Wavelike propagation of quorum activation through a spatially distributed bacterial population under natural regulation.

Many bacteria communicate using diffusible pheromone signals known as autoinducers. When the autoinducer concentration reaches a threshold, which requires a minimum population density or 'quorum', the bacteria activate specific gene regulatory pathways. Simple diffusion of autoinducer can activate quorum-dependent pathways in cells that are located at substantial distances from the secreting source. However, modeling has predicted that autoinducer diffusion, coupled with positive feedback regulation in autoinducer synthesis, could also allow a quorum-regulated behavior to spread more rapidly through a population by moving as a self-sustaining front at constant speed. Here we show that such propagation can occur in a population of bacteria whose quorum pathway operates under its own natural regulation. We find that in unstirred populations ofVibrio fischeri, introduction of autoinducer at one location triggers a wavelike traveling front of natural bioluminescence. The front moves with a well-defined speed ∼2.5 mm h-1, eventually outrunning the slower diffusional spreading of the initial stimulus. Consistent with predictions from modeling, the wave travels until late in growth, when population-wide activation occurs due to basal autoinducer production. Subsequent rounds of waves, including waves propagating in the reverse direction, can also be observed late in the growth ofV.fischeriunder natural regulation. Using an engineered,lac-dependent strain, we show that local stimuli other than autoinducers can also elicit a self-sustaining, propagating response. Our data show that the wavelike dynamics predicted by simple mathematical models of quorum signaling are readily detected in bacterial populations functioning under their own natural regulation, and that other, more complex traveling phenomena are also present. Because a traveling wave can substantially increase the efficiency of intercellular communication over macroscopic distances, our data indicate that very efficient modes of communication over distance are available to unmixed populations ofV.fischeriand other microbes.

Carbohydrate and PepO control bimodality in competence development by Streptococcus mutans.

In Streptococcus mutans, the alternative sigma factor ComX controls entry into genetic competence. Competence stimulating peptide (CSP) induces bimodal expression of comX, with only a fraction of the population becoming transformable. Curiously, the bimodality of comX is affected by peptides in the growth medium and by carbohydrate source. CSP elicits bimodal expression of comX in media rich in small peptides, but CSP elicits no response in defined media lacking small peptides. In addition, growth on certain sugars increases the proportion of the population that activates comX in response to CSP. By investigating the connection between media and comX bimodality, we find evidence for two mechanisms that modulate transcriptional positive feedback in the ComRS system, where comX bimodality originates. We find that the endopeptidase PepO suppresses the ComRS feedback loop, most likely by degrading the XIP/ComS feedback signal. Deletion of pepO eliminates comX bimodality, leading to a unimodal comX response to CSP in both defined and complex media. We also find that CSP stimulates the ComRS feedback system by upregulating comR in a carbohydrate source-dependent fashion. Our data provide mechanistic insight into how S. mutans regulates bimodality and explain the puzzle of growth medium effects on competence induction by CSP.

Characterization of LrgAB as a stationary phase-specific pyruvate uptake system in Streptococcus mutans.

BACKGROUND: Our recent '-omics' comparisons of Streptococcus mutans wild-type and lrgAB-mutant revealed that this organism undergoes dynamic cellular changes in the face of multiple exogenous stresses, consequently affecting its comprehensive virulence traits. In this current study, we further demonstrate that LrgAB functions as a S. mutans pyruvate uptake system. RESULTS: S. mutans excretes pyruvate during growth as an overflow metabolite, and appears to uptake this excreted pyruvate via LrgAB once the primary carbon source is exhausted. This utilization of excreted pyruvate was tightly regulated by glucose levels and stationary growth phase lrgAB induction. The degree of lrgAB induction was reduced by high extracellular levels of pyruvate, suggesting that lrgAB induction is subject to negative feedback regulation, likely through the LytST TCS, which is required for expression of lrgAB. Stationary phase lrgAB induction was efficiently inhibited by low concentrations of 3FP, a toxic pyruvate analogue, without affecting cell growth, suggesting that accumulated pyruvate is sensed either directly or indirectly by LytS, subsequently triggering lrgAB expression. S. mutans growth was inhibited by high concentrations of 3FP, implying that pyruvate uptake is necessary for S. mutans exponential phase growth and occurs in a Lrg-independent manner. Finally, we found that stationary phase lrgAB induction is modulated by hydrogen peroxide (H2O2) and by co-cultivation with H2O2-producing S. gordonii. CONCLUSIONS: Pyruvate may provide S. mutans with an alternative carbon source under limited growth conditions, as well as serving as a buffer against exogenous oxidative stress. Given the hypothesized role of LrgAB in cell death and lysis, these data also provide an important basis for how these processes are functionally and mechanically connected to key metabolic pathways such as pyruvate metabolism.

