RESUMO
Virtually all bacterial species synthesize (p)ppGpp (guanosine penta- or tetraphosphate), a pleiotropic regulator of the so-called stringent response, which controls many aspects of cellular physiology and metabolism. In Escherichia coli, (p)ppGpp levels are controlled by two homologous enzymes: the (p)ppGpp synthetase RelA and the bifunctional synthetase/hydrolase SpoT. We recently identified several protein candidates that can modulate (p)ppGpp levels in E. coli. In this work, we show that the putative two-component system connector protein YmgB can promote SpoT-dependent accumulation of ppGpp in E. coli. Importantly, we determined that the control of SpoT activities by YmgB is independent of its proposed role in the two-component Rcs system, and these two functions can be uncoupled. Using genetic and structure-function analysis, we show that the regulation of SpoT activities by YmgB occurs by functional and direct binding in vivo and in vitro to the TGS and Helical domains of SpoT. These results further support the role of these domains in controlling the reciprocal enzymatic states.
Assuntos
Proteínas de Escherichia coli , Escherichia coli , Escherichia coli/metabolismo , Guanosina Pentafosfato/genética , Bactérias/metabolismo , Guanosina Tetrafosfato , Hidrolases/metabolismo , Ligases/genética , Ligases/metabolismo , Regulação Bacteriana da Expressão Gênica , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismoRESUMO
Guanosine penta- or tetraphosphate (known as (p)ppGpp) serves as second messenger to respond to nutrient downshift and other environmental stresses, a phenomenon called stringent response. Accumulation of (p)ppGpp promotes the coordinated inhibition of macromolecule synthesis, as well as the activation of stress response pathways to cope and adapt to harmful conditions. In Escherichia coli, the (p)ppGpp level is tightly regulated by two enzymes, the (p)ppGpp synthetase RelA and the bifunctional synthetase/hydrolase SpoT. We recently identified the small protein YtfK as a key regulator of SpoT-mediated activation of stringent response in E. coli. Here, we further characterized the regulation of ytfK. We observed that ytfK is subjected to catabolite repression and is positively regulated by the cyclic AMP (cAMP)-cAMP receptor protein (CRP) complex. Importantly, YtfK contributes to SpoT-dependent accumulation of (p)ppGpp and cell survival in response to glucose starvation. Therefore, regulation of ytfK by the cAMP-CRP appears important to adjust (p)ppGpp level and coordinate cellular metabolism in response to glucose availability.
RESUMO
Bacteria regulate their metabolism to adapt and survive adverse conditions, in particular to stressful downshifts in nutrient availability. These shifts trigger the so-called stringent response, coordinated by the signaling molecules guanosine tetra and pentaphosphate collectively referred to as (p)ppGpp. In Escherichia coli, accumulation of theses alarmones depends on the (p)ppGpp synthetase RelA and the bifunctional (p)ppGpp synthetase/hydrolase SpoT. A tight regulation of these intracellular activities is therefore crucial to rapidly adjust the (p)ppGpp levels in response to environmental stresses but also to avoid toxic consequences of (p)ppGpp over-accumulation. In this study, we show that the small protein NirD restrains RelA-dependent accumulation of (p)ppGpp and can inhibit the stringent response in E. coli. Mechanistically, our in vivo and in vitro studies reveal that NirD directly binds the catalytic domains of RelA to balance (p)ppGpp accumulation. Finally, we show that NirD can control RelA activity by directly inhibiting the rate of (p)ppGpp synthesis.
