RESUMO
Biofilms are structured communities of bacteria that are held together by an extracellular matrix consisting of protein and exopolysaccharide. Biofilms often have a limited lifespan, disassembling as nutrients become exhausted and waste products accumulate. D-amino acids were previously identified as a self-produced factor that mediates biofilm disassembly by causing the release of the protein component of the matrix in Bacillus subtilis. Here we report that B. subtilis produces an additional biofilm-disassembly factor, norspermidine. Dynamic light scattering and scanning electron microscopy experiments indicated that norspermidine interacts directly and specifically with exopolysaccharide. D-amino acids and norspermidine acted together to break down existing biofilms and mutants blocked in the production of both factors formed long-lived biofilms. Norspermidine, but not closely related polyamines, prevented biofilm formation by B. subtilis, Escherichia coli, and Staphylococcus aureus.
Assuntos
Bacillus subtilis/fisiologia , Biofilmes , Polissacarídeos Bacterianos/metabolismo , Espermidina/análogos & derivados , Aminoácidos/biossíntese , Aminoácidos/metabolismo , Bacillus subtilis/genética , Escherichia coli/fisiologia , Mutação , Poliaminas/metabolismo , Espermidina/biossíntese , Espermidina/metabolismo , Staphylococcus aureus/fisiologiaRESUMO
For bacteria, the transition from unicellular entities to multicellular biofilm communities generates distinct metabolic microenvironments. Dynamic and programmed metabolic responses allow the biofilms to react to local changes in nutrient levels. Moreover, metabolic adaptations contribute to phenotypic antibiotic resistance of the community, suggesting novel therapeutic approaches to target biofilms.
Assuntos
Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Bactérias/crescimento & desenvolvimento , Biofilmes/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Microambiente Celular , Resistência Microbiana a Medicamentos , Animais , Bactérias/metabolismo , Tolerância a Medicamentos , Humanos , Metaboloma , Viabilidade MicrobianaRESUMO
Members of the NDR (nuclear Dbf2-related) protein-kinase family are essential for cell differentiation and polarized morphogenesis. However, their functions in plant pathogenic fungi are not well understood. Here, we characterized the NDR kinase FgCot1 and its activator FgMob2 in Fusarium graminearum, a major pathogen causing Fusarium head blight (FHB) in wheat. FgCot1 and FgMob2 formed a NDR kinase-MOB protein complex. Localization assays using FgCot1-GFP or FgMob2-RFP constructs showed diverse subcellular localizations, including cytoplasm, septum, nucleus and hyphal tip. ΔFgcot1 and ΔFgmob2 exhibited serious defects in hyphal growth, polarity, fungal development and cell wall integrity as well as reduced virulence in planta. In contrast, lipid droplet accumulation was significantly increased in these two mutants. Phosphorylation of FgCot1 at two highly conserved residues (S462 and T630) as well as five new sites synergistically contributed its role in various cellular processes. In addition, non-synonymous mutations in two MAPK (mitogen-activated protein kinase) proteins, FgSte11 and FgGpmk1, partially rescued the growth defect of ΔFgmob2, indicating a functional link between the FgCot1-Mob2 complex and the FgGpmk1 signalling pathway in regulating filamentous fungal growth. These results indicated that the FgCot1-Mob2 complex is critical for polarity, fungal development, cell wall organization, lipid metabolism and virulence in F. graminearum.
Assuntos
Fusarium , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Fusarium/genética , Fusarium/metabolismo , Metabolismo dos Lipídeos , Doenças das Plantas , VirulênciaRESUMO
Bacillus subtilis forms organized multicellular communities known as biofilms wherein the individual cells are held together by a self-produced extracellular matrix. The environmental signals that promote matrix synthesis remain largely unknown. We discovered that one such signal is impaired respiration. Specifically, high oxygen levels suppressed synthesis of the extracellular matrix. In contrast, low oxygen levels, in the absence of an alternative electron acceptor, led to increased matrix production. The response to impaired respiration was blocked in a mutant lacking cytochromes caa3 and bc and markedly reduced in a mutant lacking kinase KinB. Mass spectrometry of proteins associated with KinB showed that the kinase was in a complex with multiple components of the aerobic respiratory chain. We propose that KinB is activated via a redox switch involving interaction of its second transmembrane segment with one or more cytochromes under conditions of reduced electron transport. In addition, a second kinase (KinA) contributes to the response to impaired respiration. Evidence suggests that KinA is activated by a decrease in the nicotinamide adenine dinucleotide (NAD(+))/NADH ratio via binding of NAD(+) to the kinase in a PAS domain A-dependent manner. Thus, B. subtilis switches from a unicellular to a multicellular state by two pathways that independently respond to conditions of impaired respiration.
