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Cells that reside within a community can cooperate and also compete with each other for resources. It remains unclear how these opposing interactions are resolved at the population level. Here we investigate such an internal conflict within a microbial ( Bacillus subtilis ) biofilm community: cells in the biofilm periphery not only protect interior cells from external attack but also starve them through nutrient consumption. We discover that this conflict between protection and starvation is resolved through emergence of long-range metabolic co-dependence between peripheral and interior cells. As a result, biofilm growth halts periodically, increasing nutrient availability for the sheltered interior cells. We show that this collective oscillation in biofilm growth benefits the community in the event of a chemical attack. These findings indicate that oscillations support population-level conflict resolution by coordinating competing metabolic demands in space and time, suggesting new strategies to control biofilm growth. The emergence of long-range metabolic co-dependence within a biofilm drives oscillations in growth that resolve a social conflict between cooperation and competition, thereby increasing community-level fitness against chemical attack. Give-and-take within microbial biofilms During growth in a biofilm, cells at the periphery protect interior cells from external attack but can also starve them through nutrient consumption by the peripheral cells. Here Gürol Süel and colleagues find that this conflict between protection and starvation is resolved by the emergence of long-range metabolic co-dependence between cells at the periphery and the interior. In particular, they show in Bacillus subtilis biofilms that growth periodically halts, increasing the availability of nutrients to the sheltered interior cells, which in turn provide the metabolites necessary for growth at the periphery.

Elongation of rod-shaped bacteria is mediated by a dynamic peptidoglycan-synthetizing machinery called the Rod complex. Here we report that, in Bacillus subtilis , this complex is functional in the absence of all known peptidoglycan polymerases. Cells lacking these enzymes survive by inducing an envelope stress response that increases the expression of RodA, a widely conserved core component of the Rod complex. RodA is a member of the SEDS (shape, elongation, division and sporulation) family of proteins, which have essential but ill-defined roles in cell wall biogenesis during growth, division and sporulation. Our genetic and biochemical analyses indicate that SEDS proteins constitute a family of peptidoglycan polymerases. Thus, B. subtilis and probably most bacteria use two distinct classes of polymerase to synthesize their exoskeleton. Our findings indicate that SEDS family proteins are core cell wall synthases of the cell elongation and division machinery, and represent attractive targets for antibiotic development. SEDS proteins are core peptidoglycan polymerases involved in bacterial cell wall elongation and division. SEDS proteins key to bacterial cell wall integrity It has been generally accepted that the cell wall peptidoglycans of the bacterial exoskeleton are synthesized by penicillin binding proteins (PBPs) known as class A PBPs. Now, using genetic manipulation, phylogenetic analysis and functional experiments in Bacillus subtilis , David Rudner and colleagues have identified SEDS family proteins as the main peptidoglycan polymerases more broadly conserved than class A PBPs. Specifically in B. subtilis , they show that the SEDS protein RodA, a widely conserved component of the Rod complex involved in elongation of rod-shaped bacteria, acts with class B PBPs as the core cell wall synthase of the cell elongation and division machinery. The authors conclude that B. subtilis and probably most bacteria use two distinct classes of polymerases to synthesize their exoskeleton. This work also suggests that SEDS family proteins should be attractive targets for antibiotic development.

The primary structural component of the bacterial cell wall is peptidoglycan, which is essential for viability and the synthesis of which is the target for crucial antibiotics 1 , 2 . Peptidoglycan is a single macromolecule made of glycan chains crosslinked by peptide side branches that surrounds the cell, acting as a constraint to internal turgor 1 , 3 . In Gram-positive bacteria, peptidoglycan is tens of nanometres thick, generally portrayed as a homogeneous structure that provides mechanical strength 4 – 6 . Here we applied atomic force microscopy 7 – 12 to interrogate the morphologically distinct Staphylococcus aureus and Bacillus subtilis species, using live cells and purified peptidoglycan. The mature surface of live cells is characterized by a landscape of large (up to 60 nm in diameter), deep (up to 23 nm) pores constituting a disordered gel of peptidoglycan. The inner peptidoglycan surface, consisting of more nascent material, is much denser, with glycan strand spacing typically less than 7 nm. The inner surface architecture is location dependent; the cylinder of B. subtilis has dense circumferential orientation, while in S. aureus and division septa for both species, peptidoglycan is dense but randomly oriented. Revealing the molecular architecture of the cell envelope frames our understanding of its mechanical properties and role as the environmental interface 13 , 14 , providing information complementary to traditional structural biology approaches. Using high-resolution atomic force microscopy of live cells, the authors present an updated view of the cell walls of both Staphylococcus aureus and Bacillus subtilis .