Assuntos
Proteínas de Escherichia coli/genética , Escherichia coli/genética , GTP Pirofosfoquinase/genética , Nitrito Redutases/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , GTP Pirofosfoquinase/metabolismo , Guanosina Pentafosfato/metabolismo , Nitrito Redutases/metabolismo , Estresse FisiológicoRESUMO
The stringent response is a general bacterial stress response that allows bacteria to adapt and survive adverse conditions. This reprogramming of cell physiology is caused by the accumulation of the alarmone (p)ppGpp which, in Escherichia coli, depends on the (p)ppGpp synthetase RelA and the bifunctional (p)ppGpp synthetase/hydrolase SpoT. Although conditions that control SpoT-dependent (p)ppGpp accumulation have been described, the molecular mechanisms regulating the switching from (p)ppGpp degradation to synthesis remain poorly understood. Here, we show that the protein YtfK promotes SpoT-dependent accumulation of (p)ppGpp in E. coli and is required for activation of the stringent response during phosphate and fatty acid starvation. Our results indicate that YtfK can interact with SpoT. We propose that YtfK activates the stringent response by tilting the catalytic balance of SpoT toward (p)ppGpp synthesis.
Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/fisiologia , Guanosina Pentafosfato/biossíntese , Pirofosfatases/metabolismo , Estresse FisiológicoRESUMO
Microcin C (McC) is a peptide-nucleotide antibiotic that inhibits aspartyl-tRNA synthetase. Here, we show that McC is a strong inducer of persistence in Escherichia coli. Persistence induced by McC is mediated by (p)ppGpp and requires chromosomally encoded toxin-antitoxin modules. McC-producing cells have increased persistence levels due to a combined effect of McC imported from the cultured medium and intracellularly synthesized antibiotic. McC-producing cells also induce persistence in sensitive cells during co-cultivation, underscoring complex interactions in bacterial communities where an antagonistic compound produced by one community member can benefit other members by increasing their ability to withstand antibiotics.
Assuntos
Bacteriocinas/farmacologia , Escherichia coli/efeitos dos fármacos , Antibacterianos/farmacologia , Aspartato-tRNA Ligase/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , FosforilaçãoRESUMO
Bacterial persister cells avoid antibiotic-induced death by entering a physiologically dormant state and are considered a major cause of antibiotic treatment failure and relapsing infections. Such dormant cells form stochastically, but also in response to environmental cues, by various pathways that are usually controlled by the second messenger (p)ppGpp. For example, toxin-antitoxin modules have been shown to play a major role in persister formation in many model systems. More generally, the diversity of molecular mechanisms driving persister formation is increasingly recognized as the cause of physiological heterogeneity that underlies collective multistress and multidrug tolerance of persister subpopulations. In this Review, we summarize the current state of the field and highlight recent findings, with a focus on the molecular basis of persister formation and heterogeneity.
Assuntos
Adaptação Fisiológica/fisiologia , Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Infecções Bacterianas/microbiologia , Farmacorresistência Bacteriana Múltipla , Estresse Fisiológico/fisiologia , Adaptação Fisiológica/genética , Antibacterianos/uso terapêutico , Bactérias/metabolismo , Infecções Bacterianas/tratamento farmacológico , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/antagonistas & inibidores , Toxinas Bacterianas/metabolismo , Biofilmes , Dano ao DNA , Guanosina Pentafosfato/metabolismo , Força Próton-Motriz , Resposta SOS em Genética , Fator sigma/metabolismo , Transdução de Sinais , Processos Estocásticos , Estresse Fisiológico/genéticaRESUMO
In this issue of Molecular Cell, Verstraeten et al. (2015) demonstrate that the conserved GTPase Obg and the second messenger ppGpp mediate persistence by activation of a type I toxin-antitoxin module (hokB/sokB) in E. coli.
