Functional analysis of glucan binding protein B from Streptococcus mutans

Abstract

Mutans streptococci are major etiological agents of dental caries, and several of their secreted products contribute to bacterial accumulation on teeth. Of these, Streptococcus mutans glucan binding protein B (GbpB) is a novel, immunologically dominant protein. Its biological function is unclear, although GbpB shares homology with a putative peptidoglycan hydrolase from S. agalactiae and S. pneumoniae, indicative of a role in murein biosynthesis. To determine the cellular function of GbpB, we used several approaches to inactivate the gene, analyze its expression, and identify interacting proteins. None of the transformants analyzed were true gbpB mutants, since they all contained both disrupted and wild-type gene copies, and expression of functional GbpB was always conserved. Thus, the inability to obtain viable gbpB null mutants supports the notion that gbpB is an essential gene. Northern blot and real-time PCR analyses suggested that induction of gbpB expression in response to stress was a strain-dependent phenomenon. Proteins that interacted with GbpB were identified in pull-down and coimmunoprecipitation assays, and these data suggest that GbpB interacts with ribosomal protein L7/L12, possibly as part of a protein complex involved in peptidoglycan synthesis and cell division.

FIG. 1.

Phenotypes of transformants obtained by integration of pRMG2. (A) Parent strain UA130 growing in liquid medium. (B) A representative transformant growing under the same condition. (C) Scanning electron micrographs showing chains of cells of parent strain UA130. (D and E) Abnormally shaped and larger transformant cells. (F) Transformant cells with altered septa.

FIG. 2.

Analysis of representative transformants obtained by integration of pRMG2 into the S. mutans genome. (A) Predicted integration of pRMG2 into the genome by Campbell-like homologous recombination within gbpB. PCR primer sites are depicted as arrows. Primers sets are described in Table 2. , S. mutans genome; shaded boxes, gbpB and Erm^r genes; straight lines, vector sequences. (B) PCR amplicons (1.6 kb) generated with primer set 3 in UA130 (wild type [WT]) and representative transformant MB1. (C) Amplification with primer sets 4 and 5 to confirm linkage of gbpB with Erm^r in transconjugants; the wild-type DNA template was used as a negative control. (D) Map of pRMG2 integration deduced from sequencing of PCR amplicons. (E) Western blot of cell extracts from transformants (1 to 3) probed with anti-GbpB antibody. Control fractions from the parent were cell extract (WTa) and culture supernatant (WTb).

FIG. 3.

Analysis of S. mutans UA130 transformants obtained by allelic exchange. (A) Predicted replacement of the wild-type gene by the disrupted gbpB by double crossover recombination. (B) PCR amplicons generated with primer sets 2 and 6 demonstrated linkage of gbpB and Erm^r in transconjugants (MC). Amplicons were also detected in pRMG3, from which the transforming fragment was isolated, but not in the parent strain. WT, wild type. (C) A second wild-type copy of gbpB was obtained using primer set 3. (D) Map of gbpB region after recombination and regeneration of wild-type gbpB, deduced from PCR and sequence data. (E) Southern blot analysis of HindIII-digested transformant genomic DNA probed with gbpB confirmed the presence of two copies of the gene; the larger fragment also hybridized with the Erm^r probe, proving it to be the mutated version. (F) Western blots of culture supernatants of transformant and parent strains indicated expression of GbpB.

FIG. 4.

Northern blot analysis of gbpB transcripts from S. mutans UA130. A schematic map of the gbpB region is depicted above the blot. Genes are represented by arrows that indicate the direction of transcription. Northern blot lanes: M, RNA size markers; 1, RNA purified from a log-phase culture of UA130; 2, an RNA transcript that hybridized with gbpB was approximately 1.3 kb, consistent with monocistronic transcription. nt, nucleotide.

FIG. 5.

Relative expression of gbpB under stress and control conditions. Values were calculated from Northern dot blot intensities of triplicate spots. (A) Expression of gbpB in response to incubation with 0.5 M NaCl. Strains in which gbpB induction was statistically significant are shown with calculated P values. (B) Expression of gbpB in response to incubation at pH 5.5.

FIG. 6.

Pull-down assay with GbpB. (A) Silver-stained SDS-PAGE gel showing purified GST-GbpB that was used in the assay and the proteins recovered from the pull-down fraction. The indicated 14.4-kDa protein was excised and sequenced. (B) Control pull-down assay carried out with GST-FimR from P. gingivalis.

FIG. 7.

Coimmunoprecipitation assays. (A) Western blot of S. mutans cell wall (CW) and cell membrane (CM) proteins probed with anti-GbpB antibody. (B) Western blot of the same fractions probed with anti-L7/L12 antibody. (C) Zinc-stained SDS-PAGE of either cell wall or cell membrane proteins dissociated from uncoupled columns (lanes C [control]) and from anti-GbpB antibody-coupled columns. (D) Western blot of dissociated proteins after probing with anti-L7/L12 antibody.