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Spore-forming probiotic bacteria offer interesting properties as they have an intrinsic high stability, and when consumed, they are able to survive the adverse conditions encountered during the transit thorough the host gastrointestinal (GI) tract. A traditional healthy food, natto, exists in Japan consisting of soy fermented by the spore-forming bacterium Bacillus subtilis natto. The consumption of natto is linked to many beneficial health effects, including the prevention of high blood pressure, osteoporosis, and cardiovascular-associated disease. We hypothesize that the bacterium B. subtilis natto plays a key role in the beneficial effects of natto for humans. Here, we present the isolation of B. subtilis DG101 from natto and its characterization as a novel spore-forming probiotic strain for human consumption. B. subtilis DG101 was non-hemolytic and showed high tolerance to lysozyme, low pH, bile salts, and a strong adherence ability to extracellular matrix proteins (i.e., fibronectin and collagen), demonstrating its potential application for competitive exclusion of pathogens. B. subtilis DG101 forms robust liquid and solid biofilms and expresses several extracellular enzymes with activity against food diet-associated macromolecules (i.e., proteins, lipids, and polysaccharides) that would be important to improve food diet digestion by the host. B. subtilis DG101 was able to grow in the presence of toxic metals (i.e., chromium, cadmium, and arsenic) and decreased their bioavailability, a feature that points to this probiotic as an interesting agent for bioremediation in cases of food and water poisoning with metals. In addition, B. subtilis DG101 was sensitive to antibiotics commonly used to treat infections in medical settings, and at the same time, it showed a potent antimicrobial effect against pathogenic bacteria and fungi. In mammalians (i.e., rats), B. subtilis DG101 colonized the GI tract, and improved the lipid and protein serum homeostasis of animals fed on the base of a normal- or a deficient-diet regime (dietary restriction). In the animal model for longevity studies, Caenorhabditis elegans, B. subtilis DG101 significantly increased the animal lifespan and prevented its age-related behavioral decay. Overall, these results demonstrate that B. subtilis DG101 is the key component of natto with interesting probiotic properties to improve and protect human health.
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A basic function of the nervous system is to confer the ability to detect external stimuli and generate appropriate behavioral and physiological responses. These can be modulated when parallel streams of information are provided to the nervous system and neural activity is appropriately altered. The nematode Caenorhabditis elegans utilizes a simple and well characterized neural circuit to mediate avoidance or attraction responses to stimuli, such as the volatile odorant octanol or diacetyl (DA), respectively. Aging and neurodegeneration constitute two important factors altering the ability to detect external signals and, therefore, changing behavior. Here, we present a modified protocol to assess avoidance or attraction responses to diverse stimuli in healthy and worm models associated with neurodegenerative diseases.
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Aging and neuronal deterioration constitute important risk factors for the development of neuronal-related diseases, such as different dementia. The nematode Caenorhabditis elegans has emerged as a popular model system for studying neurodegeneration diseases, due to its complete neuronal connectivity map. DiI is a red ï¬uorescent dye that can ï¬ll the worm amphid neurons and enables the visualization of their neurodegeneration over time. This protocol provides an efficient, fast, and safe method to stain worm amphid neurons to highlight the chemosensory structures of live nematodes.
