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Biofilms are commonly defined as accumulations of microbes, embedded in a self-secreted, polysaccharide-rich extra-cellular matrix. This study aimed to characterize specific morphological changes that occur in Bacillus subtilis biofilms under nutrient-limiting growth conditions. Under varying levels of nutrient depletion, colony-type biofilms were found to exhibit different rates of spatial expansion and green fluorescent protein production. Specifically, colony-type biofilms grown on media with decreased lysogeny broth content exhibited increased spatial expansion and more stable GFP production over the entire growth period. By modeling the surface morphology of colony-type biofilms using confocal and multiphoton microscopy, we analyzed the appearance of distinctive folds or "wrinkles" that form as a result of lysogeny broth content reduction in the solid agar growth media. When subjected to varying nutritional conditions, the channel-like folds were shown to alter their morphology; growth on nutrient-depleted media was found to trigger the formation of large and straight wrinkles connecting the colony core to its periphery. To test a possible functional role of the formed channels, a fluorescent analogue of glucose was used to demonstrate preferential native uptake of the molecules into the channels' interiors which supports their possible role in the transport of molecules throughout biofilm structures.

Biofilms are commonly defined as accumulations of microbes, embedded in a self-secreted, polysaccharide-rich extra-cellular matrix. This study aimed to characterize specific morphological changes that occur in Bacillus subtilis biofilms under nutrient-limiting growth conditions. Under varying levels of nutrient depletion, colony-type biofilms were found to exhibit different rates of spatial expansion and green fluorescent protein production. Specifically, colony-type biofilms grown on media with decreased lysogeny broth content exhibited increased spatial expansion and more stable GFP production over the entire growth period. By modeling the surface morphology of colony-type biofilms using confocal and multiphoton microscopy, we analyzed the appearance of distinctive folds or "wrinkles" that form as a result of lysogeny broth content reduction in the solid agar growth media. When subjected to varying nutritional conditions, the channel-like folds were shown to alter their morphology; growth on nutrient-depleted media was found to trigger the formation of large and straight wrinkles connecting the colony core to its periphery. To test a possible functional role of the formed channels, a fluorescent analogue of glucose was used to demonstrate preferential native uptake of the molecules into the channels' interiors which supports their possible role in the transport of molecules throughout biofilm structures.

The purpose of the present study was to investigate the mechanism of action of our newly developed green sanitizer formulation comprising a natural phenolic compound, gallic acid (GA), strengthened by the Generally Recognized as Safe (GRAS) materials hydrogen peroxide (H2O2) and DL-lactic acid (LA). Combining 8 mM GA with 1 mM H2O2 resulted in an abundant generation of reactive oxygen species (ROS) and a bactericidal effect towards Gram-negative (Escherichia coli, Pseudomonas syringae, and Pectobacterium brasiliense) and Gram-positive (Bacillus subtilis) bacteria (4 to 8 log CFU mL−1 reduction). However, the exposure to this dual formulation (DF) caused only a modest 0.7 log CFU mL−1 reduction in the Gram-positive L. innocua population. Amending the DF with 20 mM LA to yield a triple formulation (TF) resulted in the efficient synergistic control of L. innocua proliferation without increasing ROS production. Despite the inability to grow on plates (>7 log CFU mL−1 population reduction), the TF-exposed L. innocua maintained high intracellular ATP pools and stable membrane integrity. The response of L. innocua to TF could be qualified as a "viable but nonculturable" (VBNC) phenomenon, while with the other species tested this formulation caused cell death. This research system may offer a platform for exploring the VBNC phenomenon, a critical food safety topic. [ABSTRACT FROM AUTHOR]

