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 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 beta-galactosidase, and colonies on agar plates turned blue when sprayed with X-Gal. The beta-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.
Gregor
Dagmar
D
Institut für Mikrobiologie und Genetik, Technische Universität Darmstadt, Schnittspahnstr. 10, D-64287 Darmstadt, Germany1.
Pfeifer
Felicitas
F
Institut für Mikrobiologie und Genetik, Technische Universität Darmstadt, Schnittspahnstr. 10, D-64287 Darmstadt, Germany1.
D019843:Archaeal Proteins / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D019848:Gene Expression Regulation, Archaeal; D017930:Genes, Reporter / Q000235:genetics*; D019617:Halobacterium salinarum / Q000235:genetics* / Q000378:metabolism; D019621:Haloferax / Q000235:genetics* / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000235:genetics; D011506:Proteins; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014158:Transcription, Genetic; D014169:Transformation, Bacterial; D001616:beta-Galactosidase / Q000235:genetics* / Q000378:metabolism
D019843:Archaeal Proteins; D008565:Membrane Proteins; D011506:Proteins; D011993:Recombinant Fusion Proteins; C038524:gas vesicle protein; D001616:beta-Galactosidase