Justin Kaspar

Growth Phase and pH Influence Peptide Signaling for Competence Development in Streptococcus mutans

The development of competence by the dental caries pathogen Streptococcus mutans is mediated primarily through the alternative sigma factor ComX (SigX), which is under the control of multiple regulatory systems and activates the expression of genes involved in DNA uptake and recombination. Here we report that the induction of competence and competence gene expression by XIP (sigX-inducing peptide) and CSP (competence-stimulating peptide) is dependent on the growth phase and that environmental pH has a potent effect on the responses to XIP. A dramatic decline in comX and comS expression was observed in mid- and late-exponential-phase cells. XIP-mediated competence development and responses to XIP were optimal around a neutral pH, although mid-exponential-phase cells remained refractory to XIP treatment, and acidified late-exponential-phase cultures were resistant to killing by high concentrations of XIP. Changes in the expression of the genes for the oligopeptide permease (opp), which appears to be responsible for the internalization of XIP, could not entirely account for the behaviors observed. Interestingly, comS and comX expression was highly induced in response to endogenously overproduced XIP or ComS in mid-exponential-phase cells. In contrast to the effects of pH on XIP, competence induction and responses to CSP in complex medium were not affected by pH, although a decreased response to CSP in cells that had exited early-exponential phase was observed. Collectively, these results indicate that competence development may be highly sensitive to microenvironments within oral biofilms and that XIP and CSP signaling in biofilms could be spatially and temporally heterogeneous.

A unique open reading frame within the comX gene of Streptococcus mutans regulates genetic competence and oxidative stress tolerance

Streptococcus mutans displays complex regulation of genetic competence, with ComX controlling late competence gene transcription. The rcrRPQ operon has been shown to link oxidative stress tolerance, (p)ppGpp metabolism and competence in S. mutans. Importantly, an rcrR polar (ΔrcrR‐P) mutant is hyper‐transformable, but an rcrR non‐polar (ΔrcrR‐NP) mutant cannot be transformed. Transcriptome comparisons of the rcrR mutants using RNA‐Seq and quantitative real‐time polymerase chain reaction revealed little expression in the 5′ region of comX in ΔrcrR‐NP, but high level expression in the 3′ region. Northern blotting with comX probes revealed two distinct transcripts in the ΔrcrR‐P and ΔrcrR‐NP strains, and 5′ Rapid Amplification of cDNA Ends mapped the 5′ terminus of the shorter transcript to nt +140 of the comX structural gene, where a unique 69‐aa open reading frame, termed XrpA, was encoded in a different reading frame than ComX. Two single‐nucleotide substitution mutants (comX::T162C; comX::T210A) were introduced to disrupt XrpA without affecting the sequence of ComX. When the mutations were in the ΔrcrR‐NP genetic background, ComX production and transformation were restored. Overexpression of xrpA led to impaired growth in aerobic conditions and decreased transformability. These results reveal an unprecedented mechanism for competence regulation and stress tolerance by a gene product encoded within the comX gene that appears unique to S. mutans.

An Essential Role for (p)ppGpp in the Integration of Stress Tolerance, Peptide Signaling, and Competence Development in Streptococcus mutans

The microbes that inhabit the human oral cavity are subjected to constant fluctuations in their environment. To overcome these challenges and gain a competitive advantage, oral streptococci employ numerous adaptive strategies, many of which appear to be intertwined with the development of genetic competence. Here, we demonstrate that the regulatory circuits that control development of competence in Streptococcus mutans, a primary etiological agent of human dental caries, are integrated with key stress tolerance pathways by the molecular alarmone (p)ppGpp. We first observed that the growth of a strain that does not produce (p)ppGpp (ΔrelAPQ, (p)ppGpp0) is not sensitive to growth inhibition by comX inducing peptide (XIP), unlike the wild-type strain UA159, even though XIP-dependent activation of the alternative sigma factor comX by the ComRS pathway is not impaired in the (p)ppGpp0 strain. Overexpression of a (p)ppGpp synthase gene (relP) in the (p)ppGpp0 mutant restored growth inhibition by XIP. We also demonstrate that exposure to micromolar concentrations of XIP elicited changes in (p)ppGpp accumulation in UA159. Loss of the RelA/SpoT homolog (RSH) enzyme, RelA, lead to higher basal levels of (p)ppGpp accumulation, but to decreased sensitivity to XIP and to decreases in comR promoter activity and ComX protein levels. By introducing single amino acid substitutions into the RelA enzyme, the hydrolase activity of the enzyme was shown to be crucial for full com gene induction and transformation by XIP. Finally, loss of relA resulted in phenotypic changes to ΔrcrR mutants, highlighted by restoration of transformation and ComX protein production in the otherwise non-transformable ΔrcrR-NP mutant. Thus, RelA activity and its influence on (p)ppGpp pools appears to modulate competence signaling and development through RcrRPQ and the peptide effectors encoded within rcrQ. Collectively, this study provides new insights into the molecular mechanisms that integrate intercellular communication with the physiological status of the cells and the regulation of key virulence-related phenotypes in S. mutans.

