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Abstract
Gas vesicle formation in haloarchaea involves 14 gas vesicle protein (gvp) genes. The strong promoter PA drives the expression of gvpACNO, which encodes 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 UASD 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 consists of two 8 nt portions (PA , 1A+2A; 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 with 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. The TATA box and/or transcription factor B recognition element (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 ’ chimeras were less active, and BRED was not functional in the environment of ‘PA’. 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 TATAD and the transcriptional start site of gvpD.
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Funding
- German Research Foundation DFG (Award Pf 165/10-1)