Assuntos
Bacillus subtilis/fisiologia , Proteínas de Bactérias/metabolismo , Biofilmes , Citocromos/metabolismo , Proteínas Quinases/metabolismo , Bacillus subtilis/efeitos dos fármacos , Bacillus subtilis/enzimologia , Bacillus subtilis/genética , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/metabolismo , Regulação Bacteriana da Expressão Gênica , Histidina Quinase , Ferro/farmacologia , Mutação , NAD/metabolismo , Oxigênio/metabolismo , Ligação Proteica , Oligoelementos/farmacologiaRESUMO
Non-caloric artificial sweeteners (NAS) are among the most widely used food additives worldwide, regularly consumed by lean and obese individuals alike. NAS consumption is considered safe and beneficial owing to their low caloric content, yet supporting scientific data remain sparse and controversial. Here we demonstrate that consumption of commonly used NAS formulations drives the development of glucose intolerance through induction of compositional and functional alterations to the intestinal microbiota. These NAS-mediated deleterious metabolic effects are abrogated by antibiotic treatment, and are fully transferrable to germ-free mice upon faecal transplantation of microbiota configurations from NAS-consuming mice, or of microbiota anaerobically incubated in the presence of NAS. We identify NAS-altered microbial metabolic pathways that are linked to host susceptibility to metabolic disease, and demonstrate similar NAS-induced dysbiosis and glucose intolerance in healthy human subjects. Collectively, our results link NAS consumption, dysbiosis and metabolic abnormalities, thereby calling for a reassessment of massive NAS usage.
Assuntos
Trato Gastrointestinal/efeitos dos fármacos , Trato Gastrointestinal/microbiologia , Intolerância à Glucose/induzido quimicamente , Intolerância à Glucose/microbiologia , Microbiota/efeitos dos fármacos , Edulcorantes/efeitos adversos , Animais , Antibacterianos/farmacologia , Aspartame/efeitos adversos , Peso Corporal/efeitos dos fármacos , Dieta Hiperlipídica , Gorduras na Dieta/farmacologia , Fezes/microbiologia , Feminino , Vida Livre de Germes , Glucose/metabolismo , Intolerância à Glucose/metabolismo , Humanos , Masculino , Síndrome Metabólica/induzido quimicamente , Síndrome Metabólica/metabolismo , Síndrome Metabólica/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Sacarina/administração & dosagem , Sacarina/efeitos adversos , Sacarose/efeitos adversos , Sacarose/análogos & derivados , Relação Cintura-QuadrilRESUMO
Biofilms formed by bacteria on plant roots play an important role in maintaining an optimal rhizosphere environment that supports plant growth and fitness. Bacillus subtilis is a potent plant growth promoter, forming biofilms that play a key role in protecting the host from fungal and bacterial infections. In this work, we demonstrate that the development of B. subtilis biofilms is antagonized by specific indole derivatives that accumulate during symbiotic interactions with plant hosts. Indole derivatives are more potent signals when the plant polysaccharide xylan serves as a carbon source, a mechanism to sustain beneficial biofilms at a biomass that can be supported by the plant. Moreover, B. subtilis biofilms formed by mutants resistant to indole derivatives become deleterious to the plants due to their capacity to consume and recycle plant polysaccharides. These results demonstrate how a dynamic metabolite-based dialogue can promote homeostasis between plant hosts and their beneficial biofilm communities.