Biofilm is commonly defined as accumulation of microbes, embedded in a self-secreted extra-cellular matrix, on solid surfaces or liquid interfaces. In this study, we analyze several aspects of Bacillus subtilis biofilm formation using tools from the field of image processing. Specifically, we characterize the growth kinetics and morphological features of B. subtilis colony type biofilm formation and compare these in colonies grown on two different types of solid media. Additionally, we propose a model for assessing B. subtilis biofilm complexity across different growth conditions. GFP-labeled B. subtilis cells were cultured on agar surfaces over a 4-day period during which microscopic images of developing colonies were taken at equal time intervals. The images were used to perform a computerized analysis of few aspects of biofilm development, based on features that characterize the different phenotypes of B. subtilis colonies. Specifically, the analysis focused on the segmented structure of the colonies, consisting of two different regions of sub-populations that comprise the biofilm - a central "core" region and an "expanding" region surrounding it. Our results demonstrate that complex biofilm of B. subtillis grown on biofilm-promoting medium [standard lysogeny broth (LB) supplemented with manganese and glycerol] is characterized by rapidly developing three-dimensional complex structure observed at its core compared to biofilm grown on standard LB. As the biofilm develops, the core size remains largely unchanged during development and colony expansion is mostly attributed to the expansion in area of outer cell sub-populations. Moreover, when comparing the bacterial growth on biofilm-promoting agar to that of colonies grown on LB, we found a significant decrease in the GFP production of colonies that formed a more complex biofilm. This suggests that complex biofilm formation has a diminishing effect on cell populations at the biofilm core, likely due to a combination of reduced metabolic rate and increased levels of cell death within this region.

Despite regulatory and technological measures, edible sprouts are still often involved in foodborne illness and are considered a high-risk food. The present study explored the potential of spore-forming Bacillus isolates to mitigate Salmonella and Escherichia coli contamination of alfalfa sprouts. Food-derived Bacillus strains were screened for antagonistic activity against S. enterica serovar Typhimurium SL1344 (STm) and enteropathogenic E. coli O55:H7. Over 4 days of sprouting, levels of STm and E. coli on contaminated seeds increased from 2.0 log CFU/g to 8.0 and 3.9 log CFU/g, respectively. Treatment of the contaminated seeds with the most active Bacillus isolate, strain BX77, at 7 log CFU/g seeds resulted in substantial reductions in the levels of STm (5.8 CFU/g) and E. coli (3.9 log CFU/g) in the sprouted seeds, compared to the control. Similarly, coculturing STm and BX77 in sterilized sprout extract at the same ratio resulted in growth inhibition and killed the Salmonella. Confocal-microscopy experiments using seeds supplemented with mCherry-tagged Salmonella revealed massive colonization of the seed coat and the root tip of 4-day-old sprouted seeds. In contrast, very few Salmonella cells were observed in sprouted seeds grown with BX77. Ca-hypochlorite disinfection of seeds contaminated with a relatively high concentration of Salmonella (5.0 log CFU/g) or treated with BX77 revealed a mild inhibitory effect. However, disinfection followed by the addition of BX77 had a synergistic effect, with a substantial reduction in Salmonella counts (7.8 log CFU/ g) as compared to untreated seeds. These results suggest that a combination of chemical and biological treatments warrants further study, toward its potential application as a multi-hurdle strategy to mitigate Salmonella contamination of sprouted alfalfa seeds. [ABSTRACT FROM AUTHOR]

Staphylococcus caprae is a Gram-positive, coagulase-negative staphylococci (CoNS), which appears as commensal in the skin, as well as a prevalent mastitis pathogen of goats. Occasionally, it is also associated with infections in humans. Biofilm formation has been identified as a putative virulence factor in S. caprae. Biofilms are multicellular communities protected by a self-produced extracellular matrix (ECM), which facilitates the resistance of bacterial cells to antimicrobial treatments. The ECM is constructed by exopolysaccharides, including the major exopolysaccharide—polysaccharide intercellular adhesion (PIA), regulated by the ica operon in Staphylococcus species. The aim of this study was to characterize the expression of the ica operon in relation to biofilm formation in S. caprae. Results showed that within a few hours of growth, S. caprae could adhere to polystyrene surfaces, start to accumulate, and form biofilm. Peak biofilm biomass and maturation were reached after 48 h, followed by a reduction in biomass after 72 h. Confocal laser scanning microscopy showed the expression of matrix-associated proteins and polysaccharides at various time points. The expression dynamics of the ica operon were investigated using real-time reverse transcriptase PCR (RT)-qPCR, which showed elevated expression during the early stages of biofilm formation and subsequent downregulation throughout the biofilm aging process. In conclusion, our results show that the ica operon is essential in regulating biofilm formation in S. caprae, similar to other Staphylococcus species. Furthermore, the robustness of the observed biofilm phenotype could account for the successful intramammary colonization and may explain disease persistence caused by this pathogenic bacterium. [ABSTRACT FROM AUTHOR]