Bacteria within communities can interact to organize their behavior. It has been unclear whether such interactions can extend beyond a single community to coordinate the behavior of distant populations. We discovered that two Bacillus subtilis biofilm communities undergoing metabolic oscillations can become coupled through electrical signaling and synchronize their growth dynamics. Coupling increases competition by also synchronizing demand for limited nutrients. As predicted by mathematical modeling, we confirm that biofilms resolve this conflict by switching from in-phase to antiphase oscillations. This results in time-sharing behavior, where each community takes turns consuming nutrients. Time-sharing enables biofilms to counterintuitively increase growth under reduced nutrient supply. Distant biofilms can thus coordinate their behavior to resolve nutrient competition through time-sharing, a strategy used in engineered systems to allocate limited resources.

Bacteria can become dormant or form spores when they are starved for nutrients. Here, we find that non-sporulating Bacillus subtilis cells can survive deep starvation conditions for many months. During this period, cells adopt an almost coccoid shape and become tolerant to antibiotics. Unexpectedly, these cells appear to be metabolically active and show a transcriptome profile very different from that of stationary phase cells. We show that these starved cells are not dormant but are growing and dividing, albeit with a doubling time close to 4 days. Very low nutrient levels, comparable to 10,000-fold diluted lysogeny broth (LB), are sufficient to sustain this growth. This extreme slow growth, which we propose to call ‘oligotrophic growth state’, provides an alternative strategy for B. subtilis to endure nutrient depletion and environmental stresses. Further work is warranted to test whether this state can be found in other bacterial species to survive deep starvation conditions. Bacteria can become dormant or form spores when starved for nutrients. Here, Gray et al. describe an alternative strategy, or ‘oligotrophic growth state’, showing that non-sporulating Bacillus subtilis cells can survive deep starvation conditions by adopting an almost coccoid shape and extremely low growth rates.

The study of bacterial ion channels has provided fundamental insights into the structural basis of neuronal signalling; however, the native role of ion channels in bacteria has remained elusive. Here we show that ion channels conduct long-range electrical signals within bacterial biofilm communities through spatially propagating waves of potassium. These waves result from a positive feedback loop, in which a metabolic trigger induces release of intracellular potassium, which in turn depolarizes neighbouring cells. Propagating through the biofilm, this wave of depolarization coordinates metabolic states among cells in the interior and periphery of the biofilm. Deletion of the potassium channel abolishes this response. As predicted by a mathematical model, we further show that spatial propagation can be hindered by specific genetic perturbations to potassium channel gating. Together, these results demonstrate a function for ion channels in bacterial biofilms, and provide a prokaryotic paradigm for active, long-range electrical signalling in cellular communities. Ion channels in bacterial biofilms are shown to conduct long-range electrical signals within the biofilm community through the propagation of potassium ions; as predicted by a simple mathematical model, potassium channel gating is shown to coordinate metabolic states between distant cells via electrical communication. Well-connected bacterial films Gürol Suel and colleagues show that ion channels in bacterial biofilms, which have no known functional role, conduct long-range electrical signals within the biofilm community through the propagation of potassium ions. Metabolic coordination between spatially segregated cells in a Bacillus subtilis biofilm is shown to be dependent on ion channel activity. Metabolic limitation triggers activation of the YugO potassium channel, which also propagates the extracellular potassium signal within the biofilm, resulting in a wave of depolarization that coordinates metabolic states among cells in the interior and periphery of the biofilm. Using a simple mathematical model the authors demonstrate that YugO channel gating is sufficient to promote efficient electrical communication between distant cells.

Bacterial-fungal interactions influence microbial community performance of most ecosystems and elicit specific microbial behaviours, including stimulating specialised metabolite production. Here, we use a co-culture experimental evolution approach to investigate bacterial adaptation to the presence of a fungus, using a simple model of bacterial-fungal interactions encompassing the bacterium Bacillus subtilis and the fungus Aspergillus niger . We find in one evolving population that B. subtilis was selected for enhanced production of the lipopeptide surfactin and accelerated surface spreading ability, leading to inhibition of fungal expansion and acidification of the environment. These phenotypes were explained by specific mutations in the DegS-DegU two-component system. In the presence of surfactin, fungal hyphae exhibited bulging cells with delocalised secretory vesicles possibly provoking an RlmA-dependent cell wall stress. Thus, our results indicate that the presence of the fungus selects for increased surfactin production, which inhibits fungal growth and facilitates the competitive success of the bacterium. Bacterial-fungal interactions can stimulate the production of specialised microbial metabolites. Here, Richter et al. use co-culture experimental evolution to show that the presence of a fungus selects for increased surfactin production in the bacterium Bacillus subtilis , which inhibits fungal growth and facilitates the competitive success of the bacterium.