Assuntos
Bacillus subtilis , Biofilmes , Indóis , Plantas , Bacillus subtilis/fisiologia , Proteínas de Bactérias/genética , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Interações Hospedeiro-Patógeno/fisiologia , Indóis/química , Indóis/farmacologia , Raízes de Plantas/microbiologia , Plantas/microbiologiaRESUMO
Bacteria in nature often reside in differentiated communities termed biofilms. These communities, which are composed of a number of functionally-distinct cell types, are an interesting example of division of labour in microbes, and as such have been used as a system to study the evolution of cooperation. The structured population of the biofilm also plays a critical role in the persistence of infections, and biofouling of medical and industrial devices. Biofilm formation involves several stages of differentiation, which are mediated by extracellular factors secreted by cells composing the biofilm. The developmental model of biofilm formation describes this process mechanistically: specific subpopulations of cells synthesize signals within the biofilm, and promote the differentiation of other subpopulations. The handicap principle suggests that signals function because they provide reliable information regarding the state of the signaller; here, we apply the handicap principle to signalling among cells composing Bacillus subtilis biofilms, emphasizing the perspective of secreted factors as sources of information rather than solely as mediators of development. Such information could facilitate competition among phenotypically-similar cells composing the biofilm, affecting local organizational patterns within defined subpopulations.
Assuntos
Bacillus subtilis/metabolismo , Biofilmes/crescimento & desenvolvimento , Transdução de Sinais/fisiologia , Microbiota/fisiologiaRESUMO
A hallmark of the Gram-positive bacteria, such as the soil-dwelling bacterium Bacillus subtilis, is their cell wall. Here, we report that d-leucine and flavomycin, biofilm inhibitors targeting the cell wall, activate the ß-lactamase PenP. This ß-lactamase contributes to ampicillin resistance in B. subtilis under all conditions tested. In contrast, both Spo0A, a master regulator of nutritional stress, and the general cell wall stress response, differentially contribute to ß-lactam resistance under different conditions. To test whether ß-lactam resistance and ß-lactamase genes are widespread in other Bacilli, we isolated Bacillus species from undisturbed soils, and found that their genomes can encode up to five ß-lactamases with differentiated activity spectra. Surprisingly, the activity of environmental ß-lactamases and PenP, as well as the general stress response, resulted in a similarly reduced lag phase of the culture in the presence of ß-lactam antibiotics, with little or no impact on the logarithmic growth rate. The length of the lag phase may determine the outcome of the competition between ß-lactams and ß-lactamases producers. Overall, our work suggests that antibiotic resistance genes in B. subtilis and related species are ancient and widespread, and could be selected by interspecies competition in undisturbed soils.
Assuntos
Bacillus subtilis/enzimologia , Rizosfera , beta-Lactamases/fisiologia , Bacillus subtilis/fisiologia , Parede Celular/fisiologia , Resistência Microbiana a Medicamentos , Ativação Enzimática , Estresse Fisiológico , Resistência beta-Lactâmica , beta-Lactamases/genética , beta-Lactamas/metabolismoRESUMO
Microbial ecosystems tightly associated with a eukaryotic host are widespread in nature. The genetic and metabolic networks of the eukaryotic hosts and the associated microbes have coevolved to form a symbiotic relationship. Both the Gram-positive Bacillus subtilis and the Gram-negative Serratia plymuthica can form biofilms on plant roots and thus can serve as a model system for the study of interspecies interactions in a host-associated ecosystem. We found that B. subtilis biofilms expand collectively and asymmetrically toward S. plymuthica, while expressing a nonribosomal antibiotic bacillaene and an extracellular protease. As a result, B. subtilis biofilms outcompeted S. plymuthica for successful colonization of the host. Strikingly, the plant host was able to enhance the efficiency of this killing by inducing bacillaene synthesis. In turn, B. subtilis biofilms increased the resistance of the plant host to pathogens. These results provide an example of how plant-bacterium symbiosis promotes the immune response of the plant host and the fitness of the associated bacteria.IMPORTANCE Our study sheds mechanistic light on how multicellular biofilm units compete to successfully colonize a eukaryote host, using B. subtilis microbial communities as our lens. The microbiota and its interactions with its host play various roles in the development and prevention of diseases. Using competing beneficial biofilms that are essential microbiota members on the plant host, we found that B. subtilis biofilms activate collective migration to capture their prey, followed by nonribosomal antibiotic synthesis. Plant hosts increase the efficiency of antibiotic production by B. subtilis biofilms, as they activate the synthesis of polyketides; therefore, our study provides evidence of a mechanism by which the host can indirectly select for beneficial microbiota members.
