Cloning of the Streptococcus mutans Gene Encoding Glucan Binding Protein B and Analysis of Genetic Diversity and Protein Production in Clinical Isolates

Abstract

Streptococcus mutans, the primary etiological agent of dental caries, produces several activities that promote its accumulation within the dental biofilm. These include glucosyltransferases, their glucan products, and proteins that bind glucan. At least three glucan binding proteins have been identified, and GbpB, the protein characterized in this study, appears to be novel. The gbpB gene was cloned and the predicted protein sequence contained several unusual features and shared extensive homology with a putative peptidoglycan hydrolase from group B streptococcus. Examination of gbpB genes from clinical isolates of S. mutans revealed that DNA polymorphisms, and hence amino acid changes, were limited to the central region of the gene, suggesting functional conservation within the amino and carboxy termini of the protein. The GbpB produced by clinical isolates and laboratory strains showed various distributions between cells and culture medium, and amounts of protein produced by individual strains correlated positively with their ability to grow as biofilms in an in vitro assay.

Two-component signal transduction systems in oral bacteria

“…Consistently, atypical long-chain phenotypes are found in v icK isogenic mutants obtained in S. pnemoniae , S. mutans , and S. sanguinis [90,105,106,111]. In VicR regulons, the most conserved target gene encodes PcsB (protein required for cell separation of group B Streptococcus ) that was first identified in group B streptococci [114] and orthologue GbpB (glucan-binding protein B in S. mutans ) [115,116]. Table 2 shows comparisons of VicR gene targets among streptococcal species of the oral cavity and oropharynx.…”

Glucan-binding Proteins of the Oral Streptococci

“…The instability of the gbpB mutant indicates that it may be an essential protein; its homology to peptidoglycan hydrolase may mean that GbpB is essential for cell-wall cycling and synthesis (Mattos-Graner et al, 2001. Recently, it became apparent that GbpB was identical to the immunodominant glycoprotein IDG-60 and the general stress protein GSP-781 (Chia et al, 2001a,b;Mattos-Graner et al, 2001).…”

“…The null mutant of GbpB is unstable (Mattos-Graner et al, 2002), precluding in vitro investigation. However, the amount of GbpB produced by clinical isolates correlates with their biofilm-forming abilities (Mattos-Graner et al, 2001). The loss of GbpC in the same model system as used with GbpA results in a biofilm about twice as thick as normal (Fig.).…”

Genotypic diversity of S. mutans in dental biofilm formed in situ under sugar stress exposure

“…Integrities of the genomic DNA samples were checked in samples electrophoretically resolved in 1% agarose gel (Invitrogen, Barcelona, Spain) and stained with ethidium bromide (5 μg/mL). Isolates were confirmed for species identity in PCR reactions with primers specific for gtfB, enconding glucosyltransferase B (5'-ACTACACTTTCGGGTGGCTTGG-3' and 5'-CAGTATAAGCGCCAGTTTCATC-3') (17) (Invitrogen) and specific to gbpB, enconding glucan-binding protein B (5'-CAACAGAAGCACAACCATCA-3' and 5'-TGTCCACCATTACCCCAGT-3') (18). The reactions were performed as described elsewhere (17,18) and the PCR products were analyzed by electrophoresis.…”