Contag P R, Williams M G, Rogers P
Department of Microbiology, University of Minnesota, Minneapolis 55455.
Appl Environ Microbiol. 1990 Dec;56(12):3760-5. doi: 10.1128/aem.56.12.3760-3765.1990.
A lactate dehydrogenase (LDH) gene of Clostridium acetobutylicum B643 was cloned on two recombinant plasmids, pPC37 and pPC58, that were selected by complementation of Escherichia coli PRC436 (acd), a fermentation-defective mutant that does not grow anaerobically on glucose. E. coli PRC436(pPC37) and PRC436(pPC58) grew anaerobically and fermented glucose to mostly lactate. When pPC37 and pPC58 were transformed into E. coli FMJ39 (ldh pfl), an LDH-deficient strain, the resulting strains grew anaerobically on glucose and produced lactate. Crude extracts of E. coli FMJ39(pPC37) and FMJ39(pP58) contained high LDH activity only when assayed for pyruvate reduction to lactate, and the LDH activity was activated 15- to 30-fold by the addition of fructose 1,6-diphosphate (FDP). E. coli FMJ39 had no detectable LDH activity, and E. coli LDH from a wild-type strain was not activated by FDP. Maxicell analysis showed that both plasmids pPC37 and pPC58 expressed a protein with an apparent Mr of 38,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Restriction endonuclease mapping of pPC37 and pPC58 and DNA hybridization studies indicated that a 2.1-kb region of these two clones of C. acetobutylicum DNA encodes the FDP-activated LDH.
丙酮丁醇梭菌B643的乳酸脱氢酶（LDH）基因克隆在两个重组质粒pPC37和pPC58上，这两个质粒是通过对大肠杆菌PRC436（acd）进行互补筛选得到的，PRC436是一种发酵缺陷型突变体，不能在葡萄糖上进行厌氧生长。大肠杆菌PRC436(pPC37)和PRC436(pPC58)能够厌氧生长，并将葡萄糖发酵产生大量乳酸。当将pPC37和pPC58转化到大肠杆菌FMJ39（ldh pfl）（一种LDH缺陷型菌株）中时，得到的菌株能够在葡萄糖上厌氧生长并产生乳酸。只有在检测丙酮酸还原为乳酸时，大肠杆菌FMJ39(pPC37)和FMJ39(pP58)的粗提取物才含有高LDH活性，并且通过添加1,6-二磷酸果糖（FDP），LDH活性可被激活15至30倍。大肠杆菌FMJ39没有可检测到的LDH活性，野生型菌株的大肠杆菌LDH也不能被FDP激活。最大细胞分析表明，在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳中，质粒pPC37和pPC58均表达一种表观分子量为38,000的蛋白质。pPC37和pPC58的限制性内切酶图谱分析以及DNA杂交研究表明，丙酮丁醇梭菌DNA的这两个克隆中的一个2.1 kb区域编码FDP激活的LDH。
