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Transposon mutagenesis in Caulobacter crescentus

Transposons Tn5 (Km) and Tn7 (Tp and Sm) were transferred to Caulobacter crescentus via P-type antibiotic resistance factors. Transposition was demonstrated by the isolation of chromosomal insertions of each transposon. With C. crescentus strains harboring RP4 aphA::Tn7, the introduction of a wild-type RP4 resulted in the loss of the resident plasmid. Simultaneous selection for Kmr and Smr yielded colonies with chromosomal insertions of Tn7. Examination of over 10,000 chromosomal insertions of Tn7 indicated no auxotrophic or motility mutants. Thus, Tn7 appears to have a high specificity of insertion in C. crescentus. The Mu-containing plasmid pJB4JI transferred Tn5 to C. crescentus, but the plasmid was not maintained. Control experiments showed that recovery of Mu-containing plasmids occurred at very low frequencies in C. crescentus and that the plasmids which were recovered had undergone extensive deletion of plasmid DNA. Presumably, some part of the Mu genome was not tolerated by C. crescentus. The instability of the Mu-containing plasmids makes them excellent vectors for the introduction of transposons, and we have used pJB4JI to isolated chromosomal insertions of Tn5. When several thousand of these insertion mutants were examined, we found auxotrophic and motility mutants at frequencies of 1 and 2%, respectively. These results indicate that Tn5 had a low specificity of insertion in C. crescentus and therefore would be a useful mutagen for obtaining a variety of mutant phenotypes.