In vivo analyses of constitutive and regulated promoters in halophilic archaea

The two gvpA promoters P-cA and P-pA of Halobacterium salinarum, and the P-mcA promoter of Haloferax mediterranei were investigated with respect to growth-phase-dependent expression and regulation in Haloferax volcanii transformants using the bgaH reading frame encoding BgaH, an enzyme with beta-galactosidase activity, as reporter. For comparison, the P-fdx promoter of the ferredoxin gene of Hbt. salinarum and the P-bgaH promoter of Haloferax lucentense (formerly Haloferax alicantei) were analysed. P-fdx, driving the expression of a house-keeping gene, was highly active during the exponential growth phase, whereas P-bgaH and the three gvpA promoters yielded the largest activities during the stationary growth phase. Compared to P-fdx, the basal promoter activities of PpA and PcA were rather low, and larger activities were only detected in the presence of the endogenous transcriptional activator protein GvpE. The P-cA promoter does not yield a detectable basal promoter activity and is only active in the presence of the homologous cGvpE. To investigate whether the P-cA-TATA box and the BRE element were the reason for the lack of the basal PcA activity, these elements and also sequences further upstream were substituted with the respective sequences of the stronger PpA promoter and investigated in H-fx. volcanii transformants. All these promoter chimera did not yield a detectable basal promoter activity. However, whenever the P-pA-BRE element was substituted for the P-cA-BRE, an enhanced cGvpE-mediated activation was observed. The promoter chimeras harbouring PpA-BRE plus 5 (or more) bp further upstream also gained activation by the heterologous pGvpE and mcGvpE proteins. The sequence required for the GvpE-mediated activation was determined by a 4 bp scanning mutagenesis with the 45 bp region upstream of P-mcA-BRE. None of these alterations influenced the basal promoter activity, but the sequence TGAAACGG-n4-TGAACCAA was important for the GvpE-mediated activation of P-mcA.