA regions of the epsilon-toxin gene. The gel retardation assay using these did not show the specific interaction between the DNA and LrpC probably because of impurity of protein samples or the presence of LrpC in the wild type as well as in the transformant. To solve theseproblems, we are currently purifying LrpC-his by using Ni-chelating Sepharose from large-scale culture of the C. perfringens transformant We have also attempted an alternative method involving an E coli expression system as reported for purification of B. subtilis LrpC. Since we constructed a LrpC gene-disrupted mutant, we have undertaken the gel retardation assay by using lysates from a wild-type strain and its isogenic LrpC(-)mntant. Less