Klapatch T R, Guerinot M L, Lynd L R
Department of Biological Sciences, Dartmouth College, Hanover, NH 03755, USA.
J Ind Microbiol. 1996 Jun;16(6):342-7. doi: 10.1007/BF01570112.
Transformation of the thermophile Clostridium thermosaccharolyticum ATCC 31960 was achieved using plasmid pCTC1 and electroporation. Evidence supporting transformation was provided by Southern blots, detection of the plasmid in 10 out of 10 erythromycin-resistant clones, retransformation of E. coli and C. thermosaccharolyticum with plasmid DNA isolated from C. thermosaccharolyticum, and a proportional relationship between the number of transformants and the amount of DNA added. Transformation efficiencies were very low for plasmid DNA prepared from E. coli (0.6 transformants mg-1 DNA), although somewhat higher for plasmid DNA prepared from C. thermosaccharolyticum (52 transformants mg-1 DNA). Transformation-dependent erythromycin resistance indicates that an adenosine methylase gene originating from Enterococcus faecalis, a mesophile, is expressed in C. thermosaccharolyticum. The plasmid pCTC1 appears to be replicated independently of the chromosome, as indicated by visualization of recovered plasmid on gels, and retransformation using recovered plasmid. pCTC1 is maintained in C. thermosaccharolyticum at both 45 and 60 degrees C. Restriction analysis showed little or no rearrangement occurred upon passage through the thermophile.
利用质粒pCTC1和电穿孔技术实现了嗜热菌嗜热解糖梭菌ATCC 31960的转化。Southern印迹法、在10个抗红霉素克隆中的10个中检测到该质粒、用从嗜热解糖梭菌分离的质粒DNA对大肠杆菌和嗜热解糖梭菌进行再次转化以及转化子数量与添加的DNA量之间的比例关系，均提供了支持转化的证据。从大肠杆菌制备的质粒DNA的转化效率非常低（0.6个转化子/毫克DNA），而从嗜热解糖梭菌制备的质粒DNA的转化效率略高一些（52个转化子/毫克DNA）。依赖转化的红霉素抗性表明，源自嗜温菌粪肠球菌的腺苷甲基化酶基因在嗜热解糖梭菌中表达。如在凝胶上观察到回收的质粒以及使用回收的质粒进行再次转化所示，质粒pCTC1似乎独立于染色体进行复制。pCTC1在45℃和60℃下均可在嗜热解糖梭菌中维持。限制性分析表明，该质粒在通过嗜热菌传代时几乎没有发生重排。
