Insertional specificity of transposon Tn5 in Acinetobacter sp.

Abstract

Suicide plasmid pJB4JI, containing transposon Tn5 and phage Mu, was introduced from Escherichia coli 1830 into Acinetobacter sp. strain HO1-N and Acinetobacter calcoaceticus BD413. Kanamycin-resistant (Km(r)) exconjugants of HO1-N and BD413, isolated on complex medium, were screened for auxotrophic requirements. Over 10,000 Km(r) clones were examined, but no auxotrophs were detected. Several Km(r) exconjugants of BD413 and HO1-N, obtained from independent matings, were chosen for further study. All Tn5-containing strains exhibited kanamycin phosphotransferase activity. Km(r) strains lacked plasmid DNA as determined by three plasmid screening procedures, and the Km(r) phenotype was not transferable by conjugal matings to kanamycin-sensitive BD413, HO1-N, or E. coli HB101. The chromosomal location of Tn5 insertions in independently isolated Km(r) exconjugants of BD413 and HO1-N was characterized by restriction endonuclease mapping and hybridization studies. Results obtained from Southern hybridization studies were consistent with a single Tn5-specific insertion site in HO1-N and two sites in BD413. Phage Mu sequences were not detected in Tn5-containing Acinetobacter sp.