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Alternative sigma factors have led the core RNA polymerase (RNAP) to recognize different sets of promoters to those recognized by the housekeeping sigma A-directed RNAP. This change in RNAP promoter selectivity allows a rapid and flexible reformulation of the genetic program to face environmental and metabolic stimuli that could compromise bacterial fitness. The model bacterium Bacillus subtilis constitutes a matchless living system in the study of the role of alternative sigma factors in gene regulation and physiology. SigB from B. subtilis was the first alternative sigma factor described in bacteria. Studies of SigB during the last 40 years have shown that it controls a genetic universe of more than 150 genes playing crucial roles in stress response, adaption, and survival. Activation of SigB relies on three separate pathways that specifically respond to energy, environmental, and low temperature stresses. SigB homologs, present in other Gram-positive bacteria, also play important roles in virulence against mammals. Interestingly, during recent years, other unexpected B. subtilis responses were found to be controlled by SigB. In particular, SigB controls the efficiencies of spore and biofilm formation, two important features that play critical roles in adaptation and survival in planktonic and sessile B. subtilis communities. In B. subtilis, SigB induces the expression of the Spo0E aspartyl-phosphatase, which is responsible for the blockage of sporulation initiation. The upregulated activity of Spo0E connects the two predominant adaptive pathways (i.e., sporulation and stress response) present in B. subtilis. In addition, the RsbP serine-phosphatase, belonging to the energy stress arm of the SigB regulatory cascade, controls the expression of the key transcription factor SinR to decide whether cells residing in the biofilm remain in and maintain biofilm growth or scape to colonize new niches through biofilm dispersal. SigB also intervenes in the recognition of and response to surrounding microorganisms, a new SigB role that could have an agronomic impact. SigB is induced when B. subtilis is confronted with phytopathogenic fungi (e.g., Fusarium verticillioides) and halts fungal growth to the benefit of plant growth. In this article, we update and review literature on the different regulatory networks that control the activation of SigB and the new roles that have been described the recent years.
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Multiple causes, apart from genetic inheritance, predispose to the production and aggregation of amyloid-ß (Aß) peptide and Alzheimer's disease (AD) development in the older population. There is currently no therapy or medicine to prevent or delay AD progression. One novel strategy against AD might involve the use of psychobiotics, probiotic gut bacteria with specific mental health benefits. Here, we report the neuronal and behavioral protective effects of the probiotic bacterium Bacillus subtilis in a Caenorhabditis elegans AD model. Aging and neuronal deterioration constitute important risk factors for AD development, and we showed that B. subtilis significantly delayed both detrimental processes in the wild-type C. elegans strain N2 compared with N2 worms colonized by the non-probiotic Escherichia coli OP50 strain. Importantly, B. subtilis alleviated the AD-related paralysis phenotype of the transgenic C. elegans strains CL2120 and GMC101 that express, in body wall muscle cells, the toxic peptides Aß3-42 and Aß1-42, respectively. B. subtilis-colonized CL2355 worms were protected from the behavioral deficits (e.g., poor chemotactic response and decreased body bends) produced by pan-neuronal Aß1-42 expression. Notably, B. subtilis restored the lifespan level of C. elegans strains that express Aß to values similar to the life expectancy of the wild-type strain N2 fed on E. coli OP50 cells. The B. subtilis proficiencies in quorum-sensing peptide (i.e., the Competence Sporulation Factor, CSF) synthesis and gut-associated biofilm formation (related to the anti-aging effect of the probiotic) play a crucial role in the anti-AD effects of B. subtilis. These novel results are discussed in the context of how B. subtilis might exert its beneficial effects from the gut to the brain of people with or at risk of developing AD.
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Doença de Alzheimer/tratamento farmacológico , Bacillus subtilis , Comportamento Animal/efeitos dos fármacos , Caenorhabditis elegans/efeitos dos fármacos , Longevidade/efeitos dos fármacos , Degeneração Neural/tratamento farmacológico , Probióticos/uso terapêutico , Envelhecimento/efeitos dos fármacos , Envelhecimento/genética , Envelhecimento/metabolismo , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/genética , Peptídeos beta-Amiloides/metabolismo , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Quimiotaxia/efeitos dos fármacos , Modelos Animais de Doenças , Progressão da Doença , Degeneração Neural/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Probióticos/farmacologiaRESUMO
Surface-associate motility on biotic and abiotic environments is a key mechanism used by the model bacterium Bacillus subtilis and its closest relatives (i.e., B. amyloliquefaciens, B. thuringiensis, B. cereus, B. pumilus) for surface colonization and spreading across surfaces. The study of this mechanism in a research, industrial or clinic laboratory is essential; however, precautions should be taken for the reproducibility of the results, for example, the procedure to inoculate the bacteria on the testing plate, the humidity of the plate and the agar concentration. In this protocol, we describe, using Bacillus subtilis, how to perform these assays and, in addition, we show how by varying the agar concentration in the plate, you can make a first approximation of what type of motility has other bacterial species.