Assuntos
Proteínas de Bactérias/genética , Toxinas Bacterianas/biossíntese , Farmacorresistência Bacteriana/fisiologia , Proteínas de Escherichia coli/biossíntese , Escherichia coli/genética , Proteínas de Ligação ao GTP/genética , Pseudomonas aeruginosa/genéticaRESUMO
The model organism Escherichia coli codes for at least 11 type II toxin-antitoxin (TA) modules, all implicated in bacterial persistence (multidrug tolerance). Ten of these encode messenger RNA endonucleases (mRNases) inhibiting translation by catalytic degradation of mRNA, and the 11th module, hipBA, encodes HipA (high persister protein A) kinase, which inhibits glutamyl tRNA synthetase (GltX). In turn, inhibition of GltX inhibits translation and induces the stringent response and persistence. Previously, we presented strong support for a model proposing (p)ppGpp (guanosine tetra and penta-phosphate) as the master regulator of persistence. Stochastic variation of [(p)ppGpp] in single cells induced TA-encoded mRNases via a pathway involving polyphosphate and Lon protease. Polyphosphate activated Lon to degrade all known type II antitoxins of E. coli. In turn, the activated mRNases induced persistence and multidrug tolerance. However, even though it was known that activation of HipA stimulated (p)ppGpp synthesis, our model did not explain how hipBA induced persistence. Here we show that, in support of and consistent with our initial model, HipA-induced persistence depends not only on (p)ppGpp but also on the 10 mRNase-encoding TA modules, Lon protease, and polyphosphate. Importantly, observations with single cells convincingly show that the high level of (p)ppGpp caused by activation of HipA does not induce persistence in the absence of TA-encoded mRNases. Thus, slow growth per se does not induce persistence in the absence of TA-encoded toxins, placing these genes as central effectors of bacterial persistence.
Assuntos
Endonucleases/metabolismo , Proteínas de Escherichia coli/metabolismo , Guanosina Pentafosfato/metabolismo , Guanosina Tetrafosfato/metabolismo , Alelos , Antitoxinas/metabolismo , Toxinas Bacterianas/metabolismo , Ativação Enzimática , Modelos Moleculares , RNA Mensageiro/metabolismo , Processos Estocásticos , Imagem com Lapso de TempoRESUMO
All bacteria form persisters, cells that are multidrug tolerant and therefore able to survive antibiotic treatment. Due to the low frequencies of persisters in growing bacterial cultures and the complex underlying molecular mechanisms, the phenomenon has been challenging to study. However, recent technological advances in microfluidics and reporter genes have improved this scenario. Here, we summarize recent progress in the field, revealing the ubiquitous bacterial stress alarmone ppGpp as an emerging central regulator of multidrug tolerance and persistence, both in stochastically and environmentally induced persistence. In several different organisms, toxin-antitoxin modules function as effectors of ppGpp-induced persistence.
Assuntos
Bactérias/efeitos dos fármacos , Farmacorresistência Bacteriana Múltipla , Guanosina Tetrafosfato/metabolismo , Bactérias/metabolismo , Fenômenos Fisiológicos Bacterianos , Escherichia coli/efeitos dos fármacos , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Fungos/efeitos dos fármacos , Transdução de SinaisRESUMO
Using Bacillus subtilis as a model organism, we investigated thermotolerance development by analysing cell survival and in vivo protein aggregate formation in severely heat-shocked cells primed by a mild heat shock. We observed an increased survival during severe heat stress, accompanied by a strong reduction of heat-induced cellular protein aggregates in cells lacking the ClpXP protease. We could demonstrate that the transcription factor Spx, a regulatory substrate of ClpXP, is critical for the prevention of protein aggregate formation because its regulon encodes redox chaperones, such as thioredoxin, required for protection against thiol-specific oxidative stress. Consequently B. subtilis cells grown in the absence of oxygen were more protected against severe heat shock and much less protein aggregates were detected compared to aerobically grown cells. The presented results indicate that in B. subtilisâ Spx and its regulon plays not only an important role for oxidative but also for heat stress response and thermotolerance development. In addition, our experiments suggest that the protection of misfolded proteins from thiol oxidation during heat shock can be critical for the prevention of cellular protein aggregation in vivo.
