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 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.