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.
The cidAB and lrgAB operons of Streptococcus mutans encode proteins that are structurally similar to the bacteriophage lambda family of holin-antiholin proteins, which are believed to facilitate cell death in other bacterial species. Although their precise function is not known, cidAB and lrgAB are linked to multiple virulence traits of S. mutans, including oxidative stress tolerance, biofilm formation, and autolysis. Here we investigate the regulation of lrgAB which in S. mutans shows a complex dependence on growth conditions that is not fully understood. By combining single-cell imaging of a fluorescent gene reporter with microfluidic control of the extracellular environment, we identify specific environmental cues that trigger lrgA expression and characterize cell-to-cell heterogeneity in lrgA activity. We find that the very abrupt activation of lrgA at stationary phase is tightly synchronized across the population. This activation is controlled by a small number of inputs that are sensitive to growth phase: extracellular pyruvate, glucose, and molecular oxygen. Activation of lrgA appears to be self-limiting, so that strong expression of lrgA is confined to a short interval of time. lrgA is programmed to switch on briefly at the end of exponential growth, as glucose and molecular oxygen are exhausted and extracellular pyruvate is available. Our findings are consistent with studies of other bacteria showing that homologs of lrgAB participate, with input from lytST, in the reimport of pyruvate for anaerobic fermentative growth.
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.
Streptococcus mutans is an important pathogen in the human oral biofilm. It expresses virulent behaviors that are linked to its genetic competence regulon, which is controlled by comX. Expression of comX is modulated by two diffusible signaling peptides, denoted CSP and XIP, and by other environmental cues such as pH and oxidative stress. The sensitivity of S. mutans competence to environmental inputs that may vary on microscopic length scales raises the question of whether the biofilm environment creates microniches where competence and related phenotypes are concentrated, leading to spatial clustering of S. mutans virulence behaviors. We have used two-photon microscopy to characterize the spatial distribution of comX expression among individual S. mutans cells in biofilms. By analyzing correlations in comX activity, we test for spatial clustering that may suggest localized competence microenvironments. Our data indicate that both competence-signaling peptides diffuse efficiently through the biofilm. XIP elicits a population-wide response. CSP triggers a Poisson-like, spatially random comX response from a subpopulation of cells that is homogeneously dispersed. Our data indicate that competence microenvironments if they exist are small enough that the phenotypes of individual cells are not clustered or correlated to any greater extent than occurs in planktonic cultures.
20The lrgAB and cidAB operons of Streptococcus mutans encode proteins that are 21 structurally similar to the bacteriophage lambda family of holin-antiholin proteins, which 22 are believed to facilitate cell death in other bacterial species. Although their precise 23 function is not known, cidAB and lrgAB are linked to multiple virulence traits of S. mutans, 24 including oxidative stress tolerance, biofilm formation, and autolysis. The regulation of 25 cidAB and lrgAB is still not understood, as these operons show opposite patterns of 26 expression as well as a complex dependence on growth conditions. We have used a 27 microfluidic approach, together with single-cell imaging of a fluorescent gene reporter, to 28 identify with greater precision the cues that trigger lrgA expression and characterize cell- 29 to-cell heterogeneity in lrgA activity. lrgA activates very abruptly at stationary phase, with 30 a high degree of synchrony across the population. We find this activation is controlled by 31 a small number of inputs that are sensitive to growth phase: Extracellular pyruvate, 32 glucose, and molecular oxygen. Further, activation of lrgA appears to be self-limiting, so 33 that lrgA is strongly expressed only for a very short interval of time. Consequently, lrgA is 34 programmed to switch on briefly at the end of exponential growth, as glucose and 35 molecular oxygen are exhausted and extracellular pyruvate is available. Our findings are 36 consistent with studies showing that homologs of lrgAB are linked, together with lytST, to 37 the reimport of pyruvate for anaerobic fermentative growth. 38 39 40 41 42 3 Importance 43The function and regulation of cidAB and lrgAB in Streptococcus mutans is not 44 understood, although these operons have been clearly linked to stress responses and 45 they show a complex dependence on environmental inputs and growth phase. Identifying 46 specific environmental cues that trigger activation of lrgAB has been difficult owing to the 47 cells' own modification of key inputs such as glucose and oxygen: In S. mutans the lrgAB 48 operon is strongly upregulated at the end of exponential phase, where growth conditions 49 in a bulk culture become poorly defined. Here we have used microfluidics to apply precise 50 control of environmental inputs to S. mutans and identify specific chemical cues that 51 activate lrgAB. We find that rigorously anaerobic conditions and the presence of 52 extracellular pyruvate are sufficient to induce lrgAB expression, suggesting that lrgAB is 53 timed to activate just as pyruvate fermentation becomes favorable. 55The oral pathogen Streptococcus mutans (1) possesses two operons designated 56 cidAB (SMU.1701/1700) and lrgAB (SMU.575/574) (2), which are closely homologous to 57 the lrgAB and cidAB operons that have been extensively studied in organisms such as 58 Bacillus subtilis and Staphylococcus aureus (3-10). Sequence homology indicates that 59 cidAB and lrgAB encode membrane proteins that are similar to holin-antiholin membrane 60 prote...
Streptococcus mutans is an important pathogen in the human oral biofilm. It expresses virulent behaviors that are linked to its genetic competence regulon, which is controlled by comX. Expression of comX is modulated by two diffusible signaling peptides, denoted CSP and XIP, and by other environmental cues such as pH and oxidative stress. The sensitivity of S. mutans competence to environmental inputs that may vary on microscopic length scales raises the question of whether the biofilm environment causes spatial clustering of S. mutans virulence behaviors, by creating microniches where competence and related phenotypes are concentrated. We have used two-photon microscopy to characterize the spatial distribution of comX expression among individual S. mutans cells in biofilms. By analyzing correlations in comX activity, we test for spatial clustering that may suggest localized, competence microenvironments. Our data indicate that both competence-signaling peptides diffuse efficiently through the biofilm. CSP triggers a Poisson-like, spatially random, comX response from a subpopulation of cells that is homogeneously dispersed. XIP elicits a population-wide response. Our data indicate that competence microenvironments if they exist are small enough that the phenotypes of individual cells are not clustered or correlated to any greater extent than occurs in planktonic cultures.
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