Assuntos
Bacillus subtilis/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Resposta ao Choque Térmico , Temperatura Alta , Estresse Oxidativo , Compostos de Sulfidrila/metabolismo , Adaptação Fisiológica , Anaerobiose , Bacillus subtilis/crescimento & desenvolvimento , Homeostase , Viabilidade Microbiana , Modelos Biológicos , Mutação/genética , Oxirredução , Estrutura Quaternária de ProteínaRESUMO
Persistence refers to the phenomenon in which isogenic populations of antibiotic-sensitive bacteria produce rare cells that transiently become multidrug tolerant. Whether slow growth in a rare subset of cells underlies the persistence phenotype has not be examined in wild-type bacteria. Here, we show that an exponentially growing population of wild-type Escherichia coli cells produces rare cells that stochastically switch into slow growth, that the slow-growing cells are multidrug tolerant, and that they are able to resuscitate. The persistence phenotype depends hierarchically on the signaling nucleotide (p)ppGpp, Lon protease, inorganic polyphosphate, and toxin-antitoxins. We show that the level of (p)ppGpp varies stochastically in a population of exponentially growing cells and that the high (p)ppGpp level in rare cells induces slow growth and persistence. (p)ppGpp triggers slow growth by activating toxin-antitoxin loci through a regulatory cascade depending on inorganic polyphosphate and Lon protease.
Assuntos
Escherichia coli/crescimento & desenvolvimento , Escherichia coli/metabolismo , Guanosina Pentafosfato/metabolismo , Antitoxinas/metabolismo , Toxinas Bacterianas/metabolismo , Farmacorresistência Bacteriana Múltipla , Escherichia coli/efeitos dos fármacos , Polifosfatos/metabolismo , Protease La/metabolismo , Transcrição GênicaRESUMO
Bacterial persistence is caused by the presence of rare, slowly growing bacteria among populations of rapidly growing cells. The slowly growing bacteria are tolerant of antibiotics and other environmental insults, whereas their isogenic, rapidly growing siblings are sensitive. Recent research has shown that persistence of the model organism Escherichia coli depends on toxin-antitoxin (TA) loci. Deletion of type II TA loci reduces the level of persistence significantly. Lon protease but no other known ATP-dependent proteases is required for persistence. Polyphosphate and (p)ppGpp also are required for persistence. These observations led to the proposal of a simple and testable model that explains the persistence of E. coli. It is now important to challenge this model and to test whether the persistence of pathogenic bacteria also depends on TA loci.
Assuntos
Toxinas Bacterianas/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/patogenicidade , Toxinas Bacterianas/genética , Proteínas de Escherichia coli/genética , Guanosina Pentafosfato/metabolismo , Guanosina Tetrafosfato/metabolismo , Humanos , Viabilidade Microbiana , Modelos Biológicos , Polifosfatos/metabolismo , Protease La/metabolismo , VirulênciaRESUMO
Bacteria form persisters, individual cells that are highly tolerant to different types of antibiotics. Persister cells are genetically identical to nontolerant kin but have entered a dormant state in which they are recalcitrant to the killing activity of the antibiotics. The molecular mechanisms underlying bacterial persistence are unknown. Here, we show that the ubiquitous Lon (Long Form Filament) protease and mRNA endonucleases (mRNases) encoded by toxin-antitoxin (TA) loci are required for persistence in Escherichia coli. Successive deletion of the 10 mRNase-encoding TA loci of E. coli progressively reduced the level of persisters, showing that persistence is a phenotype common to TA loci. In all cases tested, the antitoxins, which control the activities of the mRNases, are Lon substrates. Consistently, cells lacking lon generated a highly reduced level of persisters. Moreover, Lon overproduction dramatically increased the levels of persisters in wild-type cells but not in cells lacking the 10 mRNases. These results support a simple model according to which mRNases encoded by TA loci are activated in a small fraction of growing cells by Lon-mediated degradation of the antitoxins. Activation of the mRNases, in turn, inhibits global cellular translation, and thereby induces dormancy and persistence. Many pathogenic bacteria known to enter dormant states have a plethora of TA genes. Therefore, in the future, the discoveries described here may lead to a mechanistic understanding of the persistence phenomenon in pathogenic bacteria.
