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-galactosid ase from Haloferax alicantei was purified and several peptide sequences det ermined. The peptide sequences have now been used to clone the entire beta- galactosidase gene (designated bgaH) along with some flanking chromosomal D NA. The deduced amino acid sequence of BgaH was 665 amino acids (74 kDa) an d showed greatest amino acid similarity to members of glycosyl hydrolase fa mily 42 [classification of Henrissat, B., and Bairoch, A. (1993) New famili es in the classification of glycosyl hydrolases based on amino acid sequenc e similarities. Biochem J 293: 781-788]. Within this family, BgaH was most similar (42-43% aa identity) to enzymes from extremely thermophilic bacteri a such as Thermotoga and Thermus. Family 42 enzymes are only distantly rela ted to the Sulfolobus LacS and Escherichia coli LacZ enzymes (families one and two respectively). Three open reading frames (ORFs) upstream of bgaH we re readily identified by database searches as glucose-fructose oxidoreducta se, 2-dehydro-3-deoxyphosphogluconate aldolase and 2-keto-3-deoxygluconate kinase, enzymes that are also involved in carbohydrate metabolism. Downstre am of bgaH there was an ORF which contained a putative fibronectin III moti f. The bgaH gene was engineered into a halobacterial plasmid vector and int roduced into Haloferax volcanii, a widely used strain that lacks detectable beta-galactosidase activity. Transformants were shown to express the enzym e; colonies turned blue when sprayed with Xgal and enzyme activity could be easily quantitated using a standard ONPG assay. In an accompanying publica tion, Patenge et al. (2000) have demonstrated the utility of bgaH as a prom oter reporter in Halobacterium salinarum.