CovR and VicRK Regulate Cell Surface Biogenesis Genes Required for Biofilm Formation in Streptococcus mutans

Abstract

The two-component system VicRK and the orphan regulator CovR of Streptococcus mutans co-regulate a group of virulence genes associated with the synthesis of and interaction with extracellular polysaccharides of the biofilm matrix. Knockout mutants of vicK and covR display abnormal cell division and morphology phenotypes, although the gene function defects involved are as yet unknown. Using transcriptomic comparisons between parent strain UA159 with vicK (UAvic) or covR (UAcov) deletion mutants together with electrophoretic motility shift assays (EMSA), we identified genes directly regulated by both VicR and CovR with putative functions in cell wall/surface biogenesis, including gbpB, wapE, smaA, SMU.2146c, and lysM. Deletion mutants of genes regulated by VicR and CovR (wapE, lysM, smaA), or regulated only by VicR (SMU.2146c) or CovR (epsC) promoted significant alterations in biofilm initiation, including increased fragility, defects in microcolony formation, and atypical cell morphology and/or chaining. Significant reductions in mureinolytic activity and/or increases in DNA release during growth were observed in knockout mutants of smaA, wapE, lysM, SMU.2146c and epsC, implying roles in cell wall biogenesis. WapE and lysM mutations also affected cell hydrophobicity and sensitivity to osmotic or oxidative stress. Finally, vicR, covR and VicRK/CovR-targets (gbpB, wapE, smaA, SMU.2146c, lysM, epsC) are up-regulated in UA159 during biofilm initiation, in a sucrose-dependent manner. These data support a model in which VicRK and CovR coordinate cell division and surface biogenesis with the extracellular synthesis of polysaccharides, a process apparently required for formation of structurally stable biofilms in the presence of sucrose.

Two-component signal transduction systems in oral bacteria

“…VicR is in the OmpR/PhoB family of regulators in which the output domain forms a DNA-binding winged-helix domain. In B. subtilis and Staphylococcus aureus , both VicR and VicK are essential for bacterial viability [100]; on the other hand, only vicR is essential in S. pneumoniae [105], S. mutans [106,107], and S. sanguinis [90], and both vicR and vicK can be deleted in S. gordonii [91]. In S. pyogenes , insertional inactivation of vicR was possible, but the mutant was not viable in a mice model of infection [108].…”

“…In S. pyogenes , insertional inactivation of vicR was possible, but the mutant was not viable in a mice model of infection [108]. The exact reasons for VicR and/or VicK essentially in different species are unknown, but a possibility is their role in the regulation of peptidoglycan (murein) biosynthesis, cell division and cell wall homeostasis [90,98,103,107,109–111]. Because vicK can be deleted in S. pneumoniae, S. mutans , and S. sanguinis , one possibility is that the VicR of these species could be phosphorylated by alternative pathways.

Figure 3.VicRK orthologues of oral streptococcal species.

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“…VicRK plays an important role in modulating cell division, cell-wall biosynthesis and homeostasis, and membrane integrity, as evidenced by the presence of several genes encoding autolysins and other murein hydrolases in the VicR regulons of B. subtilis [112], S. aureus [109], S. pneumoniae [105], S. mutans [107,113], and S. sanguinis [90]. 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].…”

“…ND: not determined. DB: evidence of VicR binding to the promoter region, without transcriptional analysis. a No direct binding to covR promoter region observed in EMSA assays [107]. …”

“…In addition to GbpB/PcsB, several other non-essential murein hydrolases are directly regulated by VicR in S. mutans , including SMU_2146c, SMU.2157c (LysM), SmaA, SMU.367 [107], and the autolysin A (AtlA; smu_689) [117,123]. However, these proteins are species-specific.…”