Assuntos
Endorribonucleases/metabolismo , Escherichia coli K12/enzimologia , RNA Bacteriano/metabolismo , Antitoxinas/metabolismo , Toxinas Bacterianas/metabolismo , Ativação Enzimática , Escherichia coli K12/citologia , Proteínas de Escherichia coli/metabolismo , Protease La/metabolismo , RNA Mensageiro/metabolismoRESUMO
Reactive oxygen species (ROS) are harmful because they can oxidize biological macromolecules. We show here that atmospheric CO(2) (concentration range studied: 40-1,000 p.p.m.) increases death rates due to H(2)O(2) stress in Escherichia coli in a dose-specific manner. This effect is correlated with an increase in H(2)O(2)-induced mutagenesis and, as shown by 8-oxo-guanine determinations in cells, DNA base oxidation rates. Moreover, the survival of mutants that are sensitive to aerobic conditions (Hpx(-) dps and recA fur), presumably because of their inability to tolerate ROS, seems to depend on CO(2) concentration. Thus, CO(2) exacerbates ROS toxicity by increasing oxidative cellular lesions.
Assuntos
Dióxido de Carbono/toxicidade , Escherichia coli/efeitos dos fármacos , Escherichia coli/metabolismo , Peróxido de Hidrogênio/toxicidade , Espécies Reativas de Oxigênio/metabolismo , Mutagênese/efeitos dos fármacos , Oxirredução/efeitos dos fármacosRESUMO
The ability of Staphylococcus aureus to adapt to various conditions of stress is the result of a complex regulatory response. Among them, ClpC, belonging to the Hsp100/Clp ATPase family, seems to play an important role. For instance, we previously demonstrated that a functional clpC deletion resulted in enhanced survival in the late stationary phase (death phase period) compared to the parental S. aureus strain. However, the mechanisms for the enhanced survival of a S. aureus clpC mutant during the death phase period are still elusive. In Escherichia coli, among the factors that might lead to bacterial cell death during stationary phase, the amount of protein aggregates and/or oxidized proteins appears to be of major importance. Thus, in the present study, we have evaluated protein aggregates and carbonylated protein (as a marker of protein oxidation) contents both in the wild type and in an S. aureus clpC mutant during the exponential growth phase and the death phase. Whereas at all time points the tested clpC mutant exhibits the same amount of protein aggregates as the WT strain, the total amount of carbonylated proteins appears to be lower in the clpC mutant. Moreover, we observed that at the entrance of the death phase carbon-metabolizing enzymes [such as the TCA cycle enzymes Mqo2 (malate: quinone oxidoreductase) and FumC/CitG (fumarate hydratase)] albeit not the bulk proteins are carbonylated to a larger extent in the clpC mutant. Reduced activity of the TCA cycle due to specific carbonylation of these proteins will result in a decrease of endogenous oxidative stress which in turn might confer enhanced survival of the clpC mutant during the death phase period thus contributing to bacterial longevity and chronic infection.
Assuntos
Proteínas de Bactérias/metabolismo , Enzimas/metabolismo , Proteínas de Choque Térmico/metabolismo , Processamento de Proteína Pós-Traducional , Staphylococcus aureus/fisiologia , Proteínas de Bactérias/genética , Carbono/metabolismo , Ciclo do Ácido Cítrico , Deleção de Genes , Proteínas de Choque Térmico/genética , Humanos , Redes e Vias Metabólicas , Viabilidade Microbiana , Carbonilação Proteica , Staphylococcus aureus/enzimologia , Staphylococcus aureus/crescimento & desenvolvimentoRESUMO
BACKGROUND: Carbonyl derivatives are mainly formed by direct metal-catalysed oxidation (MCO) attacks on the amino-acid side chains of proline, arginine, lysine and threonine residues. For reasons unknown, only some proteins are prone to carbonylation. METHODOLOGY/PRINCIPAL FINDINGS: we used mass spectrometry analysis to identify carbonylated sites in: BSA that had undergone in vitro MCO, and 23 carbonylated proteins in Escherichia coli. The presence of a carbonylated site rendered the neighbouring carbonylatable site more prone to carbonylation. Most carbonylated sites were present within hot spots of carbonylation. These observations led us to suggest rules for identifying sites more prone to carbonylation. We used these rules to design an in silico model (available at http://www.lcb.cnrs-mrs.fr/CSPD/), allowing an effective and accurate prediction of sites and of proteins more prone to carbonylation in the E. coli proteome. CONCLUSIONS/SIGNIFICANCE: We observed that proteins evolve to either selectively maintain or lose predicted hot spots of carbonylation depending on their biological function. As our predictive model also allows efficient detection of carbonylated proteins in Bacillus subtilis, we believe that our model may be extended to direct MCO attacks in all organisms.
