Mutant analysis of the gene encoding glucan binding protein B indicates an essential role in Streptococcus mutans

Active or passive immunity to glucan binding protein B (GbpB) from S. mutans has caries protective effects. From a functional standpoint, GbpB shares homology to proteins involved in peptidoglycan biosynthesis in addition to a modest affinity for dextran. Objectives: The goal of this study was to generate a gbpB knock-out mutant in S. mutans. Methods: Integrating constructs based on suicide vector pVA891.2 carried different gbpB sequences and were transformed into several S. mutans strains. Recombinant clones were then selected for resistance to erythromycin (Em). Allelic exchange was also carried out with a linear fragment of gbpB containing an Em resistance cassette. Results: All strategies yielded unstable mutants. PCR, Southern blot, and sequence analysis of a large number of mutants revealed that the emr gene, and mutant versions of gbpB were associated with wild type copies, and all mutants produced GbpB as detected by Western blot. Apart from Em resistance, the majority of mutants showed no other phenotypic changes. However, one class co-expressed 100-190 kDa proteins that reacted with anti-GbpB antibody, grew poorly, and scanning electron microscopy showed they had abnormal cell shape. Conclusions: Thus attempts to inactivate gbpB yielded unstable mutant strains in which GbpB production was conserved, indicating that the protein has an essential function in S. mutans. Supported by NIDCR (DE06153) and FAPESP (99/08278-9).