Holubová I, Brenner V, Hubácek J
Institute of Microbiology, Academy of Sciences of the Czech Republic, Prague.
Folia Microbiol (Praha). 1993;38(4):277-80. doi: 10.1007/BF02898593.
To develop a host-vector system for Methylobacterium sp. using a construct based on a small indigenous methylotrophic plasmid, the E. coli--Methylobacterium sp. shuttle vector pWUBR (12.7 kb, Apr, Tcr) was constructed by joining the E. coli plasmid pBR328 and the cryptic plasmid pWU7 (7.8 kb), isolated from the soil facultative methylotrophic bacterium, Methylobacterium sp. strain M17. Via mobilization by the pDPT51 R plasmid, belonging to the IncP-1 incompatibility group, plasmid pWUBR was transferred into the original host of cryptic plasmid pWU7, strain M17, where a competition between the introduced hybrid plasmid and the indigenous cryptic plasmid took place, and into the plasmidless Methylobacterium sp. strain R2b. The stability of pWUBR in Tcr methylotrophic transconjugants after 25 generations of growth under nonselective conditions was more than 90% in both hosts. The ability to replicate in R2b strain demonstrates that the host spectrum of pWUBR is not restricted to the original host of pWU7 and indicates the possibility to use the present system for other methylotrophs.
为了利用基于小型本地甲基营养型质粒的构建体开发甲基杆菌属的宿主 - 载体系统，通过连接大肠杆菌质粒pBR328和从土壤兼性甲基营养细菌甲基杆菌属菌株M17中分离出的隐蔽质粒pWU7（7.8 kb），构建了大肠杆菌 - 甲基杆菌属穿梭载体pWUBR（12.7 kb，Apr，Tcr）。通过属于IncP - 1不相容群的pDPT51 R质粒的转移作用，将质粒pWUBR转入隐蔽质粒pWU7的原始宿主菌株M17以及无质粒的甲基杆菌属菌株R2b中，在M17中导入的杂交质粒与本地隐蔽质粒之间发生了竞争。在非选择性条件下生长25代后，pWUBR在Tcr甲基营养型转接合子中的稳定性在两种宿主中均超过90％。在R2b菌株中复制的能力表明pWUBR的宿主谱不限于pWU7的原始宿主，并表明使用本系统用于其他甲基营养菌的可能性。