Genome-Wide Screens Reveal New Gene Products That Influence Genetic Competence in Streptococcus mutans.

A network of genes and at least two peptide signaling molecules tightly control when Streptococcus mutans becomes competent to take up DNA from its environment. Widespread changes in the expression of genes occur when S. mutans is presented with competence signal peptides in vitro, including the increased production of the alternative sigma factor, ComX, which activates late competence genes. Still, the way that gene products that are regulated by competence peptides influence DNA uptake and cellular physiology are not well understood. Here, we developed and employed comprehensive transposon mutagenesis of the S. mutans genome, with a screen to identify mutants that aberrantly expressed comX, coupled with transposon sequencing (Tn-seq) to gain a more thorough understanding of the factors modulating comX expression and progression to the competent state. The screens effectively identified genes known to affect competence, e.g., comR, comS, comD, comE, cipB, clpX, rcrR, and ciaH, but disclosed an additional 20 genes that were not previously competence associated. The competence phenotypes of mutants were characterized, including by fluorescence microscopy to determine at which stage the mutants were impaired for comX activation. Among the novel genes studied were those implicated in cell division, the sensing of cell envelope stress, cell envelope biogenesis, and RNA stability. Our results provide a platform for determining the specific chemical and physical cues that are required for genetic competence in S. mutans, while highlighting the effectiveness of using Tn-seq in S. mutans to discover and study novel biological processes.IMPORTANCEStreptococcus mutans acquires DNA from its environment by becoming genetically competent, a physiologic state triggered by cell-cell communication using secreted peptides. Competence is important for acquiring novel genetic traits and has a strong influence on the expression of virulence-associated traits of S. mutans Here, we used transposon mutagenesis and genomic technologies to identify novel genes involved in competence development. In addition to identifying genes previously known to be required for comX expression, 20 additional genes were identified and characterized. The findings create opportunities to diminish the pathogenic potential of S. mutans, while validating technologies that can rapidly advance our understanding of the physiology, biology, and genetics of S. mutans and related pathogens.

Intercellular Communication via the comX-Inducing Peptide (XIP) of Streptococcus mutans.

Gram-positive bacteria utilize exported peptides to coordinate genetic and physiological processes required for biofilm formation, stress responses, and ecological competitiveness. One example is activation of natural genetic competence by ComR and the com X -inducing peptide (XIP) in Streptococcus mutans Although the competence pathway can be activated by the addition of synthetic XIP in defined medium, the hypothesis that XIP is able to function as an intercellular signaling molecule has not been rigorously tested. Coculture model systems were developed that included a "sender" strain that overexpressed the XIP precursor (ComS) and a "responder" strain harboring a green fluorescent protein (GFP) reporter fused to a ComR-activated gene (comX) promoter. The ability of the sender strain to provide a signal to activate GFP expression was monitored at the individual cell and population levels using (i) planktonic culture systems, (ii) cells suspended in an agarose matrix, or (iii) cells growing in biofilms. XIP was shown to be freely diffusible, and XIP signaling between the S. mutans sender and responder strains did not require cell-to-cell contact. The presence of a sucrose-derived exopolysaccharide matrix diminished the efficiency of XIP signaling in biofilms, possibly by affecting the spatial distribution of XIP senders and potential responders. Intercellular signaling was greatly impaired in a strain lacking the primary autolysin, AtlA, and was substantially greater when the sender strain underwent lysis. Collectively, these data provide evidence that S. mutans XIP can indeed function as a peptide signal between cells and highlight the importance of studying signaling with an endogenously produced peptide(s) in populations in various environments and physiologic states.IMPORTANCE The comX-inducing peptide (XIP) of Streptococcus mutans is a key regulatory element in the activation of genetic competence, which allows cells to take up extracellular DNA. XIP has been found in cell culture fluids, and the addition of synthetic XIP to physiologically receptive cells can robustly induce competence gene expression. However, there is a lack of consensus as to whether XIP can function as an intercellular communication signal. Here, we show that XIP indeed signals between cells in S. mutans, but that cell lysis may be a critical factor, as opposed to a dedicated secretion/processing system, in allowing for release of XIP into the environment. The results have important implications in the context of the ecology, virulence, and evolution of a ubiquitous human pathogen and related organisms.

