Transposon mutagenesis in halophilic eubacteria: conjugal transfer and insertion of transposon Tn5 and Tn1732inHalomonas elongata

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Abstract
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 Tn7732 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 In 1732 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.
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