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Bacterial adherence to extracellular matrix proteins (ECMp) plays important roles during host-pathogen interaction, however its genetic regulation remains poorly understood. yloA of the model bacterium Bacillus subtilis shows high homology to genes encoding fibronectin-binding proteins of Gram-positive pathogens. Here, we characterized the regulatory network of YloA-dependent adhesive properties of the probiotic B. subtilis natto (Bsn). YloA-proficient, but not YloA-deficient, Bsn specifically bound to ECMp in a concentration-dependent manner and were proficient in biofilm formation. yloA expression showed a continuous increase in activity during the growth phase and decreased during the stationary phase. The transcription factors AbrB and DegU downregulated yloA expression during the logarithmic and stationary growth phases respectively. Analysis of the yloA promoter region revealed the presence of AT-rich direct and inverted repeats previously reported to function as DegU-recognized binding sites. In spo0A cells, yloA expression was completely turned off because of upregulation of AbrB throughout growth. Accordingly, DNase I footprinting analysis confirmed that AbrB bound to the promoter region of yloA. Interestingly, Bsn bound fibronectin with higher affinity, lower Kd, than several bacterial pathogens and competitively excluded them from binding to immobilized-fibronectin, a finding that might be important for the anti-infective properties of B. subtilis and its relatives.
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Bacillus subtilis/metabolismo , Proteínas de Transporte/metabolismo , Matriz Extracelular/metabolismo , Fibronectinas/metabolismo , Bacillus subtilis/citologia , Bacillus subtilis/genética , Aderência Bacteriana/fisiologia , Proteínas de Bactérias/metabolismo , Sequência de Bases , Proteínas de Ligação a DNA/metabolismo , Matriz Extracelular/microbiologia , Proteínas da Matriz Extracelular/metabolismo , Regulação Bacteriana da Expressão Gênica , Interações Hospedeiro-Patógeno , Humanos , Filogenia , Regiões Promotoras Genéticas , Elementos Reguladores de Transcrição , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
Beneficial bacteria have been shown to affect host longevity, but the molecular mechanisms mediating such effects remain largely unclear. Here we show that formation of Bacillus subtilis biofilms increases Caenorhabditis elegans lifespan. Biofilm-proficient B. subtilis colonizes the C. elegans gut and extends worm lifespan more than biofilm-deficient isogenic strains. Two molecules produced by B. subtilis - the quorum-sensing pentapeptide CSF and nitric oxide (NO) - are sufficient to extend C. elegans longevity. When B. subtilis is cultured under biofilm-supporting conditions, the synthesis of NO and CSF is increased in comparison with their production under planktonic growth conditions. We further show that the prolongevity effect of B. subtilis biofilms depends on the DAF-2/DAF-16/HSF-1 signalling axis and the downregulation of the insulin-like signalling (ILS) pathway.
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Bacillus subtilis/fisiologia , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/fisiologia , Longevidade/fisiologia , Transdução de Sinais/fisiologia , Animais , Biofilmes , Regulação para Baixo , Comportamento Alimentar/fisiologia , Fatores de Transcrição Forkhead/metabolismo , Insulina/metabolismo , Óxido Nítrico/metabolismo , Comportamento Predatório/fisiologia , Receptor de Insulina/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Determining an accurate count of intestinal bacteria from Caenorhabditis elegans is one critical way to assess colonization proficiency by a given bacteria. This can be accomplished by culturing appropriate dilutions of worm gut bacteria on selective or differential agarized media. Because of the high concentration of bacteria in gut worm, dilution is necessary before plating onto growth media. Serial dilutions can reduce the concentration of the original intestinal sample to levels low enough for single colonies to be grown on media plates, allowing for the calculation of the initial counts of bacteria in the intestinal sample.