Competence Inhibition by the XrpA Peptide Encoded Within the comX Gene of Streptococcus mutans

Streptococcus mutans displays complex regulation of natural genetic competence. Competence development in S. mutans is controlled by a peptide derived from ComS (XIP); which along with the cytosolic regulator ComR controls the expression of the alternative sigma factor comX, the master regulator of competence development. Recently, a gene embedded within the coding region of comX was discovered and designated xrpA (comX regulatory peptide A). XrpA was found to be an antagonist of ComX, but the mechanism was not established. In this study, we reveal through both genomic and proteomic techniques that XrpA is the first describe negative regulator of ComRS systems in streptococci. Transcriptomic and promoter activity assays in the ΔxrpA strain revealed an up-regulation of genes controlled by both the ComR- and ComX-regulons. An in vivo protein crosslinking and in vitro fluorescent polarization assays confirmed that the N-terminal region of XrpA were found to be sufficient in inhibiting ComR-XIP complex binding to ECom-box located within the comX promoter. This inhibitory activity was sufficient for decreases in PcomX activity, transformability and ComX accumulation. XrpA serving as a modulator of ComRS activity ultimately results in changes to subpopulation behaviors and cell fate during competence activation. ABBREVIATED SUMMARY Streptococcus mutans displays complex regulation of natural genetic competence, highlighted by a novel gene, xrpA, embedded within the coding region for the master regulator ComX. We show that XrpA modulates ComRS-dependent activation of comX expression, resulting in changes to sub-population behaviors, including cell lysis. XrpA is the first described inhibitor of a ComRS system and, because it is unique to S. mutans it may be targetable to prevent diseases caused by this pathogen.

Threshold regulation and stochasticity from the MecA/ClpCP proteolytic system in Streptococcus mutans competence

Many bacterial species use the MecA/ClpCP proteolytic system to block entry into genetic competence. In Streptococcus mutans, MecA/ClpCP degrades ComX (also called SigX), an alternative sigma factor for the comY operon and other late competence genes. Although the mechanism of MecA/ClpCP has been studied in multiple Streptococcus species, its role within noisy competence pathways is poorly understood. S. mutans competence can be triggered by two different peptides, CSP and XIP, but it is not known whether MecA/ClpCP acts similarly for both stimuli, how it affects competence heterogeneity, and how its regulation is overcome. We have studied the effect of MecA/ClpCP on the activation of comY in individual S. mutans cells. Our data show that MecA/ClpCP is active under both XIP and CSP stimulation, that it provides threshold control of comY, and that it adds noise in comY expression. Our data agree quantitatively with a model in which MecA/ClpCP prevents adventitious entry into competence by sequestering or intercepting low levels of ComX. Competence is permitted when ComX levels exceed a threshold, but cell-to-cell heterogeneity in MecA levels creates variability in that threshold. Therefore MecA/ClpCP provides a stochastic switch, located downstream of the already noisy comX, that enhances phenotypic diversity.

Intracellular signaling through the comRS system in Streptococcus mutans genetic competence