Oxidative Stressors Modify the Response of Streptococcus mutans to Its Competence Signal Peptides.

The dental caries pathogen Streptococcus mutans is continually exposed to several types of stress in the oral biofilm environment. Oxidative stress generated by reactive oxygen species has a major impact on the establishment, persistence, and virulence of S. mutans Here, we combined fluorescent reporter-promoter fusions with single-cell imaging to study the effects of reactive oxygen species on activation of genetic competence in S. mutans Exposure to paraquat, which generates superoxide anion, produced a qualitatively different effect on activation of expression of the gene for the master competence regulator, ComX, than did treatment with hydrogen peroxide (H2O2), which can yield hydroxyl radical. Paraquat suppressed peptide-mediated induction of comX in a progressive and cumulative fashion, whereas the response to H2O2 displayed a strong threshold behavior. Low concentrations of H2O2 had little effect on induction of comX or the bacteriocin gene cipB, but expression of these genes declined sharply if extracellular H2O2 exceeded a threshold concentration. These effects were not due to decreased reporter gene fluorescence. Two different threshold concentrations were observed in the response to H2O2, depending on the gene promoter that was analyzed and the pathway by which the competence regulon was stimulated. The results show that paraquat and H2O2 affect the S. mutans competence signaling pathway differently, and that some portions of the competence signaling pathway are more sensitive to oxidative stress than others.IMPORTANCEStreptococcus mutans inhabits the oral biofilm, where it plays an important role in the development of dental caries. Environmental stresses such as oxidative stress influence the growth of S. mutans and its important virulence-associated behaviors, such as genetic competence. S. mutans competence development is a complex behavior that involves two different signaling peptides and can exhibit cell-to-cell heterogeneity. Although oxidative stress is known to influence S. mutans competence, it is not understood how oxidative stress interacts with the peptide signaling or affects heterogeneity. In this study, we used fluorescent reporters to probe the effect of reactive oxygen species on competence signaling at the single-cell level. Our data show that different reactive oxygen species have different effects on S. mutans competence, and that some portions of the signaling pathway are more acutely sensitive to oxidative stress than others.

Origins of heterogeneity in Streptococcus mutans competence: interpreting an environment-sensitive signaling pathway.

Bacterial pathogens rely on chemical signaling and environmental cues to regulate disease-causing behavior in complex microenvironments. The human pathogen Streptococcus mutans employs a particularly complex signaling and sensing scheme to regulate genetic competence and other virulence behaviors in the oral biofilms it inhabits. Individual S. mutans cells make the decision to enter the competent state by integrating chemical and physical cues received from their microenvironment along with endogenously produced peptide signals. Studies at the single-cell level, using microfluidics to control the extracellular environment, provide physical insight into how the cells process these inputs to generate complex and often heterogeneous outputs. Fine changes in environmental stimuli can dramatically alter the behavior of the competence circuit. Small shifts in pH can switch the quorum sensing response on or off, while peptide-rich media appear to switch the output from a unimodal to a bimodal behavior. Therefore, depending on environmental cues, the quorum sensing circuitry can either synchronize virulence across the population, or initiate and amplify heterogeneity in that behavior. Much of this complex behavior can be understood within the framework of a quorum sensing system that can operate both as an intercellular signaling mechanism and intracellularly as a noisy bimodal switch.

Effects of Carbohydrate Source on Genetic Competence in Streptococcus mutans.

