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 P_A drives the expression of gvpACNO, which encodes the major gas vesicle structural proteins GvpA and GvpC, whereas the oppositely oriented promoter P_D initiates the synthesis of the two regulator proteins, GvpD and GvpE. GvpE activates P_A and P_D, and requires a 20 nt upstream activator sequence (UAS). UAS_A and UAS_D partially overlap in the centre of the 35 bp intergenic region. The basal and GvpE-induced activities of P_A and P_D were investigated in Haloferax volcanii transformants. Each UAS consists of two 8 nt portions (P_A, 1A+2A; P_D, 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 P_A was more efficient compared with P_D. Promoter P_A with UAS_A in configuration 1A+1A was still activated by GvpE, but P_D was not inducible with UAS_D in configuration 1D+1D. The TATA box and/or transcription factor B recognition element (BRE) were exchanged between P_A and P_D. All elements of P_A functioned well in the environment of ‘P_D’ and transferred the stronger P_A activity to ‘P_D’. In contrast, the respective ‘P_A’ chimeras were less active, and BRE_D was not functional in the environment of ‘P_A’. The relative strengths of the two promoters were substantially determined by the BRE. A 4 nt scanning mutagenesis uncovered an additional regulatory element in the region between TATA_D and the transcriptional start site of gvpD.