Transposon mutagenesis in halophilic eubacteria : conjugal transfer and insertion of transposon Tn5 and Tn1732 in Halomonas elongata

• Published in: FEMS microbiology letters Vol. 128; no. 3; pp. 293 - 299
• Main Authors: KUNTE, H. J, GALINSKI, E. A
• Format: Journal Article
• Language: English
• Published: Oxford Blackwell 15.05.1995
• Subjects: Bacteriology; Biological and medical sciences; Blotting, Southern; Conjugation, Genetic; DNA Transposable Elements - genetics; Escherichia coli - genetics; Fundamental and applied biological sciences. Psychology; Genetics; Gram-Negative Aerobic Bacteria - drug effects; Gram-Negative Aerobic Bacteria - genetics; Halomonas elongata; Kanamycin Resistance - genetics; Microbiology; Mutagenesis, Insertional - methods; Sodium Chloride - pharmacology; Halophily; Transposon; Site directed mutagenesis; Bacteria; Transposable element; Conjugation; Halomonadaceae; Genetic transfer
• ISSN: 0378-1097; 1574-6968
• DOI: 10.1016/0378-1097(95)00121-K

Molecular genetic studies of halophilic eubacteria have been limited by the lack of a suitable method for mutagenesis. To overcome this, we established a transposon mutagenesis procedure for the ectoine-producing, halophilic bacterium Halomonas elongata. We used suicide plasmids pSUP101 and pSUP102-Gm to introduce the transposons Tn5 and Tn1732 respectively into H. elongata via Escherichia coli SM10 mediated conjugation. Our finding that H. elongata is sensitive to aminoglycoside antibiotics at low salinity enabled us to apply transposons that mediate kanamycin resistance. The insertions of transposon Tn1732 occurred at different sites in the chromosome of H. elongata, as proved by Southern hybridization analysis. Phenotypic analysis revealed that different auxotrophic and salt sensitive mutants were generated by mutagenesis with transposon Tn1732. To our knowledge this is the first report of a successful application of a transposon for direct generalized mutagenesis in a halophilic eubacterium.