A dual promoter region with overlapping activator sequences drives the expression of gas vesicle protein genes in haloarchaea

Abstract

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.