A new simvastatin (mevinolin)-resistance marker from Haloarcula hispanica and a new Haloferax volcanii strain cured of plasmid pHV2.
The mevinolin-resistance determinant, hmg, encodes the enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase and is a commonly used selectable marker in halobacterial genetics. Plasmids bearing this marker suffer from instability in Haloferax volcanii because the resistance gene was derived from the genome of this species and is almost identical in sequence to the chromosomal copy. In order to reduce the level of homologous recombination between introduced plasmid vectors and the chromosome of Haloferax, a homologue of the hmg determinant was obtained from the distantly related organism, Haloarcula hispanica. The nucleotide sequences of the wild-type genes (hmgA) of these two species are only 78% identical, and the predicted protein sequences show 71% identity. In comparison to the wild-type hmgA gene, the resistance gene from a mutant resistant to simvastatin (an analogue of mevinolin) showed a single base substitution in the putative promoter. Plasmids constructed using the new resistance determinant were stably maintained under selection in Hfx. volcanii and possessed very low recombination rates with the chromosome of this species. In addition, an improved strain of Hfx. volcanii was developed to overcome the plasmid instability and growth reduction observed in the commonly used WFD11 strain.
Department of Microbiology and Immunology, University of Melbourne, Melbourne, VIC 3010, Australia1.
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Wendoloski
Department of Microbiology and Immunology, University of Melbourne, Melbourne, VIC 3010, Australia1.
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Ferrer
Department of Microbiology and Immunology, University of Melbourne, Melbourne, VIC 3010, Australia1.
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Dyall-Smith
D004269:DNA, Bacterial / Q000032:analysis; D004352:Drug Resistance, Microbial; D005798:Genes, Bacterial; D005819:Genetic Markers; D019613:Haloarcula / Q000187:drug effects / Q000235:genetics* / Q000254:growth & development; D019622:Haloferax volcanii / Q000187:drug effects / Q000235:genetics*; D006903:Hydroxymethylglutaryl CoA Reductases / Q000235:genetics* / Q000378:metabolism; D019161:Hydroxymethylglutaryl-CoA Reductase Inhibitors / Q000494:pharmacology*; D008148:Lovastatin / Q000494:pharmacology*; D008969:Molecular Sequence Data; D010957:Plasmids; D017354:Point Mutation; D011401:Promoter Regions, Genetic; D011995:Recombination, Genetic; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D019821:Simvastatin / Q000494:pharmacology*
D004269:DNA, Bacterial; D005819:Genetic Markers; D019161:Hydroxymethylglutaryl-CoA Reductase Inhibitors; D008148:Lovastatin; D019821:Simvastatin; D006903:Hydroxymethylglutaryl CoA Reductases