Bacillus subtilis forms biofilms in response to signals that remain poorly defined. We report that biofilm formation is stimulated by sublethal doses of chlorine dioxide (Cl[O.sub.2]), an extremely effective and fast-acting biocide. Cl[O.sub.2] accelerated biofilm formation in B. subtilis as well as in other bacteria, suggesting that biofilm formation is a widely conserved response to sublethal doses of the agent. Biofilm formation depends on the synthesis of an extracellular matrix that holds the constituent cells together. We show that the transcription of the major operons responsible for the matrix production in B. subtilis, epsA-epsO and yqxM-sipW-tasA, was enhanced by Cl[O.sub.2], in a manner that depended on the membrane-bound kinase KinC. Activation of KinC appeared to be due to the ability of Cl[O.sub.2] to collapse the membrane potential. Importantly, strains unable to make a matrix were hypersensitive to Cl[O.sub.2], indicating that biofilm formation is a defensive response that helps protect cells from the toxic effects of the biocide. doi: 10.1128/JB.01025-10

Streptococcus mutans often adopts a sessile biofilm lifestyle that differs greatly from that of free-living cells. Biofilm formation represents a protected mode of growth that allows cells to survive in hostile environments. In this study, in vitro comparative transcriptome analysis was carried out to identify genes that are differentially expressed in biofilm of S. mutans compared with free-living cells. DNA-microarray analyses indicated that about 12% of genes showed significant differential expression: 139 were activated and 104 were repressed in biofilm vs the planktonic environment. The differential expression of 20 selected genes was confirmed by real-time RT-PCR. In addition, regulation of expression of these genes during biofilm development was tested in 100 and 400 [micro]m deep biofilms. Direct comparison of optical images consistently demonstrated that changes in biofilm thickness are accompanied by significant shifts in cell viability. From evaluation of gene expression patterns, it was shown that the majority of the genes tested were significantly down-regulated in 400 vs 100 [micro]m deep biofilms. This study provides a genome-scale synopsis and adds important insights into gene expression in biofilm development processes of S. mutans, which are strongly associated with the pathogenesis of dental diseases. DOI 10.1099/mic.0.2006/002030-0

Secreted molecules from probiotic Bacilli have often been considered potential pharmaceuticals to fight infections caused by bacterial or yeast pathogens. In the present study, we investigated the antagonistic potential of secreted probiotic filtrates (hereafter, postbiotics) derived from Lactobacillus plantarum cells against pathogenic microorganisms, such as Escherichia coli, Staphylococcus aureus, and Candida albicans. We found that the postbiotics mitigate the biofilms of the tested pathogens with no notable effect on their planktonic growth. In addition, the postbiotics suppressed some virulence traits, for instance, the dendrite swarming motility of E. coli and yeast-to-hyphal switch in C. albicans. Further assays with an active constituent produced by the L. plantarum cells–2-undecanone revealed two significant findings: (i) 2-undecanone inhibits C. albicans biofilms and hyphae in vitro and in a Caenorhabditis elegans model, and (ii) it interacts specifically with Gln 58 amino acid residue of hyphal wall protein-1 (Hwp-1) in molecular docking analysis. The results suggest the targeted mode of antagonistic action of 2-undecanone against C. albicans biofilm. In total, the findings of the study depict an appealing strategy to use postbiotics, including specific ketone molecules, produced by L. plantarum for developing novel antibiofilm and anti-hyphal pharmaceuticals. [ABSTRACT FROM AUTHOR]

Summary: Beneficial biofilms may confer effective adaptation to food matrices that assist bacteria in enduring hostile environmental conditions. The matrices, for instance, dietary fibres of various food products, might serve as a natural scaffold for bacterial cells to adhere and grow as biofilms. Here, we report on a unique interaction of Bacillus subtilis cells with the resistant starch fibresof chickpea milk (CPM), herein CPM fibres, along with the production of a reddish‐pink pigment. Genetic analysis identified the pigment as pulcherrimin, and also revealed the involvement of Spo0A/SinI pathway in modulating the observed phenotypes. Besides, through successful colonization of the CPM fibres, the wild‐type cells of B. subtilis displayed enhanced survivability and resilience to environmental stress, such as heat and in vitro gastrointestinal treatments. In total, we infer that the biofilm formation on CPM fibres is an adaptation response of B. subtilis for strategic survival. [ABSTRACT FROM AUTHOR]

