Cloning of a Streptococcus mutans glycosyltransferase gene coding for insoluble glucan synthesis

Abstract

The gtfB gene coding for a glucosyltransferase (GTF) activity of Streptococcus mutans GS-5 was isolated on a 15.4-kilobase DNA fragment by using a λL47.1 gene library. The activity was catalyzed by gene products of 150 and 145 kilodaltons which reacted with antibodies directed against both soluble and insolbule glucansynthesizing GTFs. The enzyme present in crude Escherichia coli extracts synthesized both soluble and insolbule glucans. The enzyme was partially purified from lysates of the λDS-76 clone and synthesized both types of glucans in a primer-independent fasion. In addition, the purified enzyme exhibited a pI of approximately 5.0. Southern blot analysis indicated that the cloned GTF gene represented a contiguous nucleotide sequence on the strain GS-5 chromosome. Furthermore, evidence for the existence of a distinct gene sharing partial homology with gtfB was also obtained. The gtfB gene was subcloned into plasmid pACYC184 into E. coli and exhibited GTF activity when carried on GS-5 inserts as small as 5 kilobases. The approximate location of the GTF promoter and the direction of gene transcription were also determined. The cloned enzyme was not secreted through the cytoplasmic membrane of E. coli, since most of the activity was found in the cytoplasm and, in lesser amounts, associated with the cytoplasmic membrane. The gtfB gene was insertionally inactivated by introducing a gene fragment coding for erythromycin resistance into the GTF coding region. After transformation of strain GS-5 with the altered gene, transformants defective in insoluble glucan synthesis were identified. These results indicate that the gtfB gene codes for a GTF involved in insoluble glucan synthesis in strain GS-5.