Assuntos
Antibacterianos/biossíntese , Bacillus subtilis/metabolismo , Bacillus subtilis/genética , Bacillus subtilis/crescimento & desenvolvimento , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biofilmes , Brassicaceae/microbiologia , Ecossistema , Interações Hospedeiro-Patógeno , Raízes de Plantas/microbiologia , Polienos/metabolismo , Serratia/genética , Serratia/crescimento & desenvolvimento , Serratia/fisiologiaRESUMO
The linkage between regulatory elements of transcription, such as promoters, and their protein products is central to gene function. Promoter-protein coevolution is therefore expected, but rarely observed, and the manner by which these two regulatory levels are linked remains largely unknown. We study glutamate dehydrogenase-a hub of carbon and nitrogen metabolism. In Bacillus subtilis, two paralogues exist: GudB is constitutively transcribed whereas RocG is tightly regulated. In their active, oligomeric states, both enzymes show similar enzymatic rates. However, swaps of enzymes and promoters cause severe fitness losses, thus indicating promoter-enzyme coevolution. Characterization of the proteins shows that, compared to RocG, GudB's enzymatic activity is highly dependent on glutamate and pH Promoter-enzyme swaps therefore result in excessive glutamate degradation when expressing a constitutive enzyme under a constitutive promoter, or insufficient activity when both the enzyme and its promoter are tightly regulated. Coevolution of transcriptional and enzymatic regulation therefore underlies paralogue-specific spatio-temporal control, especially under diverse growth conditions.
Assuntos
Bacillus subtilis/enzimologia , Coevolução Biológica , Regulação Bacteriana da Expressão Gênica , Glutamato Desidrogenase/genética , Bacillus subtilis/genética , Bacillus subtilis/crescimento & desenvolvimento , Proteínas de Bactérias/genética , Aptidão Genética , Ácido Glutâmico/metabolismo , Mutação , Regiões Promotoras GenéticasRESUMO
Toxin-antitoxin modules are gene pairs encoding a toxin and its antitoxin, and are found on the chromosomes of many bacteria, including pathogens. Here, we characterize the specific contribution of the TxpA and YqcG toxins in elimination of defective cells from developing Bacillus subtilis biofilms. On nutrient limitation, defective cells accumulated in the biofilm breaking its symmetry. Deletion of the toxins resulted in accumulation of morphologically abnormal cells, and interfered with the proper development of the multicellular community. Dual physiological responses are of significance for TxpA and YqcG activation: nitrogen deprivation enhances the transcription of both TxpA and YqcG toxins, and simultaneously sensitizes the biofilm cells to their activity. Furthermore, we demonstrate that while both toxins when overexpressed affect the morphology of the developing biofilm, the toxin TxpA can act to lyse and dissolve pre-established B. subtilis biofilms.
Assuntos
Antitoxinas/metabolismo , Bacillus subtilis/fisiologia , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/metabolismo , Biofilmes , Antitoxinas/genética , Bacillus subtilis/genética , Bacillus subtilis/crescimento & desenvolvimento , Proteínas de Bactérias/genética , Toxinas Bacterianas/genética , Biofilmes/crescimento & desenvolvimentoRESUMO
In response to chemical communication, bacterial cells often organize themselves into complex multicellular communities that carry out specialized tasks. These communities are frequently referred to as biofilms, which involve collective behavior of different cell types. Like cells of multicellular eukaryotes, the biofilm cells are surrounded by self-produced polymers that constitute the extracellular matrix (ECM), which binds them to each other and to the surface. In multicellular eukaryotes, it has been evident for decades that cell-ECM interactions control multiple cellular processes during development. While cells, both in biofilms and in multicellular eukaryotes, are surrounded by ECM and activate various genetic programs, until recently it has been unclear whether cell-ECM interactions are recruited in bacterial communicative behaviors. In this review, we will describe the examples reported thus far for ECM involvement in control of cell behavior throughout the different stages of biofilm formation. The studies presented in this review provide a newly emerging perspective of the bacterial ECM as an active player in regulation of biofilm development.
RESUMO
Cultivated meat is emerging to replace traditional livestock industries, which have ecological costs, including land and water overuse and considerable carbon emissions. During cultivated meat production, mammalian cells can increase their numbers dramatically through self-renewal/proliferation and transform into mature cells, such as muscle or fat cells, through maturation/differentiation. Here, we address opportunities for introducing probiotic bacteria into the cultivated meat industry, including using them to produce renewable antimicrobials and scaffolding materials. We also offer solutions to challenges, including the growth of bacteria and mammalian cells, the effect of probiotic bacteria on production costs, and the effect of bacteria and their products on texture and taste. Our summary provides a promising framework for applying microbial composites in the cultivated meat industry.