Assuntos
Bacillus subtilis/metabolismo , Bioquímica/métodos , Metais/química , Oxigênio/química , Carbonilação Proteica , Sequência de Aminoácidos , Arginina/química , Carbono/química , Catálise , Escherichia coli/metabolismo , Humanos , Lisina/química , Espectrometria de Massas/métodos , Dados de Sequência Molecular , Prolina/química , Proteínas/química , Albumina Sérica/química , Treonina/químicaRESUMO
Most time lapse microscopy experiments studying bacterial processes ie growth, progression through the cell cycle and motility have been performed on thin nutrient agar pads. An important limitation of this approach is that dynamic perturbations of the experimental conditions cannot be easily performed. In eukaryotic cell biology, fluidic approaches have been largely used to study the impact of rapid environmental perturbations on live cells and in real time. However, all these approaches are not easily applicable to bacterial cells because the substrata are in all cases specific and also because microfluidics nanotechnology requires a complex lithography for the study of micrometer sized bacterial cells. In fact, in many cases agar is the experimental solid substratum on which bacteria can move or even grow. For these reasons, we designed a novel hybrid micro fluidic device that combines a thin agar pad and a custom flow chamber. By studying several examples, we show that this system allows real time analysis of a broad array of biological processes such as growth, development and motility. Thus, the flow chamber system will be an essential tool to study any process that take place on an agar surface at the single cell level.
Assuntos
Bactérias/metabolismo , Microscopia/instrumentação , Ágar/química , Automação , Fenômenos Fisiológicos Bacterianos , Técnicas de Cultura de Células/instrumentação , Ciclo Celular , Desenho de Equipamento , Escherichia coli/metabolismo , Técnicas Analíticas Microfluídicas , Microfluídica/instrumentação , Microscopia/métodos , Myxococcus/metabolismo , Nanotecnologia/métodosRESUMO
Carbonylation is currently used as a marker for irreversible protein oxidative damage. Several studies indicate that carbonylated proteins are more prone to degradation than their nonoxidized counterparts. In this study, we observed that in Escherichia coli, more than 95% of the total carbonyl content consisted of insoluble protein and most were cytosolic proteins. We thereby demonstrate that, in vivo, carbonylated proteins are detectable mainly in an aggregate state. Finally, we show that detectable carbonylated proteins are not degraded in vivo. Here we propose that some carbonylated proteins escape degradation in vivo by forming carbonylated protein aggregates and thus becoming nondegradable. In light of these findings, we provide evidence that the accumulation of nondegradable carbonylated protein presented in an aggregate state contributes to the increases in carbonyl content observed during senescence.
Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/fisiologia , Carbonilação Proteica , Cromatografia Líquida , Citoplasma/química , Eletroforese em Gel Bidimensional , Solubilidade , Espectrometria de Massas em TandemRESUMO
In a previous study, we demonstrated the presence of protein aggregates in an exponentially grown Escherichia coli culture. In light of these observations, protein aggregates could be considered damage to cells that is able to pass from one generation to the next. Based on the assumption that the amount of aggregate protein could represent an aging factor, we monitored this amount in a bacterial culture during senescence. In doing so, we observed (i) a significant increase in the amount of aggregate protein over time, (ii) a proportional relationship between the amount of aggregate protein and the level of dead cells, (iii) a larger amount in dead cells than in culturable cells, (iv) a heterogeneous distribution of different amounts within a homogenous population of culturable cells entering stasis, and (v) that the initial amount of aggregate protein within a culturable population conditioned the death rate of the culture. Together, the results presented in this study suggest that protein aggregates indeed represent one aging factor leading to bacterial cell death.