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

Abstract
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 β-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.

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