Abstract Background Adhesion and successful colonization of bacteria onto solid surfaces play a key role in biofilm formation. The initial adhesion and the colonization of bacteria may differ between the various types of surfaces found in oral cavity. Therefore, it is conceivable that diverse biofilms are developed on those various surfaces. The aim of the study was to investigate the molecular modifications occurring during in vitro biofilm development of Streptococcus mutans UA159 on several different dental surfaces. Results Growth analysis of the immobilized bacterial populations generated on the different surfaces shows that the bacteria constructed a more confluent and thick biofilms on a hydroxyapatite surface compared to the other tested surfaces. Using DNA-microarray technology we identified the differentially expressed genes of S. mutans, reflecting the physiological state of biofilms formed on the different biomaterials tested. Eight selected genes were further analyzed by real time RT-PCR. To further determine the impact of the tested material surfaces on the physiology of the bacteria, we tested the secretion of AI-2 signal by S. mutans embedded on those biofilms. Comparative transcriptome analyses indicated on changes in the S. mutans genome in biofilms formed onto different types of surfaces and enabled us to identify genes most differentially expressed on those surfaces. In addition, the levels of autoinducer-2 in biofilms from the various tested surfaces were different. Conclusions Our results demonstrate that gene expression of S. mutans differs in biofilms formed on tested surfaces, which manifest the physiological state of bacteria influenced by the type of surface material they accumulate onto. Moreover, the stressful circumstances of adjustment to the surface may persist in the bacteria enhancing intercellular signaling and surface dependent biofilm formation.

We show that times of spikes can be very precise. In the cerebral cortex, where each nerve cell is affected by thousands of others, it is the common belief that the exact time of a spike is random up to an averaged firing rate over tens of milliseconds. In a brain slice, precise time relations of several neurons have been observed. It remained unclear whether this phenomenon can also be observed in brains of behaving animals. Here we show, in behaving monkeys, that time intervals between spikes, measured in correspondence to a specific behavior, may be controlled to within the milliseconds range. data mining | motor cortex | neural codes | precise timing | single units

The human body is inhabited by a vast diversity of probiotic microorganisms that could positively affect human physiology. Besides, prebiotic food substances may induce symbiotic relationship among probiotic species through the successful establishment of commensal microbiota, whose connections with the host are multifaceted and multidirectional. As deliberated throughout this review, prebiotic and synbiotic foods contain the capability to stimulate numerous health characteristics in host organisms through various means. Predominantly, the normal microbiota fosters the digestion of food and may boost the innate and adaptive immune system's functionalities. Therefore, live probiotic bacteria, for instance, probiotic Bacilli obtained together with prebiotic food, can help stimulate healthiness in humans. Thus, we discuss how certain dietary fibers may preserve the probiotic efficacy by serving as the scaffold for probiotic Bacilli to colonize them through forming symbiotic interactions. The fibers can essentially promote protection by encapsulating probiotic Bacilli against various environmental and physical stresses that might kill the free-living bacterial cells. Besides, these fibers would serve as prebiotic substances that would eventually be utilized for the proliferation of probiotic cells. It is believed that applying this conceptual idea will provide a novel platform toward developing probiotic and synbiotic foods, as discussed in this review. [ABSTRACT FROM AUTHOR]

Abstract Background A biofilm is a complex community of microorganisms that develop on surfaces in diverse environments. The thickness of the biofilm plays a crucial role in the physiology of the immobilized bacteria. The most cariogenic bacteria, mutans streptococci, are common inhabitants of a dental biofilm community. In this study, DNA-microarray analysis was used to identify differentially expressed genes associated with the thickness of S. mutans biofilms. Results Comparative transcriptome analyses indicated that expression of 29 genes was differentially altered in 400- vs. 100-microns depth and 39 genes in 200- vs. 100-microns biofilms. Only 10 S. mutans genes showed differential expression in both 400- vs. 100-microns and 200- vs. 100-microns biofilms. All of these genes were upregulated. As sucrose is a predominant factor in oral biofilm development, its influence was evaluated on selected genes expression in the various depths of biofilms. The presence of sucrose did not noticeably change the regulation of these genes in 400- vs. 100-microns and/or 200- vs. 100-microns biofilms tested by real-time RT-PCR. Furthermore, we analyzed the expression profile of selected biofilm thickness associated genes in the luxS- mutant strain. The expression of those genes was not radically changed in the mutant strain compared to wild-type bacteria in planktonic condition. Only slight downregulation was recorded in SMU.2146c, SMU.574, SMU.609, and SMU.987 genes expression in luxS- bacteria in biofilm vs. planktonic environments. Conclusion These findings reveal genes associated with the thickness of biofilms of S. mutans. Expression of these genes is apparently not regulated directly by luxS and is not necessarily influenced by the presence of sucrose in the growth media.

