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

产气荚膜梭菌的一个表型毒素基因在启动子区域弯曲了DNA。已证明它在编码区具有另一个弱弯曲DNA，它与上游弯曲DNA一起调节epsilon毒素基因的表达。当两个弯曲DNA区域的DNA片段被聚合酶链式反应扩增并克隆到原核表达载体中时，位于DNA弯曲下游的由8个腺嘌呤残基组成的A-道丢失了第8个腺嘌呤。这表明克隆该片段会导致质粒不稳定。利用构建的表达载体pFF，构建了含和不含His‘的产气荚膜梭菌LrpC的表达载体。不幸的是，携带这些质粒的产气荚膜梭菌13和SM101的转化未能产生大量的重组LrpC蛋白。因此，我们未能从这些培养物中纯化LrpC。纯化困难的一个可能原因是重组产物在纯化过程中可能发生蛋白质分解。我们构建了一个梭状芽胞杆菌-…更像是缺乏蛋白酶的突变体，因为这是生物体产生的最有效的硫醇蛋白水解酶，可能会降低LrpC产物的产量。我们目前正试图通过使用这个突变体来纯化LrpC。为了评估His标记的LrpC是否正常功能，我们检测了两个转化子的生物学特性，这两个转化子都携带含有LrpC和LrpC-His基因的质粒。结果表明，LrpC和LrpC-His参与了芽胞形成的启动，就像枯草芽孢杆菌一样，证明了LrpC-His在产气荚膜芽孢杆菌中是部分的。我们制备了含有和不含有LrpC基因的质粒的菌株13的细胞裂解物，以及与含有epsilon毒素基因的两个弯曲DNA区域的片段相对应的PCR产物。凝胶滞留分析没有显示DNA与LrpC之间的特异性相互作用，这可能是由于蛋白质样品的杂质或野生型和转化子中LrpC的存在。为了解决这些问题，我们目前正在使用大规模培养产气荚膜梭菌转化子的镍螯合琼脂糖凝胶来纯化LrpC-His。我们还尝试了一种替代的方法，即利用已报道的大肠杆菌表达系统来纯化枯草杆菌LrpC。自我们构建了LrpC基因突变株以来，我们利用一株野生型菌株及其同基因LrpC(-)突变体的裂解产物进行了凝胶滞留分析。较少

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