Youngleson Jonathan S, Santangelo Joseph D, Jones David T, Woods David R
Department of Microbiology, University of Cape Town, Rondebosch 7700, South Africa.
Appl Environ Microbiol. 1988 Mar;54(3):676-682. doi: 10.1128/aem.54.3.676-682.1988.
An alcohol dehydrogenase (ADH) gene from Clostridium acetobutylicum was cloned on a recombinant plasmid, pCADH100. Escherichia coli HB101, and an allyl alcohol-resistant mutant, HB101-adh1, containing this plasmid were unable to grow aerobically or anaerobically on agar media containing sublethal concentrations of allyl alcohol. E. coli HB101 and HB101-adh1 transformed with the plasmid pCADH100 produced increased levels of ethanol when grown anaerobically under alkaline conditions in the absence of nitrate. Cell extracts from aerobically and anaerobically grown E. coli HB101(pCADH100) and HB101-adhl(pCADH100) cells exhibited increased levels of NADP-dependent ADH activity with either ethanol or butanol as the substrate. The inability of E. coli HB101(pCADH100) to grow in the presence of allyl alcohol correlated with the appearance of an NADP-dependent ADH activity band on nondenaturing polyacrylamide gel electrophoresis with either ethanol or butanol as the substrate. The position of the cloned NADP-dependent ADH activity bands in E. coli HB101(pCADH100) cell extracts with either ethanol or butanol as the substrate coincided with the position of a single NADP-dependent ADH activity band in extracts of C. acetobutylicum cells. E. coli HB101(pCADH100) cell extracts prepared from both aerobically and anaerobically grown cells exhibited an additional protein band with an apparent M(r) of approximately 33,000 on sodium dodecyl sulfate-polyacryl-amide gel electrophoresis which was absent in cell extracts of E. coli HB101. A protein band with a similar apparent M(r) was observed in cell extracts of C. acetobutylicum, and in vitro transcription and translation experiments with pCADH100 produced a major protein product with a similar apparent M(r).
从丙酮丁醇梭菌中克隆出一个乙醇脱氢酶（ADH）基因，并将其克隆到重组质粒pCADH100上。含有该质粒的大肠杆菌HB101及其烯丙醇抗性突变体HB101-adh1，在含有亚致死浓度烯丙醇的琼脂培养基上，无论有氧还是无氧条件下都无法生长。用质粒pCADH100转化的大肠杆菌HB101和HB101-adh1，在无氧且碱性条件下、无硝酸盐存在时生长，会产生更高水平的乙醇。在有氧和无氧条件下生长的大肠杆菌HB101(pCADH100)和HB101-adhl(pCADH100)细胞的提取物，以乙醇或丁醇为底物时，NADP依赖的ADH活性水平升高。大肠杆菌HB101(pCADH100)在烯丙醇存在时无法生长，这与在非变性聚丙烯酰胺凝胶电泳上出现以乙醇或丁醇为底物的NADP依赖的ADH活性条带相关。在大肠杆菌HB101(pCADH100)细胞提取物中，以乙醇或丁醇为底物时，克隆的NADP依赖的ADH活性条带位置，与丙酮丁醇梭菌细胞提取物中单一的NADP依赖的ADH活性条带位置一致。从有氧和无氧条件下生长的细胞制备的大肠杆菌HB101(pCADH100)细胞提取物，在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳上显示出一条额外的蛋白条带，其表观分子量约为33,000，而大肠杆菌HB101的细胞提取物中没有该条带。在丙酮丁醇梭菌的细胞提取物中观察到一条具有相似表观分子量的蛋白条带，用pCADH100进行的体外转录和翻译实验产生了一种具有相似表观分子量的主要蛋白产物。
