Molecular Cloning of Streptococcus bovis Lactose Catabolic Genes

Abstract

SUMMARY: The hydrolysis of lactose in Streptococcus bovis was shown to be catalysed by β-D-galactosidase. To isolate lactose catabolic genes, a gene library of S. bovis DNA was constructed in bacteriophage λ47.1 and recombinants expressing β-D-galactosidase activity were detected using 5-bromo-4-chloro-indoyl-β-D-galactoside (X-Gal). The gene was cloned on a 7·8 kb DNA restriction fragment into pBR322 to generate the recombinant plasmid pHG1, which also encoded S. bovis lactose permease, thiogalactoside transacetylase and lactose repressor protein. The position and orientation of the four genes were determined by subcloning and transposon mutagenesis. The β-D-galactosidase, lactose permease and thiogalactoside transacetylase genes constitute an operon controlled by the repressor protein. β-D-Galactosides induced expression of the S. bovis lac genes in Escherichia coli while glucose had no effect. The nucleotide sequence of the presumptive regulatory region of the S. bovis lac operon was determined and compared with the corresponding E. coli sequence. The operators and the 5’ end of the lacZ genes showed strong identity. The catabolite activator protein binding sequence, present in the E. coli promoter, was absent from the corresponding S. bovis region.