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Microbiology (2001), 147, 1745-1754.
© 2001 Society for General Microbiology

Genetics and Molecular Biology

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

Dagmar Gregor1 and Felicitas Pfeifer1

Institut für Mikrobiologie und Genetik, Technische Universität Darmstadt, Schnittspahnstr. 10, D-64287 Darmstadt, Germany1

Author for correspondence: Felicitas Pfeifer. Tel: +49 6151 162957. Fax: +49 6151 162956. e-mail: pfeifer{at}bio.tu-darmstadt.de

The bgaH reading frame encoding a ß-galactosidase of ‘Haloferax alicantei’ was used as a reporter gene to investigate three different promoter regions derived from gvpA genes of Haloferax mediterranei (mc-gvpA) and Halobacterium salinarum (c-gvpA and p-gvpA) in Haloferax volcanii transformants. The fusion of bgaH at the start codon of each gvpA reading frame (A1–bgaH fusion genes) caused translational problems in some cases. Transformants containing constructs with fusions further downstream in the gvpA reading frame (A–bgaH) produced ß-galactosidase, and colonies on agar plates turned blue when sprayed with X-Gal. The ß-galactosidase activities quantified by standard ONPG assays correlated well with the mRNA data determined with transformants containing the respective gvpA genes: the cA–bgaH 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 GvpE. The cGvpE, pGvpE and mcGvpE proteins were able to activate the promoter of pA–bgaH 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 strongest transcriptional activator.

Keywords: Haloferax volcanii, ß-galactosidase reporter gene, gene regulation

Abbreviations: gvp, gas vesicle protein gene; Gvp, gas vesicle protein




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