Bacillus subtilis is a Gram-positive probiotic bacterium that successfully colonises plant roots due to its ability to utilise various sugars. The vast probiotic potential of B. subtilis has been recently demonstrated in numerous host organisms under different environmental conditions. We examined the probiotic potential of B. subtilis against the pathogenic bacterium Streptococcus mutans, which is involved in various oral disorders due to its robust biofilm-forming capability. B. subtilis cells attenuated biofilm formation by S. mutans during their dual growth in the presence of sugar alcohols. Transcription of genes encoding key enzymes in the metabolism of sugar alcohols by B. subtilis were highly induced. Moreover, growth-curve analysis suggested that B. subtilis is more efficient at early utilising sugar alcohols than S. mutans, as supported by the bacterial metabolic activity rates. Similarly, a comparison of secondary metabolites of mono and mixed cultures of B. subtilis and S. mutans indicated that B. subtilis is more active metabolically in the dual culture. Finally, knock-out mutations of the genes encoding key enzymes in the central metabolic pathway significantly reduced B. subtilis' ability to mitigate biofilm formation by S. mutans. We conclude that effective metabolism of sugar alcohols by B. subtilis reinforces the probiotic potential of this bacterium against pathogenic species such as S. mutans. [ABSTRACT FROM AUTHOR]

Biofilm‐forming Bacillus species are often involved in persistent contamination and spoilage of dairy products. They therefore present a major microbiological challenge in the field of dairy food quality and safety. Due to their substantial physiological versatility, Bacillus species can survive in various parts of dairy manufacturing plants, leading to a high risk of product spoilage and potential dissemination of foodborne diseases. Furthermore, biofilm and heat‐resistant spore formation make these bacteria challenging to eliminate. Thus, some strategies have been employed to remove, prevent, or delay the formation of Bacillus biofilms in the dairy industry, but with limited success. Lack of understanding of the Bacillus biofilm structure and behavior in conditions relevant to dairy‐associated environments could partially account for this situation. The current paper reviews dairy‐associated biofilm formation by Bacillus species, with particular attention to the role of biofilm in Bacillus species adaptation and survival in a dairy processing environment. Relevant model systems are discussed for the development of novel antimicrobial approaches to improve the quality of dairy food. © 2020 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

Biofilm-forming bacteria, which colonize the surfaces of equipment in the dairy industry, may adversely affect the safety and quality of the milk and its products. Despite numerous efforts to combat biofilm formation, there is still no effective technological means to thoroughly solve the biofilm problem in the dairy industry. Here, we introduced peptide-based coating in order to modify the physical properties of the stainless steel surface by affecting its availability for bacterial adhesion. We found that the coated surface displays a notable decrease in the ability of bacterial cells to attach and to subsequently form biofilm by Gram-positive Bacillus licheniformis and Gram-negative Pseudomonas aeruginosa. Furthermore, the coated surface retained its anti-biofilm ability following its exposure to raw milk. Importantly, the modified surface did not affect the milk coagulation process or its nutritious properties and quality. Overall, this anti-biofilm approach may serve as an attractive solution for the dairy industry in its struggle against bacterial contamination. [ABSTRACT FROM AUTHOR]

Probiotics, live microbial supplements, are often incorporated into foods and beverages to provide putative health benefits. To ensure their beneficial effects, these organisms must survive processing and storage of food, its passage through the upper gastrointestinal tract (GIT), and subsequent chemical ingestion processes until they reach their target organ. However, there is considerable loss of viability of probiotic bacteria in the acidic conditions of the stomach and the high bile concentration in the small intestine. Bacillus subtilis, a spore-forming non-pathogenic bacterium, recently has gained interest in its probiotic properties; it can effectively maintain a favorable balance of microflora in the GIT. In addition, B. subtilis produces an extracellular matrix that protects it from stressful environments. We suggested that the extracellular matrix produced by B. subtilis could protect other probiotic bacteria and therefore potentially could be used as a vehicle for delivering viable probiotic cells to humans. Therefore, we developed a novel cultivation system that enables co-culturing of B. subtilis along with probiotic lactic acid bacteria (LAB) by increasing production of the extracellular matrix by B. subtilis cells. Moreover, we showed that B. subtilis improved survivability of LAB during food preparation, storage and ingestion. Therefore, we believe that the results of our study will provide a novel technique of using a natural system for preservation and delivery of probiotics to humans. [ABSTRACT FROM AUTHOR]

