RESUMO
Oxygen availability is the major determinant of the metabolic modes adopted by Escherichia coli. Although much is known about E. coli gene expression and metabolism under fully aerobic and anaerobic conditions, the intermediate oxygen tensions that are encountered in natural niches are understudied. Here, for the first time, the transcript profiles of E. coli K-12 across the physiologically significant range of oxygen availabilities are described. These suggested a progressive switch to aerobic respiratory metabolism and a remodeling of the cell envelope as oxygen availability increased. The transcriptional responses were consistent with changes in the abundance of cytochrome bd and bo' and the outer membrane protein OmpW. The observed transcript and protein profiles result from changes in the activities of regulators that respond to oxygen itself or to metabolic and environmental signals that are sensitive to oxygen availability (aerobiosis). A probabilistic model (TFInfer) was used to predict the activity of the indirect oxygen-sensing two-component system ArcBA across the aerobiosis range. The model implied that the activity of the regulator ArcA correlated with aerobiosis but not with the redox state of the ubiquinone pool, challenging the idea that ArcA activity is inhibited by oxidized ubiquinone. The amount of phosphorylated ArcA correlated with the predicted ArcA activities and with aerobiosis, suggesting that fermentation product-mediated inhibition of ArcB phosphatase activity is the dominant mechanism for regulating ArcA activity under the conditions used here.
Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Escherichia coli K12/metabolismo , Proteínas de Escherichia coli/metabolismo , Modelos Biológicos , Oxigênio/metabolismo , Proteínas Repressoras/metabolismo , Transcrição Gênica/fisiologia , Aerobiose/fisiologia , Anaerobiose/fisiologia , Proteínas da Membrana Bacteriana Externa/genética , Grupo dos Citocromos b/genética , Grupo dos Citocromos b/metabolismo , Citocromos/genética , Citocromos/metabolismo , Complexo de Proteínas da Cadeia de Transporte de Elétrons/genética , Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Escherichia coli K12/genética , Proteínas de Escherichia coli/genética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Oxirredutases/genética , Oxirredutases/metabolismo , Fosforilação/fisiologia , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Proteínas Repressoras/genética , Ubiquinona/genética , Ubiquinona/metabolismoRESUMO
Bacterial spore formers are prime organisms of concern in the food industry. Spores from the genus Bacillus are extremely stress resistant, most notably exemplified by high thermotolerance. This sometimes allows surviving spores to germinate and grow out to vegetative cells causing food spoilage and possible intoxication. Similar issues though more pending toward spore toxigenicity are observed for the anaerobic Clostridia. The paper indicates the nature of stress resistance and highlights contemporary molecular approaches to analyze the mechanistic basis of it in Bacilli. A molecular comparison between a laboratory strain and a food borne isolate, very similar at the genomic level to the laboratory strain but generating extremely heat resistant spores, is discussed. The approaches cover genome-wide genotyping, proteomics and genome-wide expression analyses studies. The analyses aim at gathering sufficient molecular information to be able to put together an initial framework for dynamic modelling of spore germination and outgrowth behaviour. Such emerging models should be developed both at the population and at the single spore level. Tools and challenges in achieving the latter are succinctly discussed.
Assuntos
Adaptação Fisiológica/fisiologia , Bacillus/fisiologia , Microbiologia de Alimentos , Temperatura Alta , Adaptação Fisiológica/genética , Bacillus/genética , Bacillus subtilis/fisiologia , Qualidade de Produtos para o Consumidor , Contaminação de Alimentos/prevenção & controle , Genômica , Genótipo , Humanos , Proteômica , Esporos Bacterianos/crescimento & desenvolvimentoRESUMO
The 'Omics' revolution has brought a wealth of new mechanistic insights in many fields of biology. It offers options to base predictions of microbial behaviour on mechanistic insight. As the cellular mechanisms involved often turn out to be highly intertwined it is crucial that model development aims at identifying the level of complexity that is relevant to work at. For the prediction of microbiologically stable foods insight in the behaviour of bacterial spore formers is crucial. Their chances of germination and likelihood of outgrowth are major food stability indicators, as well as the transition from outgrowth to first cell division and vegetative growth. Current available technology to assess these parameters in a time-resolved manner at the single spore level will be discussed. Tools to study molecular processes operative in heat induced damage will be highlighted.
