Kunte H J, Galinski E A
Institut für Mikrobiologie and Biotechnologie, Rheinische Friedrich-Wilhelms-Universität, Bonn, Germany.
FEMS Microbiol Lett. 1995 May 15;128(3):293-9. doi: 10.1111/j.1574-6968.1995.tb07539.x.
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.
嗜盐真细菌的分子遗传学研究因缺乏合适的诱变方法而受到限制。为克服这一问题，我们为产ectoine的嗜盐细菌嗜盐栖热袍菌建立了转座子诱变程序。我们使用自杀质粒pSUP101和pSUP102-Gm分别通过大肠杆菌SM10介导的接合作用将转座子Tn5和Tn1732导入嗜盐栖热袍菌。我们发现嗜盐栖热袍菌在低盐度下对氨基糖苷类抗生素敏感，这使我们能够应用介导卡那霉素抗性的转座子。Southern杂交分析证明，转座子Tn1732插入到嗜盐栖热袍菌染色体的不同位点。表型分析表明，用转座子Tn1732诱变产生了不同的营养缺陷型和盐敏感突变体。据我们所知，这是转座子在嗜盐真细菌中成功应用于直接通用诱变的首次报道。