Assuntos
Carne , Probióticos , Animais , Carne in vitro , Bactérias , MamíferosRESUMO
The anti-fungal properties of the probiotic bacterium Bacillus subtilis have been studied extensively in agriculture and ecology, but their applications in the built environment remain to be determined. Our work aims to utilize this biological component to introduce new diverse anti-mold properties into paint. "Mold" refers to the ubiquitous fungal species that generate visible multicellular filaments commonly found in household dust. The development of mold leads to severe health problems for occupants, including allergic response, hypersensitivity pneumonitis, and asthma, which have significant economic and clinical outcomes. We here demonstrate the robust effect of a commercial paint enhanced with Bacillus subtilis cells against the common mold agent, Aspergillus niger, and identify three biosynthetic clusters essential for this effect. Our results lay the foundation for bio-convergence and synthetic biology approaches to introduce renewable and environmentally friendly bio-anti-fungal agents into the built environment.
RESUMO
In their natural habitats, organisms encounter numerous external stimuli and must be able to sense and adapt to those stimuli to survive. Unlike mutations, epigenetic changes do not alter the underlying DNA sequence. Instead, they create modifications that promote or silence gene expression. Bacillus subtilis has long been a model organism in studying genetics and development. It is beneficial for numerous biotechnological applications where it is included as a probiotic, in fermentation, or in bio-concrete design. This bacterium has also emerged recently as a model organism for studying bacterial epigenetic adaptation. In this review, we examine the evolving knowledge of epigenetic regulation (restriction-modification systems (RM), orphan methyltransferases, and chromosome condensation) in B. subtilis and related bacteria, and utilize it as a case study to test their potential roles and future applications in genetic engineering and microbial biotechnology. Finally, we suggest how the implementation of these fundamental findings promotes the design of synthetic epigenetic memory circuits and their future applications in agriculture, medicine, and biotechnology.
Assuntos
Bacillus subtilis , Biotecnologia , Epigênese Genética , Probióticos , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Agricultura/métodos , Engenharia Genética/métodos , Bactérias/genética , Bactérias/metabolismoRESUMO
Bacillus subtilis is a beneficial bacterium that supports plant growth and protects plants from bacterial, fungal, and viral infections. Using a simplified system of B. subtilis and Arabidopsis thaliana interactions, we studied the fitness and transcriptome of bacteria detached from the root over generations of growth in LB medium. We found that bacteria previously associated with the root or exposed to its secretions had greater stress tolerance and were more competitive in root colonization than bacteria not previously exposed to the root. Furthermore, our transcriptome results provide evidence that plant secretions induce a microbial stress response and fundamentally alter signaling by the cyclic nucleotide c-di-AMP, a signature maintained by their descendants. The changes in cellular physiology due to exposure to plant exudates were multigenerational, as they allowed not only the bacterial cells that colonized a new plant but also their descendants to have an advance over naive competitors of the same species, while the overall plasticity of gene expression and rapid adaptation were maintained. These changes were hereditary but not permanent. Our work demonstrates a bacterial memory manifested by multigenerational reversible adaptation to plant hosts in the form of activation of the stressosome, which confers an advantage to symbiotic bacteria during competition.
Assuntos
Arabidopsis , Bacillus subtilis , Raízes de Plantas , Simbiose , Bacillus subtilis/genética , Bacillus subtilis/fisiologia , Arabidopsis/microbiologia , Arabidopsis/genética , Raízes de Plantas/microbiologia , Transcriptoma , Estresse Fisiológico , Regulação Bacteriana da Expressão Gênica , Adaptação Fisiológica/genéticaRESUMO
Biofilms are often associated with human bacterial infections, and the natural tolerance of biofilms to antibiotics challenges treatment. Compounds with antibiofilm activity could become useful adjuncts to antibiotic therapy. We used norspermidine, a natural trigger for biofilm disassembly in the developmental cycle of Bacillus subtilis , to develop guanidine and biguanide compounds with up to 20-fold increased potency in preventing biofilm formation and breaking down existing biofilms. These compounds also were active against pathogenic Staphylococcus aureus . An integrated approach involving structure-activity relationships, protonation constants, and crystal structure data on a focused synthetic library revealed that precise spacing of positively charged groups and the total charge at physiological pH distinguish potent biofilm inhibitors.