UNLABELLED: The capacity to internalize and catabolize carbohydrates is essential for dental caries pathogens to persist and cause disease. The expression of many virulence-related attributes by Streptococcus mutans, an organism strongly associated with human dental caries, is influenced by the peptide signaling pathways that control genetic competence. Here, we demonstrate a relationship between the efficiency of competence signaling and carbohydrate source. A significant increase in the activity of the promoters for comX, comS, and comYA after exposure to competence-stimulating peptide (CSP) was observed in cells growing on fructose, maltose, sucrose, or trehalose as the primary carbohydrate source, compared to cells growing on glucose. However, only cells grown in the presence of trehalose or sucrose displayed a significant increase in transformation frequency. Notably, even low concentrations of these carbohydrates in the presence of excess glucose could enhance the expression of comX, encoding a sigma factor needed for competence, and the effects on competence were dependent on the cognate sugar:phosphotransferase permease for each carbohydrate. Using green fluorescent protein (GFP) reporter fusions, we observed that growth in fructose or trehalose resulted in a greater proportion of the population activating expression of comX and comS, encoding the precursor of comX-inducing peptide (XIP), after addition of CSP, than growth in glucose. Thus, the source of carbohydrate significantly impacts the stochastic behaviors that regulate subpopulation responses to CSP, which can induce competence in S. mutans IMPORTANCE: The signaling pathways that regulate development of genetic competence in Streptococcus mutans are intimately intertwined with the pathogenic potential of the organism, impacting biofilm formation, stress tolerance, and expression of known virulence determinants. Induction of the gene for the master regulator of competence, ComX, by competence-stimulating peptide (CSP) occurs in a subpopulation of cells. Here, we show that certain carbohydrates that are common in the human diet enhance the ability of CSP to activate transcription of comX and that a subset of these carbohydrates stimulates progression to the competent state. The cognate sugar:phosphotransferase permeases for each sugar are needed for these effects. Interestingly, single-cell analysis shows that the carbohydrates that increase com gene expression do so by enhancing the proportion of cells that respond to CSP. A mathematical model is developed to explain how carbohydrates modulate bistable behavior in the system via the ComRS pathway and ComX stability.

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

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 (PcomX) 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 PcomX response from a subpopulation of cells, whereas outside this range the proportion of cells expressing comX declines sharply. Likewise, PcomX 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.

Folding dynamics and pathways of the trp-cage miniproteins.

Using alternate measures of fold stability for a wide variety of Trp-cage mutants has raised the possibility that prior dynamics T-jump measures may not be reporting on complete cage formation for some species. NMR relaxation studies using probes that only achieve large chemical shift difference from unfolded values on complete cage formation indicate slower folding in some but not all cases. Fourteen species have been examined, with cage formation time constants (1/kF) ranging from 0.9-7.5 µs at 300 K. The present study does not change the status of the Trp-cage as a fast folding, essentially two-state system, although it does alter the stage at which this description applies. A diversity of prestructuring events, depending on the specific analogue examined, may appear in the folding scenario, but in all cases, formation of the N-terminal helix is complete either at or before the cage-formation transition state. In contrast, the fold-stabilizing H-bonding interactions of the buried Ser14 side chain and the Arg/Asp salt bridge are post-transition state features on the folding pathway. The study has also found instances in which a [P12W] mutation is fold destabilizing but still serves to accelerate the folding process.

Entropy-driven motility of Sinorhizobium meliloti on a semi-solid surface.

Sinorhizobium meliloti growing on soft agar can exhibit an unusual surface spreading behaviour that differs from other bacterial surface motilities. Bacteria in the colony secrete an exopolysaccharide-rich mucoid fluid that expands outward on the surface, carrying within it a suspension of actively dividing cells. The moving slime disperses the cells in complex and dynamic patterns indicative of simultaneous bacterial growth, swimming and aggregation. We find that while flagellar swimming is required to maintain the cells in suspension, the spreading and the associated pattern formation are primarily driven by the secreted exopolysaccharide EPS II, which creates two entropy-increasing effects: an osmotic flow of water from the agar to the mucoid fluid and a crowding or depletion attraction between the cells. Activation of these physical/chemical phenomena may be a useful function for the high molecular weight EPS II, a galactoglucan whose biosynthesis is tightly regulated by the ExpR/SinI/SinR quorum-sensing system: unlike bacterial colonies that spread via bacterium-generated, physical propulsive forces, S. meliloti under quorum conditions may use EPS II to activate purely entropic forces within its environment, so that it can disperse by passively 'surfing' on those forces.