Entry into genetic competence in streptococci is controlled by ComX, an alternative sigma factor for genes that enable the import of exogenous DNA. In Streptococcus mutans, the immediate activator of comX is the ComRS signaling system, which consists of the cytosolic receptor ComR and the 7-residue signal peptide XIP, which is derived from ComS. Extracellular XIP imported by an oligopeptide permease interacts with ComR to form a transcriptional activator for both comX and comS. Therefore, extracellular XIP can function as an exogenous signal to trigger S. mutans competence. However, the mechanisms that process ComS and export it as XIP are not fully known in S. mutans. The observation that comX is expressed bimodally under some environmental conditions suggests that ComR may also interact with endogenously produced XIP or ComS, creating an intracellular positive feedback loop in comS transcription. Here we use single cell and microfluidic methods to compare the effects of the native comS gene and extracellular XIP on comX expression. We find that deletion of comS reduces the response of comX to extracellular XIP. We also find that comS-overexpressing cells autoactivate their comX even when their growth medium is rapidly exchanged, although this autoactivation requires an intact copy of comS under control of its own promoter. However comS-overexpressing cells do not activate comS-deficient mutants growing in coculture. These data show that individual cells can activate comX without exporting or importing the XIP or ComS signal, and that endogenously and exogenously produced ComS/XIP have inequivalent effects on comX behavior. These data are fully consistent with a model in which intracellular positive feedback in comS transcription plays a role in ComRS signaling, and is responsible for the bimodal expression of comX. Author Summary Heterogeneous gene expression in genetically identical populations plays an important role in bacterial persistence and survival under changing environmental conditions. In the oral pathogen Streptococcus mutans, the physiological state of genetic competence can exhibit bimodality, with only some cells becoming competent. S. mutans controls its entry into competence by using the ComRS signaling system to activate comX, a gene encoding the master competence regulator ComX. The ComRS system is understood as a quorum sensing system, in which the extracellular accumulation of the small signal peptide XIP, derived from ComS, induces comX expression. We coupled observation of bacteria that fluoresce when comX is active with mathematical analysis and chemical binding assays to show that activation of comX does not necessarily require extracellular XIP or ComS, and that comX-active cells do not necessarily export XIP. Our experiments and mathematical modeling indicate that a positive feedback loop in comS transcription allows a cell to activate comX in response to its own XIP or ComS in the absence of extracellular XIP, or to amplify its comX response to extracellular XIP if present. Such positive feedback loops are often the cause of bimodal gene expression like that seen in S. mutans competence.

Competence inhibition by the XrpA peptide encoded within the comX gene of Streptococcus mutans : Antagonism of the ComRS-XIP circuit

Streptococcus mutans displays complex regulation of natural genetic competence. Competence development in S. mutans is controlled by a peptide derived from ComS (XIP); which along with the cytosolic regulator ComR controls the expression of the alternative sigma factor comX, the master regulator of competence development. Recently, a gene embedded within the coding region of comX was discovered and designated xrpA (comX regulatory peptide A). XrpA was found to be an antagonist of ComX, but the mechanism was not established. In this study, we reveal through both genomic and proteomic techniques that XrpA is the first described negative regulator of ComRS systems in streptococci. Transcriptomic and promoter activity assays in the ΔxrpA strain revealed an up‐regulation of genes controlled by both the ComR‐ and ComX‐regulons. An in vivo protein crosslinking and in vitro fluorescent polarization assays confirmed that the N‐terminal region of XrpA were found to be sufficient in inhibiting ComR‐XIP complex binding to ECom‐box located within the comX promoter. This inhibitory activity was sufficient for decreases in PcomX activity, transformability and ComX accumulation. XrpA serving as a modulator of ComRS activity ultimately results in changes to subpopulation behaviors and cell fate during competence activation.

Threshold regulation and stochasticity from the MecA/ClpCP proteolytic system in Streptococcus mutans competence: Thresholding and stochasticity from MecA/ClpCP

Many bacterial species use the MecA/ClpCP proteolytic system to block entry into genetic competence. In Streptococcus mutans, MecA/ClpCP degrades ComX (also called SigX), an alternative sigma factor for the comY operon and other late competence genes. Although the mechanism of MecA/ClpCP has been studied in multiple Streptococcus species, its role within noisy competence pathways is poorly understood. S. mutans competence can be triggered by two different peptides, CSP and XIP, but it is not known whether MecA/ClpCP acts similarly for both stimuli, how it affects competence heterogeneity, and how its regulation is overcome. We have studied the effect of MecA/ClpCP on the activation of comY in individual S. mutans cells. Our data show that MecA/ClpCP is active under both XIP and CSP stimulation, that it provides threshold control of comY, and that it adds noise in comY expression. Our data agree quantitatively with a model in which MecA/ClpCP prevents adventitious entry into competence by sequestering or intercepting low levels of ComX. Competence is permitted when ComX levels exceed a threshold, but cell‐to‐cell heterogeneity in MecA levels creates variability in that threshold. Therefore, MecA/ClpCP provides a stochastic switch, located downstream of the already noisy comX, that enhances phenotypic diversity.