Assuntos
Bacillus subtilis/crescimento & desenvolvimento , Microbiologia de Alimentos/métodos , Modelos Biológicos , Bacillus subtilis/ultraestrutura , Temperatura Alta , Microscopia de Contraste de Fase , Esporos Bacterianos/crescimento & desenvolvimento , Esporos Bacterianos/ultraestruturaRESUMO
The weak organic acid sorbic acid is a commonly used food preservative, as it inhibits the growth of bacteria, yeasts, and molds. We have used genome-wide transcriptional profiling of Bacillus subtilis cells during mild sorbic acid stress to reveal the growth-inhibitory activity of this preservative and to identify potential resistance mechanisms. Our analysis demonstrated that sorbic acid-stressed cells induce responses normally seen upon nutrient limitation. This is indicated by the strong derepression of the CcpA, CodY, and Fur regulon and the induction of tricarboxylic acid cycle genes, SigL- and SigH-mediated genes, and the stringent response. Intriguingly, these conditions did not lead to the activation of sporulation, competence, or the general stress response. The fatty acid biosynthesis (fab) genes and BkdR-regulated genes are upregulated, which may indicate plasma membrane remodeling. This was further supported by the reduced sensitivity toward the fab inhibitor cerulenin upon sorbic acid stress. We are the first to present a comprehensive analysis of the transcriptional response of B. subtilis to sorbic acid stress.
Assuntos
Bacillus subtilis/genética , Perfilação da Expressão Gênica/métodos , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Ácido Sórbico/farmacologia , Ânions/metabolismo , Bacillus subtilis/crescimento & desenvolvimento , Proteínas de Bactérias/genética , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Cerulenina/farmacologia , Concentração de Íons de Hidrogênio , Análise de Sequência com Séries de Oligonucleotídeos , Regulon/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Spore-forming bacteria are a special problem for the food industry as some of them are able to survive preservation processes. Bacillus spp. spores can remain in a dormant, stress resistant state for a long period of time. Vegetative cells are formed by germination of spores followed by a more extended outgrowth phase. Spore germination and outgrowth progression are often very heterogeneous and therefore, predictions of microbial stability of food products are exceedingly difficult. Mechanistic details of the cause of this heterogeneity are necessary. In order to examine spore heterogeneity we made a novel closed air-containing chamber for live imaging. This chamber was used to analyze Bacillus subtilis spore germination, outgrowth, as well as subsequent vegetative growth. Typically, we examined around 90 starting spores/cells for ≥4 hours per experiment. Image analysis with the purposely built program "SporeTracker" allows for automated data processing from germination to outgrowth and vegetative doubling. In order to check the efficiency of the chamber, growth and division of B. subtilis vegetative cells were monitored. The observed generation times of vegetative cells were comparable to those obtained in well-aerated shake flask cultures. The influence of a heat stress of 85°C for 10 min on germination, outgrowth, and subsequent vegetative growth was investigated in detail. Compared to control samples fewer spores germinated (41.1% less) and fewer grew out (48.4% less) after the treatment. The heat treatment had a significant influence on the average time to the start of germination (increased) and the distribution and average of the duration of germination itself (increased). However, the distribution and the mean outgrowth time and the generation time of vegetative cells, emerging from untreated and thermally injured spores, were similar.
Assuntos
Bacillus subtilis/fisiologia , Bacillus subtilis/citologia , Bacillus subtilis/crescimento & desenvolvimento , Meios de Cultura , Temperatura Alta , Esporos Bacterianos , Estresse FisiológicoRESUMO
Bacillus species are a spoilage and safety challenge to the food industry due to their extremely resistant endospores. To interfere with (out)growth of spores and vegetative cells, weak organic acids are suitable preservatives. To ensure their continued use while optimally preserving product quality, knowledge of resistance development is important. In Bacilli stress responses induced by weak organic acids include intracellular membrane and pH homeostasis and detoxification of reactive oxygen species. Targeted identification of inhibitors and formulation of milder antimicrobial combinations, as desired by consumers, is thereby facilitated. In addition to being food spoilers, probiotic Bacilli are known and utilized. Knowledge of weak organic acid stress resistance may be used to develop enhanced strain robustness facilitating their permanence in the gastrointestinal tract.
