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We report here on advances made in the construction of plasmid shuttle vectors suitable for genetic manipulations in both Escherichia coli and halobacteria. Starting with a 20.4-kb construct, pMDS1, new vectors were engineered which were considerably smaller yet retained several alternative cloning sites. A restriction barrier observed when plasmid DNA was transferred into Haloferax volcanii cells was found to operate via adenine methylation, resulting in a 10(3) drop in transformation efficiency and the loss of most constructs by incorporation of the resistance marker into the chromosome. Passing shuttle vectors through E. coli dam mutants effectively avoided this barrier. Deletion analysis revealed that the gene(s) for autonomous replication of pHK2 (the plasmid endogenous to Haloferax strain Aa2.2 and used in the construction of pMDS1) was located within a 4.2-kb SmaI-KpnI fragment. Convenient restriction sites were identified near the termini of the novobiocin resistance determinant (gyrB), allowing the removal of flanking sequences (including gyrA). These deletions did not appear to significantly affect transformation efficiencies or the novobiocin resistance phenotype of halobacterial transformants. Northern blot hybridization with strand- and gene-specific probes identified a single gyrB-gyrA transcript of 4.7 kb. This is the first demonstration in prokaryotes that the two subunits of DNA gyrase may be cotranscribed.