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The cell-to-cell communication and division of labour that occurs inside a beneficial biofilm produce significant differences in gene expression compared with the gene expression pattern of cells grew under planktonic conditions. In this sense, the levels of NO (nitric oxide) and CSF (Competence Sporulation Stimulating Factor) produced in Bacillus subtilis cultures have been measured only under planktonic growth conditions. We sought to determine whether NO and/or CSF production is affected in B. subtilis cells that develop as a biofilm. To measure the production levels of the two prolongevity molecules, we grew B. subtilis cells under planktonic and biofilm supporting condition.
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Here we describe the development of novel nanostructured coating systems with improved photocatalytic and antibacterial activities. These systems comprise a layer of SiO2 followed by a layer of mesoporous or dense TiO2-anatase, and doping with silver nanoparticles (Ag NPs). The coatings were synthesized via a sol-gel technique by combining colloidal Ag NPs with TiO2 and SiO2 sols. The photocatalytic activity was studied through methyl orange decomposition under UV light. Results showed a great increase of photocatalytic activity by Ag NPs doping. The most active photocatalyst corresponded to the Ag-SiO2/TiO2 mesoporous system, associated with the porosity of the coatings and with the decrease of e-h recombination for the presence of Ag NPs. All the TiO2 coatings showed a strong bactericidal activity against planktonic forms of Gram-negative (enterohemorrhagic Escherichia coli) and Gram-positive (Listeria monocytogenes) pathogens, as well as a strong germicidal effect against deadly spores of human gas gangrene- and anthrax-producing bacteria (Clostridium perfringens and Bacillus anthracis, respectively). The bactericidal and sporocidal activity was improved by doping the coatings with Ag NPs, even more when nanoparticles were in the outer layer of TiO2, because they are more accessible to the environment. The mechanisms responsible for the increase of photocatalytic and bactericidal behaviors related to Ag NP doping were studied by spectroscopic ellipsometry, UV-vis spectroscopy, photoluminescence and anodic stripping voltammetry. It was found that the separation of the electron-hole pair contributed to the enhancement of photocatalysis, whereas the effect of the local electric field reinforcement was probably present. A possible involvement of a decrease of band-gap energy and dispersion by silver nanoparticles is ruled out. bactericidal efficacy was increased by Ag(+) ion release. Overall, the results included in this article show that the architecture of the films may tune photocatalytic and bactericidal properties.
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Materiais Revestidos Biocompatíveis , Nanopartículas Metálicas , Processos Fotoquímicos , Prata/química , Titânio/química , Catálise , Microscopia Eletrônica de TransmissãoRESUMO
During sporulation in Bacillus subtilis, the committed-cell undergoes substantial membrane rearrangements to generate two cells of different sizes and fates: the mother cell and the forespore. Here, we demonstrate that the master transcription factor Spo0A reactivates lipid synthesis during development. Maximal Spo0A-dependent lipid synthesis occurs during the key stages of asymmetric division and forespore engulfment. Spo0A reactivates the accDA operon that encodes the carboxylase component of the acetyl-CoA carboxylase enzyme, which catalyses the first and rate-limiting step in de novo lipid biosynthesis, malonyl-CoA formation. The disruption of the Spo0A-binding box in the promoter region of accDA impairs its transcriptional reactivation and blocks lipid synthesis. The Spo0A-insensitive accDA(0A) cells were proficient in planktonic growth but defective in sporulation (σ(E) activation) and biofilm development (cell cluster formation and water repellency). Exogenous fatty acid supplementation to accDA(0A) cells overcomes their inability to synthesize lipids during development and restores sporulation and biofilm proficiencies. The transient exclusion of the lipid synthesis regulon from the forespore and the known compartmentalization of Spo0A and ACP in the mother cell suggest that de novo lipid synthesis is confined to the mother cell. The significance of the Spo0A-controlled de novo lipid synthesis during B. subtilis development is discussed.
