Overlapping activator sequences determined for two oppositely oriented promoters in halophilic Archaea.

Transcription of the genomic region involved in gas vesicle formation in Halobacterium salinarum (p-vac) and Haloferax mediterranei (mc-vac) is driven by two divergent promoters, P(A) and P(D), separated by only 35 nt. Both promoters are activated by the transcription activator GvpE which in the case of P(mcA) requires a 20-nt sequence (UAS) consisting of two conserved 8-nt sequence portions located upstream of BRE. Here, we determined the two UAS elements in the promoter region of p-vac by scanning mutageneses using constructs containing P(pD) (without P(pA)) fused to the bgaH reporter gene encoding an enzyme with beta-galactosidase activity, or the dual reporter construct pApD with P(pD) fused to bgaH and P(pA) to an altered version of gvpA. The two UAS elements found exhibited a similar extension and distance to BRE as previously determined for the UAS in P(mcA). Their distal 8-nt portions almost completely overlapped in the centre of P(pD)-P(pA), and mutations in this region negatively affected the GvpE-mediated activation of both promoters. Any alteration of the distance between BRE and UAS resulted in the loss of the GvpE activation, as did a complete substitution of the proximal 8-nt portion, underlining that a close location of UAS and BRE was very important.

Identification of core active site residues of the sulfur oxygenase reductase from Acidianus ambivalens by site-directed mutagenesis.

The sulfur oxygenase reductase (SOR) is the initial enzyme in the sulfur oxidation pathway of Acidianus ambivalens. The SOR is composed of 308 aa residues, three of which are cysteines, and contains a mononuclear non-heme iron site. Mutations of the suspected iron-binding residues H86, H90 and E114 to alanine resulted in inactive enzyme with no iron incorporated, whereas an E114D mutant showed 1% of wild type activity. The mutation of C31 to alanine and serine caused inactivity of the enzyme, however, the iron content was the same as in the wild type. C101A, C104S/A, and C101/104S/A double mutants caused a decrease in specific activity to 10-43% of the wild type while the C101S mutant showed only 1% activity of the wild type. The drop in activity of the C101S and E114D mutants was accompanied with a proportional decrease in iron content. In all cases the oxygenase and reductase partial reactions were equally affected. It was concluded that the Fe site with H86, H90 and E114 as ligands and C31 constitute the core active site whereas C101 and C104 optimize reaction conditions.