Sharply Tuned pH Response of Genetic Competence Regulation in Streptococcus mutans: a Microfluidic Study of the Environmental Sensitivity of comX

Abstract

Genetic competence in Streptococcus mutans is a transient state that is regulated in response to multiple environmental inputs. These include extracellular pH and the concentrations of two secreted peptides, designated CSP (competence-stimulating peptide) and XIP (comX-inducing peptide). The role of environmental cues in regulating competence can be difficult to disentangle from the effects of the organism's physiological state and its chemical modification of its environment. We used microfluidics to control the extracellular environment and study the activation of the key competence gene comX. We find that the comX promoter (P comX ) responds to XIP or CSP only when the extracellular pH lies within a narrow window, about 1 pH unit wide, near pH 7. Within this pH range, CSP elicits a strong P comX response from a subpopulation of cells, whereas outside this range the proportion of cells expressing comX declines sharply. Likewise, P comX is most sensitive to XIP only within a narrow pH window. While previous work suggested that comX may become refractory to CSP or XIP stimulus as cells exit early exponential phase, our microfluidic data show that extracellular pH dominates in determining sensitivity to XIP and CSP. The data are most consistent with an effect of pH on the ComR/ComS system, which has direct control over transcription of comX in S. mutans. Genetic competence is a transient physiological state during which a bacterial cell is able to internalize DNA from its environment. Competence occurs in many bacterial species but was first described in the streptococci, where its regulation has been the subject of intensive study (1, 2). In the oral pathogen Streptococcus mutans, competence is important not only because it contributes to genetic diversity but also because its regulation is closely intertwined with the manifestation of virulence-related behaviors, including bacteriocin production, biofilm formation, tolerance of low pH, and carbohydrate catabolism (3-7). S. mutans regulates competence in part through two secreted quorum-sensing peptides, designated competence-stimulating peptide (CSP) and comX-inducing peptide (XIP). Interestingly, the activity of these peptides depends on environmental parameters, including pH, carbohydrate, and media (8-11). Through mechanisms that are not well understood, the competence regulon integrates the peptide signals with environmental and internal parameters (12, 13) to trigger a transient state of competence during early exponential growth phase.The interaction of the extracellular environment with competence and related virulence behaviors is important in the context of oral biofilms. Heterogeneous local environments of pH and oxygen/redox, carbohydrate, and secreted-peptide concentrations in a biofilm could potentially lead to spatial variations in virulence gene expression in S. mutans (14-17). pH is particularly important because the fermentation of carbohydrates by S. mutans generates acids that can rapidly modify the pH of the environment. The pH in a biofilm ...

Natural Transformation of Oral Streptococci by Use of Synthetic Pheromones

“…Spontaneous competence development under laboratory conditions is in practice not always reliable, and subtle changes in factors such as medium batch, medium composition, pH, and growth temperatures, may dramatically affect competence levels [12,13]. The introduction of synthetic pheromones in protocols for natural transformation has overcome many of these 4 difficulties, by bypassing stringent conditions for production and secretion of natural pheromones, and by prolonging the transient nature of the competent state in streptococci [5].…”

Markerless Genome Editing in Competent Streptococci

“…In S. mutans, the recently described competence pheromone known as the sigXinducing peptide (XIP) [1,2] has the potential of stimulating development of competence in all cells in a population, in contrast to the bimodal response to another S. mutans competence pheromone, CSP [3,4,5,6]. In addition, the XIP-induced competent state is unusually persistent in S. mutans , lasting for hours and accompanied by a reduced apparent growth rate [7].…”

A Highly Arginolytic Streptococcus Species That Potently Antagonizes Streptococcus mutans

“…Instead, the Rgg-like transcriptional regulator ComR is the proximal regulator of comX expression, and ComR activity is enhanced by binding to the 7-aa comX-inducing peptide (XIP). The ComRS regulatory pathway includes processing of the 17-aa ComS to XIP, which appears extracellularly, followed by active transport of XIP into the cell by the Opp oligopeptide permease and formation of a ComR-XIP complex that binds near the comX promoter to activate transcription (35,38,39). Higher levels of XIP can also trigger the activation of the bacteriocin genes of S. mutans, at least in part because ComX can enhance the transcription of the comE gene (40,41).…”