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Abstract
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.