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Bacillus subtilis/crescimento & desenvolvimento , Bacillus subtilis/metabolismo , Proteínas de Bactérias/metabolismo , Biofilmes/crescimento & desenvolvimento , Ácidos Graxos/biossíntese , Esporos Bacterianos/crescimento & desenvolvimento , Esporos Bacterianos/metabolismo , Fatores de Transcrição/metabolismo , Regulação Bacteriana da Expressão GênicaRESUMO
Clostridium perfringens is an anaerobic, gram-positive, spore-forming bacterium responsible for the production of severe histotoxic and gastrointestinal diseases in humans and animals. In silico analysis of the three available genome-sequenced C. perfringens strains (13, SM101, and ATCC13124) revealed that genes that encode flagellar proteins and genes involved in chemotaxis are absent. However, those strains exhibit type IV pilus (TFP)-dependent gliding motility. Since carbon catabolite regulation has been implicated in the control of different bacterial behaviors, we investigated the effects of glucose and other readily metabolized carbohydrates on C. perfringens gliding motility. Our results demonstrate that carbon catabolite regulation constitutes an important physiological regulatory mechanism that reduces the proficiencies of the gliding motilities of a large number of unrelated human- and animal-derived pathogenic C. perfringens strains. Glucose produces a strong dose-dependent inhibition of gliding development without affecting vegetative growth. Maximum gliding inhibition was observed at a glucose concentration (1%) previously reported to also inhibit other important behaviors in C. perfringens, such as spore development. The inhibition of gliding development in the presence of glucose was due, at least in part, to the repression of the genes pilT and pilD, whose products are essential for TFP-dependent gliding proficiency. The inhibitory effects of glucose on pilT and pilD expression were under the control of the key regulatory protein CcpA (catabolite control protein A). The deficiency in CcpA activity of a ccpA knockout C. perfringens mutant strain restored the expressions of pilT and pilD and gliding proficiency in the presence of 1% glucose. The carbon catabolite repression of the gliding motility of the ccpA mutant strain was restored after the introduction of a complementing plasmid harboring a wild-type copy of ccpA. These results point to a central role for CcpA in orchestrating the negative effect of carbon catabolite regulation on C. perfringens gliding motility. Furthermore, we discovered a novel positive role for CcpA in pilT and pilD expression and gliding proficiency in the absence of catabolite regulation. Carbon catabolite repression of gliding motility and the dual role of CcpA, either as repressor or as activator of gliding, are analyzed in the context of the different social behaviors and diseases produced by C. perfringens.
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Clostridium perfringens/fisiologia , Clostridium perfringens/patogenicidade , Fímbrias Bacterianas/fisiologia , Anaerobiose , Animais , Proteínas de Bactérias/genética , Quimiotaxia/genética , Quimiotaxia/fisiologia , Clonagem Molecular , Clostridium perfringens/efeitos dos fármacos , Clostridium perfringens/genética , Primers do DNA , Proteínas de Ligação a DNA/genética , Deleção de Genes , Teste de Complementação Genética , Glucose/farmacologia , Glucuronidase/genética , Glucuronidase/metabolismo , Humanos , Plasmídeos , Proteínas Repressoras/genéticaRESUMO
Cell-to-cell communication in bacteria is mediated by quorum-sensing systems (QSS) that produce chemical signal molecules called autoinducers (AI). In particular, LuxS/AI-2-dependent QSS has been proposed to act as a universal lexicon that mediates intra- and interspecific bacterial behavior. Here we report that the model organism Bacillus subtilis operates a luxS-dependent QSS that regulates its morphogenesis and social behavior. We demonstrated that B. subtilis luxS is a growth-phase-regulated gene that produces active AI-2 able to mediate the interspecific activation of light production in Vibrio harveyi. We demonstrated that in B. subtilis, luxS expression was under the control of a novel AI-2-dependent negative regulatory feedback loop that indicated an important role for AI-2 as a signaling molecule. Even though luxS did not affect spore development, AI-2 production was negatively regulated by the master regulatory proteins of pluricellular behavior, SinR and Spo0A. Interestingly, wild B. subtilis cells, from the undomesticated and probiotic B. subtilis natto strain, required the LuxS-dependent QSS to form robust and differentiated biofilms and also to swarm on solid surfaces. Furthermore, LuxS activity was required for the formation of sophisticated aerial colonies that behaved as giant fruiting bodies where AI-2 production and spore morphogenesis were spatially regulated at different sites of the developing colony. We proposed that LuxS/AI-2 constitutes a novel form of quorum-sensing regulation where AI-2 behaves as a morphogen-like molecule that coordinates the social and pluricellular behavior of B. subtilis.
