Abstract
A technique of transposon mutagenesis involving the use of Tn5 on a suicide plasmid was developed for Proteus mirabilis. Analysis of the resulting exconjugants indicated that Tn5 transposed in P. mirabilis at a frequency of ca. 4.5 x 10(-6) per recipient cell. The resulting mutants were stable and retained the transposon-encoded antibiotic resistance when incubated for several generations under nonselective conditions. The frequency of auxotrophic mutants in the population, as well as DNA-DNA hybridizaiton to transposon sequences, confirmed that the insertion of the transposon was random and the Proteus chromosome did not contain significant insertional hot spots of transposition. Approximately 35% of the mutants analyzed possessed plasmid-acquired ampicillin resistance, although no extrachromosomal plasmid DNA was found. In these mutants, insertion of the Tn5 element and a part or all of the plasmid had occurred. Application of this technique to the study of swarmer cell differentiation in P. mirabilis is discussed.
Full text
PDF
Images in this article
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Belas R., Erskine D., Flaherty D. Proteus mirabilis mutants defective in swarmer cell differentiation and multicellular behavior. J Bacteriol. 1991 Oct;173(19):6279–6288. doi: 10.1128/jb.173.19.6279-6288.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Belas R., Mileham A., Simon M., Silverman M. Transposon mutagenesis of marine Vibrio spp. J Bacteriol. 1984 Jun;158(3):890–896. doi: 10.1128/jb.158.3.890-896.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Coetzee J. N. Genetics of the Proteus group. Annu Rev Microbiol. 1972;26:23–54. doi: 10.1146/annurev.mi.26.100172.000323. [DOI] [PubMed] [Google Scholar]
- Ditta G., Stanfield S., Corbin D., Helinski D. R. Broad host range DNA cloning system for gram-negative bacteria: construction of a gene bank of Rhizobium meliloti. Proc Natl Acad Sci U S A. 1980 Dec;77(12):7347–7351. doi: 10.1073/pnas.77.12.7347. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Groisman E. A., Casadaban M. J. Cloning of genes from members of the family Enterobacteriaceae with mini-Mu bacteriophage containing plasmid replicons. J Bacteriol. 1987 Feb;169(2):687–693. doi: 10.1128/jb.169.2.687-693.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Groisman E. A., Castilho B. A., Casadaban M. J. In vivo DNA cloning and adjacent gene fusing with a mini-Mu-lac bacteriophage containing a plasmid replicon. Proc Natl Acad Sci U S A. 1984 Mar;81(5):1480–1483. doi: 10.1073/pnas.81.5.1480. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kolter R., Inuzuka M., Helinski D. R. Trans-complementation-dependent replication of a low molecular weight origin fragment from plasmid R6K. Cell. 1978 Dec;15(4):1199–1208. doi: 10.1016/0092-8674(78)90046-6. [DOI] [PubMed] [Google Scholar]
- Miller V. L., Mekalanos J. J. A novel suicide vector and its use in construction of insertion mutations: osmoregulation of outer membrane proteins and virulence determinants in Vibrio cholerae requires toxR. J Bacteriol. 1988 Jun;170(6):2575–2583. doi: 10.1128/jb.170.6.2575-2583.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Way J. C., Davis M. A., Morisato D., Roberts D. E., Kleckner N. New Tn10 derivatives for transposon mutagenesis and for construction of lacZ operon fusions by transposition. Gene. 1984 Dec;32(3):369–379. doi: 10.1016/0378-1119(84)90012-x. [DOI] [PubMed] [Google Scholar]
- Williams F. D., Schwarzhoff R. H. Nature of the swarming phenomenon in Proteus. Annu Rev Microbiol. 1978;32:101–122. doi: 10.1146/annurev.mi.32.100178.000533. [DOI] [PubMed] [Google Scholar]
- de Lorenzo V., Herrero M., Jakubzik U., Timmis K. N. Mini-Tn5 transposon derivatives for insertion mutagenesis, promoter probing, and chromosomal insertion of cloned DNA in gram-negative eubacteria. J Bacteriol. 1990 Nov;172(11):6568–6572. doi: 10.1128/jb.172.11.6568-6572.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]