Traveling waves in response to a diffusing quorum sensing signal in spatially-extended bacterial colonies.

In the behavior known as quorum sensing (QS), bacteria release diffusible signal molecules known as autoinducers, which by accumulating in the environment induce population-wide changes in gene expression. Although QS has been extensively studied in well-mixed systems, the ability of diffusing QS signals to synchronize gene expression in spatially extended colonies is not well understood. Here we investigate the one-dimensional spatial propagation of QS-circuit activation in a simple, analytically tractable reaction-diffusion model for the LuxR-LuxI circuit, which regulates bioluminescence of the marine bacterium Aliivibrio fischeri. The quorum activation loop is modeled by a Hill function with a cooperativity exponent (m=2.2). The model is parameterized from laboratory data and captures the major empirical properties of the LuxR-LuxI system and its QS regulation of A. fischeri bioluminescence. Our simulations of the model show propagating waves of activation or deactivation of the QS circuit in a spatially extended colony. We further prove analytically that the model equations possess a traveling wave solution. This mathematical proof yields the rate of autoinducer degradation that is compatible with a traveling wave of gene expression as well as the critical degradation rate at which the nature of the wave switches from activation to deactivation. Our results can be used to predict the direction and activating or deactivating nature of a wave of gene expression in experimentally controlled bacterial populations subject to a diffusing autoinducer signal.

Microfluidic study of competence regulation in Streptococcus mutans: environmental inputs modulate bimodal and unimodal expression of comX.

Streptococcus mutans regulates genetic competence through a complex network that receives inputs from a number of environmental stimuli, including two signalling peptides designated as CSP and XIP. The response of the downstream competence genes to these inputs shows evidence of stochasticity and bistability and has been difficult to interpret. We have used microfluidic, single-cell methods to study how combinations of extracellular signals shape the response of comX, an alternative sigma factor governing expression of the late competence genes. We find that the composition of the medium determines which extracellular signal (XIP or CSP) can elicit a response from comX and whether that response is unimodal or bimodal across a population of cells. In a chemically defined medium, exogenous CSP does not induce comX, whereas exogenous XIP elicits a comX response from all cells. In complex medium, exogenous XIP does not induce comX, whereas CSP elicits a bimodal comX response from the population. Interestingly, bimodal behaviour required an intact copy of comS, which encodes the precursor of XIP. The comS-dependent capability for both unimodal and bimodal response suggests that a constituent - most likely peptides - of complex medium interacts with a positive feedback loop in the competence regulatory network.

Crosstalk enables mutual activation of coupled quorum sensing pathways through "jump-start" and "push-start" mechanisms.

Many quorum sensing microbes produce more than one chemical signal and detect them using interconnected pathways that crosstalk with each other. While there are many hypotheses for the advantages of sensing multiple signals, the prevalence and functional significance of crosstalk between pathways are much less understood. We explore the effect of intracellular signal crosstalk using a simple model that captures key features of typical quorum sensing pathways: multiple pathways in a hierarchical configuration, operating with positive feedback, with crosstalk at the receptor and promoter levels. We find that crosstalk enables activation or inhibition of one output by the non-cognate signal, broadens the dynamic range of the outputs, and allows one pathway to modulate the feedback circuit of the other. Our findings show how crosstalk between quorum sensing pathways can be viewed not as a detriment to the processing of information, but as a mechanism that enhances the functional range of the full regulatory system. When positive feedback systems are coupled through crosstalk, several new modes of activation or deactivation become possible.

Quorum activation at a distance: spatiotemporal patterns of gene regulation from diffusion of an autoinducer signal.

