Molecular basis of isozyme formation of β-galactosidases in Bacillus stearothermophilus: Isolation of two β-galactosidase genes, bgaA and bgaB

Abstract

Bacillus stearothermophilus IAM11001 produced three β-galactosidases, β-galactosidase I, II, and III (β-gal I, II, and III), which are detectable by polyacrylamide (nondenatured) gel electrophoresis. By connecting restriction fragments of the chromosomal DNA to plasmid vectors, followed by transformation of Escherichia coli, two β-galactosidase genes (bgaA and bgaB) located close to each other on the chromosome were isolated. Identification of the gene products and Southern hybridization analyses with a 2.7-kilobase-pair EcoRI fragment containing the bgaA gene as probe revealed that a single bgaA gene exists on the genome and that β-gal II and β-gal III consist of a common subunit (the bgaA gene product; molecular weight, 120,000), but differ in their assembly (β-gal II is a dimer, and β-gal III is a tetramer). The bgaB gene product (molecular weight, 70,000) in Bacillus subtilis harboring pHG5 (a hybrid plasmid consisting of pUB110 and a 2.9-kilobase-pair EcoRI fragment) was estimated to be the β-gal I protein from its heat stability. Southern hybridization and immunological testing indicated that the two genes have no homology.