Use of a halobacterial bgaH reporter gene to analyse the regulation of gene expression in halophilic archaea

The bgaH reading frame encoding a beta -galactosidase of 'Haloferax alicant ei' was used as a reporter gene to investigate three different promoter reg ions derived from gvpA genes of Haloferax mediterranei (mc-gvpA) and Haloba cterium salinarum (c-gvpA and p-gvpA) in Haloferax volcanii INTRODUCTION tr ansformants. The fusion of bgaH at the start codon of each gvpA reading fra me (A1-bgaH fusion genes) caused translational problems in some cases. Tran sformants containing constructs with fusions further downstream in the gvpA reading frame (A-bgaH) produced beta -galactosidase, and colonies on agar plates turned blue when sprayed with X-Gal. The beta -galactosidase activit ies quantified by standard ONPG assays correlated well with the mRNA data d etermined with transformants containing the respective gvpA genes: the cA-b gaH fusion gene was completely inactive, the mcA-bgaH transformants showed low amounts of products whereas the pA-bgaH fusion gene was constitutively expressed in the respective transformants. The transcription of each A-bgaH gene was activated by the homologous transcriptional activator protein Gvp E. The cGvpE, pGvpE and mcGvpE proteins were able to activate the promoter of pA-kgaH and mcA-bgaH, whereas the promoter of cA-bgaH was only activated by cGvpE. Among the three GvpE proteins tested, cGvpE appeared to be the s trongest transcriptional activator.