Quorum sensing (QS) bacteria regulate gene expression collectively by exchanging diffusible signal molecules known as autoinducers. Although QS is often studied in well-stirred laboratory cultures, QS bacteria colonize many physically and chemically heterogeneous environments where signal molecules are transported primarily by diffusion. This raises questions of the effective distance range of QS and the degree to which colony behavior can be synchronized over such distances. We have combined experiments and modeling to investigate the spatiotemporal patterns of gene expression that develop in response to a diffusing autoinducer signal. We embedded a QS strain in a narrow agar lane and introduced exogenous autoinducer at one terminus of the lane. We then measured the expression of a QS reporter as a function of space and time as the autoinducer diffused along the lane. The diffusing signal readily activates the reporter over distances of ~1 cm on time scales of ~10 h. However, the patterns of activation are qualitatively unlike the familiar spreading patterns of simple diffusion, as the kinetics of response are surprisingly insensitive to the distance the signal has traveled. We were able to reproduce these patterns with a mathematical model that combines simple diffusion of the signal with logistic growth of the bacteria and cooperative activation of the reporter. In a wild-type QS strain, we also observed the propagation of a unique spatiotemporal excitation. Our results show that a chemical signal transported only by diffusion can be remarkably effective in synchronizing gene expression over macroscopic distances.

Analysis of gene expression levels in individual bacterial cells without image segmentation.

Studies of stochasticity in gene expression typically make use of fluorescent protein reporters, which permit the measurement of expression levels within individual cells by fluorescence microscopy. Analysis of such microscopy images is almost invariably based on a segmentation algorithm, where the image of a cell or cluster is analyzed mathematically to delineate individual cell boundaries. However segmentation can be ineffective for studying bacterial cells or clusters, especially at lower magnification, where outlines of individual cells are poorly resolved. Here we demonstrate an alternative method for analyzing such images without segmentation. The method employs a comparison between the pixel brightness in phase contrast vs fluorescence microscopy images. By fitting the correlation between phase contrast and fluorescence intensity to a physical model, we obtain well-defined estimates for the different levels of gene expression that are present in the cell or cluster. The method reveals the boundaries of the individual cells, even if the source images lack the resolution to show these boundaries clearly.

Multi-scaled explorations of binding-induced folding of intrinsically disordered protein inhibitor IA3 to its target enzyme.

Biomolecular function is realized by recognition, and increasing evidence shows that recognition is determined not only by structure but also by flexibility and dynamics. We explored a biomolecular recognition process that involves a major conformational change - protein folding. In particular, we explore the binding-induced folding of IA3, an intrinsically disordered protein that blocks the active site cleft of the yeast aspartic proteinase saccharopepsin (YPrA) by folding its own N-terminal residues into an amphipathic alpha helix. We developed a multi-scaled approach that explores the underlying mechanism by combining structure-based molecular dynamics simulations at the residue level with a stochastic path method at the atomic level. Both the free energy profile and the associated kinetic paths reveal a common scheme whereby IA3 binds to its target enzyme prior to folding itself into a helix. This theoretical result is consistent with recent time-resolved experiments. Furthermore, exploration of the detailed trajectories reveals the important roles of non-native interactions in the initial binding that occurs prior to IA3 folding. In contrast to the common view that non-native interactions contribute only to the roughness of landscapes and impede binding, the non-native interactions here facilitate binding by reducing significantly the entropic search space in the landscape. The information gained from multi-scaled simulations of the folding of this intrinsically disordered protein in the presence of its binding target may prove useful in the design of novel inhibitors of aspartic proteinases.

Amyloid Aggregates Are Localized to the Nonadherent Detached Fraction of Aging Streptococcus mutans Biofilms.

