MUTATIONS IN DNA GYRASE RESULT IN NOVOBIOCIN RESISTANCE IN HALOPHILIC ARCHAEBACTERIA

We have developed a cloning vector for use in halophilic archaebacteria which has a novobiocin resistance determinant as a selectable marker. The resistance determinant, which was derived from the genome of a resistant mutant strain, was mapped to a site within a 6.7-kb DNA clone by using a recombination assay and was sequenced. An open reading frame of 1,920 nucleotides (640 amino acids) was identified, with the predicted protein being highly homologous to the DNA gyrase B subunit (i.e., GyrB) of eubacteria. Three mutations were identified in the GyrB protein of the resistant mutant compared with the wild type (at amino acids 82, 122, and 137) which together enable Haloferax cells to grow in concentrations of novobiocin some 1,000 times higher than that possible for cells carrying only the wild-type enzyme. One base beyond the stop codon of gyrB was the start of gyrA, coding for the gyrase A subunit.