Rod-shaped bacteria elongate by the action of cell wall synthesis complexes linked to underlying dynamic MreB filaments. To understand how the movements of these filaments relate to cell wall synthesis, we characterized the dynamics of MreB and the cell wall elongation machinery using high-precision particle tracking in Bacillus subtilis. We found that MreB and the elongation machinery moved circumferentially around the cell, perpendicular to its length, with nearby synthesis complexes and MreB filaments moving independently in both directions. Inhibition of cell wall synthesis by various methods blocked the movement of MreB. Thus, bacteria elongate by the uncoordinated, circumferential movements of synthetic complexes that insert radial hoops of new peptidoglycan during their transit, possibly driving the motion of the underlying MreB filaments.

Clostridium difficile is the primary cause of nosocomial diarrhea and pseudomembranous colitis. It produces dormant spores, which serve as an infectious vehicle responsible for transmission of the disease and persistence of the organism in the environment. In Bacillus subtilis, the sin locus coding SinR (113 aa) and SinI (57 aa) is responsible for sporulation inhibition. In B. subtilis, SinR mainly acts as a repressor of its target genes to control sporulation, biofilm formation, and autolysis. SinI is an inhibitor of SinR, so their interaction determines whether SinR can inhibit its target gene expression. The C. difficile genome carries two sinR homologs in the operon that we named sinR and sinR', coding for SinR (112 aa) and SinR' (105 aa), respectively. In this study, we constructed and characterized sin locus mutants in two different C. difficile strains R20291 and JIR8094, to decipher the locus's role in C. difficile physiology. Transcriptome analysis of the sinRR' mutants revealed their pleiotropic roles in controlling several pathways including sporulation, toxin production, and motility in C. difficile. Through various genetic and biochemical experiments, we have shown that SinR can regulate transcription of key regulators in these pathways, which includes sigD, spo0A, and codY. We have found that SinR' acts as an antagonist to SinR by blocking its repressor activity. Using a hamster model, we have also demonstrated that the sin locus is needed for successful C. difficile infection. This study reveals the sin locus as a central link that connects the gene regulatory networks of sporulation, toxin production, and motility; three key pathways that are important for C. difficile pathogenesis.

Key Points The Bacillus subtilis spore coat is a multilayered protective structure composed of more than 70 different proteins. In addition to its protective role, the spore coat influences the process of spore germination and defines the type of interactions that spores can establish with various surfaces in the environment. Fluorescence microscopy in combination with high-resolution image analysis has produced a spatially scaled coat protein interaction network indicating that the coat is organized into four distinct layers. These studies led to the discovery of the outermost layer of the coat in B. subtilis , referred to as the spore crust. Time course analyses of spore coat assembly have revealed that two main steps can be distinguished in coat morphogenesis: the initial recruitment of proteins to the spore surface as a scaffold cap, followed by spore encasement in a series of successive waves. Coat assembly is regulated at the transcriptional level by the sequential expression of individual coat genes and at the protein level by a small group of coat morphogenetic proteins that coordinate both the recruitment of coat proteins to specific coat layers and spore encasement. Sporulation in Bacillus subtilis results in the formation of an endospore surrounded by a multilayered protective structure, known as the coat. In this Review, Patrick Eichenberger and colleagues describe recent studies that have illuminated the architecture of the coat and the dynamics of coat assembly. Sporulation in Bacillus subtilis involves an asymmetric cell division followed by differentiation into two cell types, the endospore and the mother cell. The endospore coat is a multilayered shell that protects the bacterial genome during stress conditions and is composed of dozens of proteins. Recently, fluorescence microscopy coupled with high-resolution image analysis has been applied to the dynamic process of coat assembly and has shown that the coat is organized into at least four distinct layers. In this Review, we provide a brief summary of B. subtilis sporulation, describe the function of the spore surface layers and discuss the recent progress that has improved our understanding of the structure of the endospore coat and the mechanisms of coat assembly.