Assuntos
Bacillus/isolamento & purificação , Bacillus/fisiologia , Contaminação de Alimentos/prevenção & controle , Microbiologia de Alimentos , Tecnologia de Alimentos/tendências , Doenças Transmitidas por Alimentos/microbiologia , Doenças Transmitidas por Alimentos/prevenção & controle , Esterilização/tendências , HumanosRESUMO
The specific pH values of cellular compartments affect virtually all biochemical processes, including enzyme activity, protein folding and redox state. Accurate, sensitive and compartment-specific measurements of intracellular pH (pHi) dynamics in living cells are therefore crucial to the understanding of stress response and adaptation. We used the pH-sensitive GFP derivative 'ratiometric pHluorin' expressed in the cytosol and in the mitochondrial matrix of growing Saccharomyces cerevisiae to assess the variation in cytosolic pH (pHcyt) and mitochondrial pH (pHmit) in response to nutrient availability, respiratory chain activity, shifts in environmental pH and stress induced by addition of sorbic acid. The in vivo measurement allowed accurate determination of organelle-specific pH, determining a constant pHcyt of 7.2 and a constant pHmit of 7.5 in cells exponentially growing on glucose. We show that pHcyt and pHmit are differentially regulated by carbon source and respiratory chain inhibitors. Upon glucose starvation or sorbic acid stress, pHi decrease coincided with growth stasis. Additionally, pHi and growth coincided similarly in recovery after addition of glucose to glucose-starved cultures or after recovery from a sorbic acid pulse. We suggest a relation between pHi and cellular energy generation, and therefore a relation between pHi and growth.
Assuntos
Citosol/química , Proteínas de Fluorescência Verde/metabolismo , Mitocôndrias/química , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Citosol/metabolismo , Regulação Fúngica da Expressão Gênica , Glucose/metabolismo , Proteínas de Fluorescência Verde/genética , Resposta ao Choque Térmico , Concentração de Íons de Hidrogênio , Mitocôndrias/metabolismo , Ácido Sórbico/metabolismoRESUMO
Bacterial spores are the ultimate (stress) 'survival capsules'. They allow strains from the Bacillus and Clostridium species to survive harsh environmental conditions. In addition to the decision to enter sporulation the decision to do the reverse (germinate) is also a decisive event after which there is no return. Generally it is observed that the behaviour of spores towards the environment is not homogeneous. In fact in many cases it is even quite heterogeneous, certainly upon subjecting the spores to a thermal stress treatment. Genome information coupled to high resolution single-cell analysis techniques allow us currently to analyse signalling events of individual cells. In the area of food preservation the next challenge is to couple the newly acquired mechanistic data to the physiologically observed heterogeneity in spore behaviour. The current paper will introduce the background of physiological heterogeneity while discussing the molecular processes that likely contribute to the observed heterogeneity in outgrowth. The discussion is set in the framework of contemporary and future needs for single-cell data integration in order to enhance the mechanistic basis of food preservation and spoilage models targeting bacterial spores.
Assuntos
Bacillus/fisiologia , Contaminação de Alimentos/análise , Microbiologia de Alimentos , Conservação de Alimentos/métodos , Esporos Bacterianos/crescimento & desenvolvimento , Contagem de Colônia Microbiana , Qualidade de Produtos para o Consumidor , Contaminação de Alimentos/prevenção & controle , Humanos , FenótipoRESUMO
Bacillus subtilis forms dormant spores upon nutrient depletion. Under favorable environmental conditions, the spore breaks its dormancy and resumes growth in a process called spore germination and outgrowth. To elucidate the physiological processes that occur during the transition of the dormant spore to an actively growing vegetative cell, we studied this process in a time-dependent manner by a combination of microscopy, analysis of extracellular metabolites, and a genome-wide analysis of transcription. The results indicate the presence of abundant levels of late sporulation transcripts in dormant spores. In addition, the results suggest the existence of a complex and well-regulated spore outgrowth program, involving the temporal expression of at least 30% of the B. subtilis genome.