The soil bacterium Bacillus subtilis is often found in association with plants in the rhizosphere. Previously, plant polysaccharides have been shown to stimulate formation of root-associated multicellular communities, or biofilms, in this bacterium, yet the underlying mechanism is not fully understood. A five-gene gan operon (ganSPQAB) in B. subtilis has recently been shown to be involved in utilization of the plant-derived polysaccharide galactan. Despite these findings, molecular details about the regulation of the operon and the role of the operon in biofilm formation remain elusive. In this study, we performed comprehensive genetic analyses on the regulation of the gan operon. We show that this operon is regulated both by a LacI-like transcription repressor (GanR), which directly binds to pairs of inverted DNA repeats in the promoter region of the operon, and by the catabolite control protein A (CcpA). Derepression can be triggered by the presence of the inducer β-1,4-galactobiose, a hydrolysis product of galactan, or in situ when B. subtilis cells are associated with plant roots. In addition to the transcriptional regulation, the encoded ß-galactosidase GanA (by ganA), which hydrolyzes ß-1,4-galactobiose into galactose, is inhibited at the enzymatic level by the catalytic product galactose. Thus, the galactan utilization pathway is under complex regulation involving both positive and negative feedback mechanisms in B. subtilis. We discuss about the biological significance of such complex regulation as well as a hypothesis of biofilm induction by galactan via multiple mechanisms. [ABSTRACT FROM AUTHOR]

Microbial damages caused by biofilm forming bacteria in the dairy industry are a fundamental threat to safety and quality of dairy products. In order to ensure the optimal level of equipment hygiene in the dairy industry, it is necessary to determine the biofilm removal efficiency of cleaning agents used for cleaning-in-place (CIP) procedures. However, currently there is no standard method available for evaluating and comparing cleaning agents for use in CIP procedures in the dairy industry under realistic conditions. The present study aims to establish a CIP model system to evaluate the effectiveness of cleaning agents in removal of biofilm derived spores from the surfaces of stainless steel which is the predominant substrate in milking equipment on dairy farms. The system is based on Bacillus subtilis spores surrounded with exopolymeric substances produced by bacteria during biofilm formation. The spores applied on sampling plates were mounted on T-junctions protruding 1.5-11-times the milk pipe diameter from the main loop to resemble different levels of cleaning difficulty. The cleaning tests were conducted using commercial alkaline detergents and caustic soda at conditions which are relevant to actual farm environment. The spores removal effect was evaluated by comparing the number of viable spores (attached to sampling plates) before and after cleaning. Evaluation of the cleaning and disinfecting effect of cleaning agents toward biofilm derived spores was further performed, which indicates whether spores elimination effect of an agent is due to killing the spores or removing them from the surfaces of dairy equipment. Moreover, it was established that the presence of extracellular matrix is an important factor responsible for high level of cleaning difficulty characteristic for surface attached spores. In overall, the results of this study suggest that the developed model system simulates actual farm conditions for quantitative evaluation of the effectiveness of cleaning and disinfecting agents and their cleaning and disinfecting effect on removal of biofilm derived spores. [ABSTRACT FROM AUTHOR]

Streptococcus mutans activates multiple cellular processes in response to the formation of a complex between comX-inducing peptide (XIP) and the ComR transcriptional regulator. Bulk phase and microfluidic experiments previously revealed that ComRdependent activation of comX is altered by pH and by carbohydrate source. Biofilm formation is a major factor in bacterial survival and virulence in the oral cavity. Here, we sought to determine the response of S. mutans biofilm cells to XIP during different stages of biofilm maturation. Using flow cytometry and confocal microscopy, we showed that exogenous addition of XIP to early biofilms resulted in robust comX activation. However, as the biofilms matured, increasing amounts of XIP were required to activate comX expression. Single-cell analysis demonstrated that the entire population was responding to XIP with activation of comX in early biofilms, but only a sub-population was responding in mature biofilms. The sub-population response of mature biofilms was retained when the cells were dispersed and then treated with XIP. The proportion and intensity of the bi-modal response of mature biofilm cells was altered in mutants lacking the Type II toxins MazF and RelE, or in a strain lacking the (p)ppGpp synthase/hydrolase RelA. Thus, competence signaling is markedly altered in cells growing in mature biofilms, and pathways that control cell death and growth/survival decisions modulate activation of comX expression in these sessile populations. [ABSTRACT FROM AUTHOR]