The number of bacterial species recognized to utilize purposeful amyloid aggregation within biofilms continues to grow. The oral pathogen Streptococcus mutans produces several amyloidogenic proteins, including adhesins P1 (also known as AgI/II, PAc) and WapA, whose truncation products, namely, AgII and AgA, respectively, represent the amyloidogenic moieties. Amyloids demonstrate common biophysical properties, including recognition by Thioflavin T (ThT) and Congo red (CR) dyes that bind to the cross ß-sheet quaternary structure of amyloid aggregates. Previously, we observed amyloid formation to occur only after 60 h or more of S. mutans biofilm growth. Here, we extend those findings to investigate where amyloid is detected within 1- and 5-day-old biofilms, including within tightly adherent compared with those in nonadherent fractions. CR birefringence and ThT uptake demonstrated amyloid within nonadherent material removed from 5-day-old cultures but not within 1-day-old or adherent samples. These experiments were done in conjunction with confocal microscopy and immunofluorescence staining with AgII- and AgA-reactive antibodies, including monoclonal reagents shown to discriminate between monomeric protein and amyloid aggregates. These results also localized amyloid primarily to the nonadherent fraction of biofilms. Lastly, we show that the C-terminal region of P1 loses adhesive function following amyloidogenesis and is no longer able to competitively inhibit binding of S. mutans to its physiologic substrate, salivary agglutinin. Taken together, our results provide new evidence that amyloid aggregation negatively impacts the functional activity of a widely studied S. mutans adhesin and are consistent with a model in which amyloidogenesis of adhesive proteins facilitates the detachment of aging biofilms. IMPORTANCE Streptococcus mutans is a keystone pathogen and causative agent of human dental caries, commonly known as tooth decay, the most prevalent infectious disease in the world. Like many pathogens, S. mutans causes disease in biofilms, which for dental decay begins with bacterial attachment to the salivary pellicle coating the tooth surface. Some strains of S. mutans are also associated with bacterial endocarditis. Amyloid aggregation was initially thought to represent only a consequence of protein mal-folding, but now, many microorganisms are known to produce functional amyloids with biofilm environments. In this study, we learned that amyloid formation diminishes the activity of a known S. mutans adhesin and that amyloid is found within the nonadherent fraction of older biofilms. This finding suggests that the transition from adhesin monomer to amyloid facilitates biofilm detachment. Knowing where and when S. mutans produces amyloid will help in developing therapeutic strategies to control tooth decay and other biofilm-related diseases.

Dimension-reduction simplifies the analysis of signal crosstalk in a bacterial quorum sensing pathway.

Many pheromone sensing bacteria produce and detect more than one chemically distinct signal, or autoinducer. The pathways that detect these signals are typically noisy and interlocked through crosstalk and feedback. As a result, the sensing response of individual cells is described by statistical distributions that change under different combinations of signal inputs. Here we examine how signal crosstalk reshapes this response. We measure how combinations of two homoserine lactone (HSL) input signals alter the statistical distributions of individual cell responses in the AinS/R- and LuxI/R-controlled branches of the Vibrio fischeri bioluminescence pathway. We find that, while the distributions of pathway activation in individual cells vary in complex fashion with environmental conditions, these changes have a low-dimensional representation. For both the AinS/R and LuxI/R branches, the distribution of individual cell responses to mixtures of the two HSLs is effectively one-dimensional, so that a single tuning parameter can capture the full range of variability in the distributions. Combinations of crosstalking HSL signals extend the range of responses for each branch of the circuit, so that signals in combination allow population-wide distributions that are not available under a single HSL input. Dimension reduction also simplifies the problem of identifying the HSL conditions to which the pathways and their outputs are most sensitive. A comparison of the maximum sensitivity HSL conditions to actual HSL levels measured during culture growth indicates that the AinS/R and LuxI/R branches lack sensitivity to population density except during the very earliest and latest stages of growth respectively.

Bacterium in a box: sensing of quorum and environment by the LuxI/LuxR gene regulatory circuit.

The chemical signaling mechanism known as "bacterial quorum sensing" (QS) is normally interpreted as allowing bacteria to detect their own population density, in order to coordinate gene expression across a colony. However, the release of the chemical signal can also be interpreted as a means for one or a few cells to probe the local physical properties of their microenvironment. We have studied the behavior of the LuxI/LuxR QS circuit of Vibrio fischeri in tightly confining environments where individual cells detect their own released signals. We find that the lux genes become activated in these environments, although the activation onset time shows substantial cell-to-cell variability and little sensitivity to the confining volume. Our data suggest that noise in gene expression could significantly impact the utility of LuxI/LuxR as a probe of the local physical environment.