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Bacillus subtilis/citologia , Bacillus subtilis/fisiologia , Proteínas de Bactérias/fisiologia , Bacillus subtilis/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biofilmes/crescimento & desenvolvimento , Liases de Carbono-Enxofre , Proteínas de Ligação a DNA/genética , Regulação Bacteriana da Expressão Gênica , Homosserina/análogos & derivados , Homosserina/genética , Homosserina/metabolismo , Lactonas/metabolismo , Locomoção , Substâncias Luminescentes/metabolismo , Transdução de Sinais , Fatores de Transcrição/genéticaRESUMO
Clostridium perfringens enterotoxin (CPE) is an important virulence factor for food poisoning and non-food borne gastrointestinal (GI) diseases. Although CPE production is strongly regulated by sporulation, the nature of the signal(s) triggering sporulation remains unknown. Here, we demonstrated that inorganic phosphate (Pi), and not pH, constitutes an environmental signal inducing sporulation and CPE synthesis. In the absence of Pi-supplementation, C. perfringens displayed a spo0A phenotype, i.e., absence of polar septation and DNA partitioning in cells that reached the stationary phase of growth. These results received support from our Northern blot analyses which demonstrated that Pi was able to counteract the inhibitory effect of glucose at the onset of sporulation and induced spo0A expression, indicating that Pi acts as a key signal triggering spore morphogenesis. In addition to being the first study reporting the nature of a physiological signal triggering sporulation in clostridia, these findings have relevance for the development of antisporulation drugs to prevent or treat CPE-mediated GI diseases in humans.
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Clostridium perfringens/efeitos dos fármacos , Enterotoxinas/biossíntese , Fosfatos/farmacologia , Clostridium perfringens/fisiologia , Meios de Cultura , Concentração de Íons de Hidrogênio , Morfogênese/efeitos dos fármacos , Esporos Bacterianos/efeitos dos fármacos , Esporos Bacterianos/crescimento & desenvolvimentoRESUMO
Even though there is a large body of information concerning the harmful effects of alcohol on different organisms, the mechanism(s) that affects developmental programs, at a single-cell level, has not been clearly identified. In this respect, the spore-forming bacterium Bacillus subtilis constitutes an excellent model to study universal questions of cell fate, cell differentiation, and morphogenesis. Here, we demonstrate that treatment with subinhibitory concentrations of alcohol that did not affect vegetative growth inhibited the initiation of spore development through a selective blockage of key developmental genes under the control of the master transcription factor Spo0A approximately P. Isopropyl-beta-D-thiogalactopyranoside-directed expression of a phosphorylation-independent form of Spo0A (Sad67) and the use of an in vivo mini-Tn10 insertional library permitted the identification of the developmental SinR repressor and RapA phosphatase as the effectors that mediated the inhibitory effect of alcohol on spore morphogenesis. A double rapA sinR mutant strain was completely resistant to the inhibitory effects of different-C-length alcohols on sporulation, indicating that the two cell fate determinants were the main or unique regulators responsible for the spo0 phenotype of wild-type cells in the presence of alcohol. Furthermore, treatment with alcohol produced a significant induction of rapA and sinR, while the stationary-phase induction of sinI, which codes for a SinR inhibitor, was completely turned off by alcohol. As a result, a dramatic repression of spo0A and the genes under its control occurred soon after alcohol addition, inhibiting the onset of sporulation and permitting the evaluation of alternative pathways required for cellular survival.
