Most cited article - PubMed ID 2125292
An efficient method for isolation of plasmid DNA from methylotrophic bacteria
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.
Several tens of methanol-utilizing bacterial strains isolated from soil were screened for the presence of plasmids. From the obligate methylotroph Methylomonas sp. strain R103a plasmid pIH36 (36 kb) was isolated and its restriction map was constructed. In pink-pigmented facultative methylotrophs (PPFM), belonging to the genus Methylobacterium four plasmids were detected: plasmids pIB200 (200 kb) and pIB14 (14 kb) in the strain R15d and plasmids pWU14 (14 kb) and pWU7 (7.8 kb) in the strain M17. Because of the small size and the presence of several unique REN sites (HindIII, EcoRI, NcoI), plasmid pWU7 was chosen for the construction of a vector for cloning in methylotrophs. Cointegrates pKWU7A and pKWU7B were formed between pWU7 and the E. coli plasmid pK19 Kmr, which were checked for conjugative transfer from E. coli into the methylotrophic host.
- MeSH
- Anti-Bacterial Agents pharmacology MeSH
- Drug Resistance, Microbial genetics MeSH
- Escherichia coli genetics MeSH
- Genetic Vectors MeSH
- Gram-Negative Aerobic Bacteria genetics metabolism MeSH
- Cloning, Molecular MeSH
- Conjugation, Genetic MeSH
- Lactams MeSH
- Methanol metabolism MeSH
- Methylococcaceae genetics metabolism MeSH
- Plasmids genetics MeSH
- Restriction Mapping * MeSH
- Publication type
- Journal Article MeSH
- Names of Substances
- Anti-Bacterial Agents MeSH
- Lactams MeSH
- Methanol MeSH
