A Dual Promoter Region with Overlapping Activator Sequences Drives the Expression of Gas Vesicle Protein Genes in Haloarchaea.

Gas vesicle formation in haloarchaea involves 14 gas vesicle protein (gvp) genes. The strong promoter PA drives the expression of gvpACNO encoding the major gas vesicle structural proteins GvpA and GvpC, whereas the oppositely oriented promoter PD initiates the synthesis of the two regulator proteins, GvpD and GvpE. GvpE activates PA and PD, and requires a 20-nt upstream activator sequence (UAS). UASA and USAD partially overlap in the centre of the 35-bp intergenic region. The basal and GvpE-induced activities of PA and PD were investigated in Haloferax volcanii transformants. Each UAS is constituted of two 8-nt portions (PA, 1A+2A, and PD, 1D+2D), and mutations in the overlapping 1A and 1D portions affected the GvpE-induction of both promoters. Substitution of one of the UAS portions by a nonsense sequence showed that a complete UAS is required for activation. The activation of PA was more efficient compared to PD. Promoter PA with UASA in configuration 1A+1A was still activated by GvpE, but PD was not inducible with UASD in configuration 1D+1D. TATA box and/or BRE were exchanged between PA and PD. All elements of PA functioned well in the environment of "PD" and transferred the stronger PA activity to "PD". In contrast, the respective "PA" chimers were less active, and BRED was not functional in the environment of "PA". The relative strengths of the two promoters were considerably determined by the BRE. A 4-nt scanning mutagenesis uncovered an additional regulatory element in the region between TATAD and the transcription start site of gvpD