Homologous Bacterio-opsin-encoding Gene Expression Via Site-specific Vector Integration

Homologous recombination in the archaebacterium Halobacterium halobium has been investigated and exploited for the wild-type (wt) level of expression of the bacterio-opsin-encoding gene (bop). The Haloferax volcanii-Escherichia coli shuttle vector, pWL102, was used to construct a shuttle-mutagenesis vector, pEF191, bearing bop and short flanking sequences. Transformation of a bacteriorhodopsin (BR)-negative H. halobium strain with pEF191 resulted in plasmid integration at the homologous bop locus. A model for this site-specific vector integration is presented which has been confirmed by determining the arrangement of the repeated homologous sequences on the chromosome. Two different configurations are obtained after integrative transformation due to the presence of an insertion element in the genomic copy of bop. In one configuration, the functional bop cluster containing the regulatory bat and brp genes was in wt arrangement. In the second configuration, the bop cluster is interrupted by 10 kb of plasmid vector sequences, and the upstream region required for bop expression was limited to 400 bp. The BR production for both configurations was determined and found to be at wt level. These results suggest that the function of the putative bop promoter does not depend on the defined upstream positions of bat and brp. The system presented here can be easily exploited for structure-function studies on BR and introduces homologous gene targeting as a powerful tool in the study of halobacterial genetics.