Richard W. Mair

The LiaFSR System Regulates the Cell Envelope Stress Response in Streptococcus mutans

Maintaining cell envelope integrity is critical for bacterial survival, including bacteria living in a complex and dynamic environment such as the human oral cavity. Streptococcus mutans, a major etiological agent of dental caries, uses two-component signal transduction systems (TCSTSs) to monitor and respond to various environmental stimuli. Previous studies have shown that the LiaSR TCSTS in S. mutans regulates virulence traits such as acid tolerance and biofilm formation. Although not examined in streptococci, homologs of LiaSR are widely disseminated in Firmicutes and function as part of the cell envelope stress response network. We describe here liaSR and its upstream liaF gene in the cell envelope stress tolerance of S. mutans strain UA159. Transcriptional analysis established liaSR as part of the pentacistronic liaFSR-ppiB-pnpB operon. A survey of cell envelope antimicrobials revealed that mutants deficient in one or all of the liaFSR genes were susceptible to Lipid II cycle interfering antibiotics and to chemicals that perturbed the cell membrane integrity. These compounds induced liaR transcription in a concentration-dependent manner. Notably, under bacitracin stress conditions, the LiaFSR signaling system was shown to induce transcription of several genes involved in membrane protein synthesis, peptidoglycan biosynthesis, envelope chaperone/proteases, and transcriptional regulators. In the absence of an inducer such as bacitracin, LiaF repressed LiaR-regulated expression, whereas supplementing cultures with bacitracin resulted in derepression of liaSR. While LiaF appears to be an integral component of the LiaSR signaling cascade, taken collectively, we report a novel role for LiaFSR in sensing cell envelope stress and preserving envelope integrity in S. mutans.

Inactivation of VicK Affects Acid Production and Acid Survival of Streptococcus mutans

The regulation of acid production in and the tolerance to low pH of the cariogenic bacterium Streptococcus mutans have garnered considerable attention since both of these properties contribute substantially to the virulence of this organism. Frequent or prolonged exposure to acid end products, mainly lactic acid, that are present following the consumption of dietary sugars erodes the dental enamel, thereby initiating dental caries. Here we report the involvement of the S. mutans VicK sensor kinase in both the acidogenicity and the aciduricity of this bacterium. When cultures were supplemented with glucose, the glycolytic rate of a VicK null mutant was significantly decreased compared to the glycolytic rate of the wild type (P < 0.05), suggesting that there was impaired acid production. Not surprisingly, the VicK deletion mutant produced less lactic acid, while an acid tolerance response assay revealed that loss of VicK significantly enhanced the survival of S. mutans (P < 0.05). Compared to the survival rates of the wild type, the survival rates of the VicK-deficient mutant were drastically increased when cultures were grown at pH 3.5 with or without preexposure to a signal pH (pH 5.5). Global transcriptional analysis using DNA microarrays and S. mutans wild-type UA159 and VicK deletion mutant strains grown at neutral and low pH values revealed that loss of VicK significantly affected expression of 89 transcripts more than twofold at pH 5.5 (P < 0.001). The affected transcripts included genes with putative functions in transport and maintenance of cell membrane integrity. While our results provide insight into the acid-inducible regulon of S. mutans, here we imply a novel role for VicK in regulating intracellular pH homeostasis in S. mutans.

Involvement of Sortase Anchoring of Cell Wall Proteins in Biofilm Formation by Streptococcus mutans

ABSTRACT Streptococcus mutans is one of the best-known biofilm-forming organisms associated with humans. We investigated the role of the sortase gene (srtA) in monospecies biofilm formation and observed that inactivation of srtA caused a decrease in biofilm formation. Genes encoding three putative sortase-dependent proteins were also found to be up-regulated in biofilms versus planktonic cells and mutations in these genes resulted in reduced biofilm biomass.

CHARACTERIZATION OF DNA BINDING SITES OF THE COME RESPONSE REGULATOR FROM STREPTOCOCCUS MUTANS

In Streptococcus mutans, both competence and bacteriocin production are controlled by ComC and the ComED two-component signal transduction system. Recent studies of S. mutans suggested that purified ComE binds to two 11-bp direct repeats in the nlmC-comC promoter region, where ComE activates nlmC and represses comC. In this work, quantitative binding studies and DNase I footprinting analysis were performed to calculate the equilibrium dissociation constant and further characterize the binding site of ComE. We found that ComE protects sequences inclusive of both direct repeats, has an equilibrium dissociation constant in the nanomolar range, and binds to these two direct repeats cooperatively. Furthermore, similar direct repeats were found upstream of cslAB, comED, comX, ftf, vicRKX, gtfD, gtfB, gtfC, and gbpB. Quantitative binding studies were performed on each of these sequences and showed that only cslAB has a similar specificity and high affinity for ComE as that seen with the upstream region of comC. A mutational analysis of the binding sequences showed that ComE does not require both repeats to bind DNA with high affinity, suggesting that single site sequences in the genome may be targets for ComE-mediated regulation. Based on the mutational analysis and DNase I footprinting analysis, we propose a consensus ComE binding site, TCBTAAAYSGT.

Characterization of a Glutamate Transporter Operon, glnQHMP, in Streptococcus mutans and Its Role in Acid Tolerance

Glutamate contributes to the acid tolerance response (ATR) of many Gram-negative and Gram-positive bacteria, but its role in the ATR of the oral bacterium Streptococcus mutans is unknown. This study describes the discovery and characterization of a glutamate transporter operon designated glnQHMP (Smu.1519 to Smu.1522) and investigates its potential role in acid tolerance. Deletion of glnQHMP resulted in a 95% reduction in transport of radiolabeled glutamate compared to the wild-type UA159 strain. The addition of glutamate to metabolizing UA159 cells resulted in an increased production of acidic end products, whereas the glnQHMP mutant produced less lactic acid than UA159, suggesting a link between glutamate metabolism and acid production and possible acid tolerance. To investigate this possibility, we conducted a microarray analysis with glutamate and under pH 5.5 and pH 7.5 conditions which showed that expression of the glnQHMP operon was downregulated by both glutamate and mild acid. We also measured the growth kinetics of UA159 and its glnQHMP-negative derivative at pH 5.5 and found that the mutant doubled at a much slower rate than the parent strain but survived at pH 3.5 significantly better than the wild type. Taken together, these findings support the involvement of the glutamate transporter operon glnQHMP in the acid tolerance response in S. mutans.