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In vivo analyses of constitutive and regulated promoters in halophilic archaea

Institut für Mikrobiologie und Genetik, Technische Universität Darmstadt, Schnittspahnstr. 10, D-64287 Darmstadt, Germany

Correspondence
Felicitas Pfeifer
pfeifer{at}bio.tu-darmstadt.de

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 -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 P_pA and P_mcA 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 P_cA activity, these elements and also sequences further upstream were substituted with the respective sequences of the stronger P_pA promoter and investigated in Hfx. 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 P_pA-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.

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