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ε-毒素基因在启动子区具有弯曲的DNA。已显示在编码区中具有另一个弱弯曲DNA，其沿着上游弯曲DNA调节ε毒素基因表达。当PCR扩增包含这两个弯曲DNA区域的DNA片段并克隆到大肠杆菌质粒中时，位于弯曲DNA下游的由8个腺嘌呤残基组成的A段丢失了第8个腺嘌呤。这表明片段的克隆引起质粒不稳定性。我们构建了表达C.利用我们的质粒载体pFF，对带有和不带有His'的产气荚膜杆菌LrpC进行了比较。不幸的是，C.携带这些质粒的产气荚膜杆菌菌株13和SM 101不能产生大量重组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的原因是在纯化过程中重组产物可能的蛋白水解分解。我们构建了梭菌蛋白酶- ...更多信息 类似于蛋白酶缺陷突变体，因为这是生物体产生的蛋白酶中最有效的巯基蛋白酶，并且可能降低LrpC产物的产率。我们目前正试图通过使用这种突变体来纯化LrpC。为了评估His标记的LrpC是否正常发挥功能，我们检查了携带含有LrpC和LrpC-his基因的质粒的两种转化体的生物学特性。结果表明LrpC和LrpC-his参与孢子形成的开始，如枯草芽孢杆菌所证明的，证明LrpC-his在产气荚膜梭菌中是部分的。我们制备了来自菌株13的细胞裂解物，其具有和不具有含有LrpC基因的质粒以及对应于含有ε毒素基因的两个弯曲DNA区域的片段的PCR产物。使用这些的凝胶阻滞试验没有显示DNA和LrpC之间的特异性相互作用，可能是因为蛋白质样品的杂质或野生型以及野生型中存在LrpC。为了解决这些问题，我们目前正在使用镍螯合琼脂糖从大规模培养的C.产气荚膜梭菌B我们还尝试了另一种方法，该方法涉及所报道的用于纯化B的大肠杆菌表达系统。枯草芽孢杆菌由于我们构建了一个LrpC基因破坏的突变体，我们已经进行了凝胶阻滞试验，通过使用裂解物从野生型菌株和其同基因LrpC（-）mintant。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