Cloning of a Streptococcus mutans glucosyltransferase 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 lambda L47.1 gene library. The activity was catalyzed by gene products of 150 and 145 kilodaltons which reacted with antibodies directed against both soluble and insoluble glucan-synthesizing GTFs. The enzyme present in crude Escherichia coli extracts synthesized both soluble and insoluble glucans. The enzyme was partially purified from lysates of the lambda DS-76 clone and synthesized both types of glucans in a primer-independent fashion. 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.

Utilization of a mini‐mu transposon to construct defined mutants in Streptococcus mutans

“…The results presented also indicate that MudE couid insert into the GS-5 chromosome without loss of GTF-i activity (strain 101 OAR), It ts iii<eiy that these transformants were obtained foliowing insertion of MudE into a second gene which is partially homologous to the gtfB gene (Aoki et al, 1986). However, the hybridization resuits (Fig, 3) did not attow discrimination i:>etween insertions into the two genes because of the relatively large size of the MudE inserts and the resultant fcoRi fragments.…”

“…Transformants were then utilized to prepare lysates for transduction of strain M8820 (Mucts). The Km'Cm' transductants were next grown on sucrosecontaining LB agar plates in order to detect inactivation of the GTF-1 gene (Aoki et al. 1986).…”

Molecular analysis of the inhibitory effects of oolong tea polyphenols on glucan-binding domain of recombinant glucosyltransferases fromStreptococcus mutansMT8148

“…Sodium dodecyl sulfate^polyacrylamide gel electrophoresis (SDS^PAGE) and Western blot analyses were carried out to determine the purity of the rGTFs and their expression of the gtf gene, according to a method previously described by Aoki et al [6]. rGTFB and rGTFD were detected using rabbit anti-GTF antibody [13] as well as swine anti-rabbit immunoglobulin conjugated with alkaline phosphatase (Dako, Glostrup, Denmark).…”

Effects of Magnolol and Honokiol on the activities of streptococcal glucosyltransferases both in solution and adsorbed on an experimental pellicle

“…Glucosyltransferases (Gtfs), produced by cariogenic streptococci such as Streptococcus mutans (Loesche 1986;Smith 2003), are essential for the expression of virulence that allows to successfully colonize the tissue of the host mouth (Fujiwara et al 2000;Duarte et al 2003). The Gtfs synthesize soluble and insoluble a-linked glucans from sucrose and contribute significantly to the polysaccharide composition of the dental plaque matrix (Aoki et al 1986;Hanada and Kuramitsu 1988). These glucans can enhance the pathogenic potential of dental plaque by promoting the accumulation of cariogenic streptococci on the teeth of humans and experimental animals (Smith 2003).…”

“…Streptococcus mutans produces at least three GTFs: GtfB, GtfC and GtfD (Koo et al 2002). GtfB synthesizes a polymer of mostly insoluble a1,3-linked glucan (Aoki et al 1986). GtfC synthesizes a mixture of insoluble a1,3linked glucan (Hanada and Kuramitsu 1988) and soluble a1,6-linked glucan, and GtfD produces a soluble glucan that is predominantly composed of a1,6-linked glucosyl units (Hanada and Kuramitsu 1989).…”