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-m ethylglutaryl coenzyme A (HMG-CoA) reductase and is a commonly used selecta ble marker in halobacterial genetics. Plasmids bearing this marker suffer f rom instability in Haloferax volcanii because the resistance gene was deriv ed from the genome of this species and is almost identical in sequence to t he chromosomal copy. In order to reduce the level of homologous recombinati on between introduced plasmid vectors and the chromosome of Haloferax, a ho mologue of the hmg determinant was obtained from the distantly related orga nism, Haloarcula hispanics. The nucleotide sequences of the wild-type genes (hmgA) of these two species are only 78% identical, and the predicted prot ein sequences show 71% identity. In comparison to the wildtype hmgA gene, t he resistance gene from a mutant resistant to simvastatin (an analogue of m evinolin) showed a single base substitution in the putative promoter. Plasm ids constructed using the new resistance determinant were stably maintained under selection in Hfx. volcanii and possessed very low recombination rate s with the chromosome of this species. In addition, an improved strain of H fx. volcanii was developed to overcome the plasmid instability and growth r eduction observed in the commonly used WFD11 strain.