Construction of composite transposons for halophilic Archaea.
Transposons with selectable marker genes (e.g., antibiotic resistance) have been extremely useful tools in bacterial genetics but have not been found naturally in Archaea. We constructed synthetic transposons consisting of halobacterial ISH elements (ISH2, ISH26, or ISH28) flanking a mevinolin resistance determinant. Introduction of these constructs into Haloferax volcanii cells can produce drug-resistant transformants through homologous recombination between the plasmid hmgA gene and the chromosomal hmgA locus. This problem was overcome by using another host, Haloarcula hispanica, the hmgA gene of which shares little homology with that from Haloferax volcanii. Introduction of an ISH28-based transposon (ThD28) into Haloarcula hispanica cells produced numerous transformants. Each of these was shown to contain an ISH-flanked mevinolin resistance determinant integrated into the cellular DNA. Integration was not obviously site specific. Transposon ThD26 (based on ISH26a), was less mobile, relative to ThD28, and the ISH2-based construct (ThD22) did not transpose at all in these cells. The further development of halobacterial transposons may provide useful genetic tools allowing rapid isolation and analysis of halobacterial genes, particularly those with no selectable phenotype.
Dyall-Smith
M L
ML
Department of Microbiology, University of Melbourne, Australia.
Doolittle
W F
WF
D001483:Base Sequence; D015139:Blotting, Southern; D004251:DNA Transposable Elements / Q000235:genetics*; D004269:DNA, Bacterial / Q000032:analysis; D004274:DNA, Recombinant / Q000235:genetics*; D004352:Drug Resistance, Microbial; D005798:Genes, Bacterial / Q000235:genetics*; D005819:Genetic Markers; D006216:Halobacteriaceae / Q000187:drug effects / Q000235:genetics*; D006903:Hydroxymethylglutaryl CoA Reductases / Q000235:genetics; D008148:Lovastatin / Q000494:pharmacology; D008969:Molecular Sequence Data; D010957:Plasmids / Q000032:analysis; D014169:Transformation, Bacterial
D004251:DNA Transposable Elements; D004269:DNA, Bacterial; D004274:DNA, Recombinant; D005819:Genetic Markers; D008148:Lovastatin; D006903:Hydroxymethylglutaryl CoA Reductases