Abstract
An effective shotgun cloning procedure was developed for Bacillus megaterium by amplifying gene libraries in Bacillus subtilis. This technique was useful in isolating at least 11 genes from B. megaterium which are involved with cobalamin (vitamin B12) biosynthesis. Amplified plasmid banks were transformed into protoplasts of both a series of Cob mutants blocked before the biosynthesis of cobinamide and Cbl mutants blocked in the conversion of cobinamide into cobalamin. Amplification of gene libraries overcame the cloning barriers inherent in the relatively low protoplast transformation frequency of B. megaterium. A family of plasmids was isolated by complementation of seven different Cob and Cbl mutants. Each plasmid capable of complementing a Cob or Cbl mutant was transformed into each one of the series of Cob and Cbl mutants; many of the plasmids isolated by complementation of one mutation carried genetic activity for complementation of other mutations. By these criteria, four different complementation groups were resolved. At least six genes involved in the biosynthesis of cobinamide are carried on a fragment of DNA approximately 2.7 kilobase pairs in length; other genes involved in the biosynthesis of cobinamide were located in two other complementation groups. The physical and genetic data permitted an ordering of genes within several of the complementation groups. The presence of complementing plasmids in mutants blocked in cobalamin synthesis resulted in restoration of cobalamin biosynthesis.
Full text
PDF
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Brown B. J., Carlton B. C. Plasmid-mediated transformation in Bacillus megaterium. J Bacteriol. 1980 May;142(2):508–512. doi: 10.1128/jb.142.2.508-512.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Chang S., Cohen S. N. High frequency transformation of Bacillus subtilis protoplasts by plasmid DNA. Mol Gen Genet. 1979 Jan 5;168(1):111–115. doi: 10.1007/BF00267940. [DOI] [PubMed] [Google Scholar]
- Dubnau D., Davidoff-Abelson R. Fate of transforming DNA following uptake by competent Bacillus subtilis. I. Formation and properties of the donor-recipient complex. J Mol Biol. 1971 Mar 14;56(2):209–221. doi: 10.1016/0022-2836(71)90460-8. [DOI] [PubMed] [Google Scholar]
- Gryczan T., Shivakumar A. G., Dubnau D. Characterization of chimeric plasmid cloning vehicles in Bacillus subtilis. J Bacteriol. 1980 Jan;141(1):246–253. doi: 10.1128/jb.141.1.246-253.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Holmes D. S., Quigley M. A rapid boiling method for the preparation of bacterial plasmids. Anal Biochem. 1981 Jun;114(1):193–197. doi: 10.1016/0003-2697(81)90473-5. [DOI] [PubMed] [Google Scholar]
- Jackson R. H., Cornish-Bowden A., Cole J. A. Prosthetic groups of the NADH-dependent nitrite reductase from Escherichia coli K12. Biochem J. 1981 Mar 1;193(3):861–867. doi: 10.1042/bj1930861. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Jeter R. M., Olivera B. M., Roth J. R. Salmonella typhimurium synthesizes cobalamin (vitamin B12) de novo under anaerobic growth conditions. J Bacteriol. 1984 Jul;159(1):206–213. doi: 10.1128/jb.159.1.206-213.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Murphy M. J., Siegel L. M. Siroheme and sirohydrochlorin. The basis for a new type of porphyrin-related prosthetic group common to both assimilatory and dissimilatory sulfite reductases. J Biol Chem. 1973 Oct 10;248(19):6911–6919. [PubMed] [Google Scholar]
- Siegel L. M., Davis P. S., Murphy M. J. Incorporation of methionine-derived methyl groups into sirohaem by Escherichia coli. Biochem J. 1977 Dec 1;167(3):669–674. doi: 10.1042/bj1670669. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Vary P. S., Garbe J. C., Franzen M., Frampton E. W. MP13, a generalized transducing bacteriophage for Bacillus megaterium. J Bacteriol. 1982 Mar;149(3):1112–1119. doi: 10.1128/jb.149.3.1112-1119.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wolf J. B., Brey R. N. Isolation and genetic characterizations of Bacillus megaterium cobalamin biosynthesis-deficient mutants. J Bacteriol. 1986 Apr;166(1):51–58. doi: 10.1128/jb.166.1.51-58.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]