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Bacillus subtilis/fisiologia , Proteínas de Bactérias/fisiologia , Etanol/farmacologia , Regulação Bacteriana da Expressão Gênica , Fosfoproteínas Fosfatases/fisiologia , Esporos Bacterianos/fisiologia , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Esporos Bacterianos/efeitos dos fármacos , Esporos Bacterianos/crescimento & desenvolvimento , Fatores de TranscriçãoRESUMO
Spore development and stress resistance in Bacillus subtilis are governed by the master transcription factors Spo0A and sigma(B), respectively. Here we show that the coding genes for both regulatory proteins are dramatically induced, during logarithmic growth, after a temperature downshift from 37 to 20 degrees C. The loss of sigma(B) reduces the stationary-phase viability of cold-adapted cells 10- to 50-fold. Furthermore, we show that sigma(B) activity is required at a late stage of development for efficient sporulation at a low temperature. On the other hand, Spo0A loss dramatically reduces the stationary-phase viability of cold-adapted cells 10,000-fold. We show that the requirement of Spo0A for cellular survival during the cold is independent of the activity of the key transition state regulator AbrB and of the simple loss of sporulation ability. Furthermore, Spo0A, and not proficiency in sporulation, is required for the development of complete stress resistance of cold-adapted cells to heat shock (54 degrees C, 1 h), since a loss of Spo0A, but not a loss of the essential sporulation transcription factor sigma(F), reduced the cellular survival in response to heat by more than 1,000-fold. The overall results argue for new and important roles for Spo0A in the development of full stress resistance by nonsporulating cells and for sigma(B) in sporulation proficiency at a low temperature.
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Bacillus subtilis/fisiologia , Proteínas de Bactérias/fisiologia , Fator sigma/fisiologia , Fatores de Transcrição/fisiologia , Temperatura Baixa , Esporos Bacterianos/fisiologiaRESUMO
Compartmentalized gene expression during sporulation is initiated after asymmetric division by cell-specific activation of the transcription factors sigmaF and sigmaE. Synthesis of these sigma factors, and their regulatory proteins, requires the activation (phosphorylation) of Spo0A by the phosphorelay signalling system. We report here a novel regulatory function of the anti-anti-sigmaF SpoIIAA as inhibitor of Spo0A activation. This effect did not require sigmaF activity, and it was abolished by expression of the phosphorelay-independent form Spo0A-Sad67 indicating that SpoIIAA directly interfered with Spo0A approximately P generation. IPTG-directed synthesis of the SpoIIE phosphatase in a strain carrying a multicopy plasmid coding for SpoIIAA and its specific inhibitory kinase SpoIIAB blocked Spo0A activation suggesting that the active form of the inhibitor was SpoIIAA and not SpoIIAA-P. Furthermore, expression of the non-phosphorylatable mutant SpoIIAAS58A (SpoIIAA-like), but not SpoIIAAS58D (SpoIIAA-P-like), completely blocked Spo0A-dependent gene expression. Importantly, SpoIIAA expressed from the chromosome under the control of its normal spoIIA promoter showed the same negative effect regulated not only by SpoIIAB and SpoIIE but also by septum morphogenesis. These findings are discussed in relation to the potential contribution of this novel inhibitory feedback with the proper activation of sigmaF and sigmaE during development.
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Bacillus subtilis/fisiologia , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/fisiologia , Fatores de Transcrição/metabolismo , Substituição de Aminoácidos , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Compartimento Celular , Retroalimentação Fisiológica , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Genes Reporter , Isopropiltiogalactosídeo/farmacologia , Óperon Lac , Modelos Genéticos , Mutação de Sentido Incorreto , Fosforilação , Regiões Promotoras Genéticas/efeitos dos fármacos , Regiões Promotoras Genéticas/genética , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes de Fusão/fisiologia , Fator sigma/metabolismo , Esporos Bacterianos , Transcrição GênicaRESUMO
The influence of sub-MIC of ciprofloxacin on the surface properties of 25 non-P mannose-resistant uropathogenic Escherichia coli (UPEC) strains was studied. Thirteen isolates responded to antibiotic treatment with an increase in haemagglutination titre and/or surface hydrophobicity, which correlated with a higher expression of surface proteins. Only UPEC strains with ciprofloxacin-enhanced hydrophobicity increased their adhesiveness to urinary catheters that correlated, in one analysed case, with a dramatic increase in the number of fimbriae peripherally located. The overall results indicate that sub-MICs of ciprofloxacin could increase the adhesiveness, and hence the risk of colonization by UPEC strains expressing mannose-resistant adhesins different from type P.