Sequence and expression of a halobacterial beta-galactosidase gene.
Studies of gene expression in haloarchaea have been greatly hindered by the lack of a convenient reporter gene. In a previous study, a beta-galactosidase from Haloferax alicantei was purified and several peptide sequences determined. The peptide sequences have now been used to clone the entire beta-galactosidase gene (designated bgaH) along with some flanking chromosomal DNA. The deduced amino acid sequence of BgaH was 665 amino acids (74 kDa) and showed greatest amino acid similarity to members of glycosyl hydrolase family 42 [classification of Henrissat, B., and Bairoch, A. (1993) New families in the classification of glycosyl hydrolases based on amino acid sequence similarities. Biochem J 293: 781-788]. Within this family, BgaH was most similar (42-43% aa identity) to enzymes from extremely thermophilic bacteria such as Thermotoga and Thermus. Family 42 enzymes are only distantly related to the Sulfolobus LacS and Escherichia coli LacZ enzymes (families one and two respectively). Three open reading frames (ORFs) upstream of bgaH were readily identified by database searches as glucose-fructose oxidoreductase, 2-dehydro-3-deoxyphosphogluconate aldolase and 2-keto-3-deoxygluconate kinase, enzymes that are also involved in carbohydrate metabolism. Downstream of bgaH there was an ORF which contained a putative fibronectin III motif. The bgaH gene was engineered into a halobacterial plasmid vector and introduced into Haloferax volcanii, a widely used strain that lacks detectable beta-galactosidase activity. Transformants were shown to express the enzyme; colonies turned blue when sprayed with Xgal and enzyme activity could be easily quantitated using a standard ONPG assay. In an accompanying publication, Patenge et al. (2000) have demonstrated the utility of bgaH as a promoter reporter in Halobacterium salinarum.
Holmes
M L
ML
Department of Microbiology and Immunology, University of Melbourne, Parkville 3052, Australia.
Dyall-Smith
M L
ML
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D019766:Genes, Archaeal; D017930:Genes, Reporter; D019621:Haloferax / Q000201:enzymology / Q000235:genetics*; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D019642:RNA, Archaeal / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis; D015183:Restriction Mapping; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D001616:beta-Galactosidase / Q000096:biosynthesis / Q000235:genetics*
D019642:RNA, Archaeal; D012333:RNA, Messenger; D011994:Recombinant Proteins; D001616:beta-Galactosidase