RESUMO
Post-translational modifications such as phosphorylation, nitrosylation, and pupylation modulate multiple cellular processes in Mycobacterium tuberculosis. While protein methylation at lysine and arginine residues is widespread in eukaryotes, to date only two methylated proteins in Mtb have been identified. Here, we report the identification of methylation at lysine and/or arginine residues in nine mycobacterial proteins. Among the proteins identified, we chose MtrA, an essential response regulator of a two-component signaling system, which gets methylated on multiple lysine and arginine residues to examine the functional consequences of methylation. While methylation of K207 confers a marginal decrease in the DNA-binding ability of MtrA, methylation of R122 or K204 significantly reduces the interaction with the DNA. Overexpression of S-adenosyl homocysteine hydrolase (SahH), an enzyme that modulates the levels of S-adenosyl methionine in mycobacteria decreases the extent of MtrA methylation. Most importantly, we show that decreased MtrA methylation results in transcriptional activation of mtrA and sahH promoters. Collectively, we identify novel methylated proteins, expand the list of modifications in mycobacteria by adding arginine methylation, and show that methylation regulates MtrA activity. We propose that protein methylation could be a more prevalent modification in mycobacterial proteins.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteínas de Bactérias/metabolismo , DNA Bacteriano/metabolismo , Proteínas de Ligação a DNA/metabolismo , Mycobacterium tuberculosis/metabolismo , Regiões Promotoras Genéticas , Processamento de Proteína Pós-Traducional , Transportadores de Cassetes de Ligação de ATP/genética , Proteínas de Bactérias/genética , DNA Bacteriano/genética , Proteínas de Ligação a DNA/genética , Metilação , Mycobacterium tuberculosis/genéticaRESUMO
Reversible protein phosphorylation is one of the major mechanisms in the regulation of protein expression and protein activity, controlling physiological functions of the important human pathogen Staphylococcus aureus Phosphorylations at serine, threonine and tyrosine are known to influence for example protein activity in central metabolic pathways and the more energy-rich phosphorylations at histidine, aspartate or cysteine can be found as part of two component system sensor domains or mediating bacterial virulence. In addition to these well-known phosphorylations, the phosphorylation at arginine residues plays an essential role. Hence, the deletion mutant S. aureus COL ΔptpB (protein tyrosine phosphatase B) was studied because the protein PtpB is assumed to be an arginine phosphatase. A gel-free approach was applied to analyze the changes in the phosphoproteome of the deletion mutant ΔptpB and the wild type in growing cells, thereby focusing on the occurrence of phosphorylation on arginine residues. In order to enhance the reliability of identified phosphorylation sites at arginine residues, a subset of arginine phosphorylated peptides was chemically synthesized. Combined spectral libraries based on phosphoenriched samples, synthetic arginine phosphorylated peptides and classical proteome samples provide a sophisticated tool for the analysis of arginine phosphorylations. This way, 212 proteins phosphorylated on serine, threonine, tyrosine or arginine residues were identified within the mutant ΔptpB and 102 in wild type samples. Among them, 207 arginine phosphosites were identified exclusively within the mutant ΔptpB, widely distributed along the whole bacterial metabolism. This identification of putative targets of PtpB allows further investigation of the physiological relevance of arginine phosphorylations and provides the basis for reliable quantification of arginine phosphorylations in bacteria.
Assuntos
Arginina/metabolismo , Proteínas de Bactérias/metabolismo , Staphylococcus aureus/metabolismo , Biblioteca de Peptídeos , Peptídeos/metabolismo , FosforilaçãoRESUMO
Bacillus subtilis possesses two glyceraldehyde-3-phosphate dehydrogenases with opposite roles, the glycolytic NAD-dependent GapA and the NADP-dependent GapB enzyme, which is exclusively required during gluconeogenesis but not active under conditions promoting glycolysis. We propose that proteins that are no longer needed will be recognized and proteolyzed by Clp proteases and thereby recycled. To test this postulation, we analyzed the stability of the glycolytic enzyme GapA and the gluconeogenetic enzyme GapB in the presence and absence of glucose. It turned out that GapA remained rather stable under both glycolytic and gluconeogenetic conditions. In contrast, the gluconeogenetic enzyme GapB was degraded after a shift from malate to glucose (i.e., from gluconeogenesis to glycolysis), displaying an estimated half-life of approximately 3 h. Comparative in vivo pulse-chase labeling and immunoprecipitation experiments of the wild-type strain and isogenic mutants identified the ATP-dependent ClpCP protease as the enzyme responsible for the degradation of GapB. However, arginine protein phosphorylation, which was recently described as a general tagging mechanism for protein degradation, did not seem to play a role in GapB proteolysis, because GapB was also degraded in a mcsB mutant, lacking arginine kinase, in the same manner as in the wild type.IMPORTANCE GapB, the NADP-dependent glyceraldehyde-3-phosphosphate dehydrogenase, is essential for B. subtilis under gluconeogenetic conditions. However, after a shift to glycolytic conditions, GapB loses its physiological function within the cell and becomes susceptible to degradation, in contrast to GapA, the glycolytic NAD-dependent glyceraldehyde-3-phosphate dehydrogenase, which remains stable under glycolytic and gluconeogenetic conditions. Subsequently, GapB is proteolyzed in a ClpCP-dependent manner. According to our data, the arginine kinase McsB is not involved as adaptor protein in this process. ClpCP appears to be in charge in the removal of inoperable enzymes in B. subtilis, which is a strictly regulated process in which the precise recognition mechanism(s) remains to be identified.
Assuntos
Bacillus subtilis/enzimologia , Bacillus subtilis/metabolismo , Endopeptidase Clp/metabolismo , Gliceraldeído-3-Fosfato Desidrogenase (NADP+)(Fosforiladora)/metabolismo , Proteólise , Glucose/metabolismo , Glicólise , Imunoprecipitação , Marcação por Isótopo , Estabilidade ProteicaRESUMO
Protein lysine acetylation is known to regulate multiple aspects of bacterial metabolism. However, its presence in mycobacterial signal transduction and virulence-associated proteins has not been studied. In this study, analysis of mycobacterial proteins from different cellular fractions indicated dynamic and widespread occurrence of lysine acetylation. Mycobacterium tuberculosis proteins regulating diverse physiological processes were then selected and expressed in the surrogate host Mycobacterium smegmatis. The purified proteins were analyzed for the presence of lysine acetylation, leading to the identification of 24 acetylated proteins. In addition, novel lysine succinylation and propionylation events were found to co-occur with acetylation on several proteins. Protein-tyrosine phosphatase B (PtpB), a secretory phosphatase that regulates phosphorylation of host proteins and plays a critical role in Mycobacterium infection, is modified by acetylation and succinylation at Lys-224. This residue is situated in a lid region that covers the enzyme's active site. Consequently, acetylation and succinylation negatively regulate the activity of PtpB.
Assuntos
Mycobacterium tuberculosis/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Acilação , Sequência de Aminoácidos , Dados de Sequência Molecular , Mycobacterium tuberculosis/enzimologia , Monoéster Fosfórico Hidrolases/química , Fosforilação , Conformação Proteica , Homologia de Sequência de Aminoácidos , Relação Estrutura-AtividadeRESUMO
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
The clpC operon is known to regulate several processes such as genetic competence, protein degradation and stress survival in bacteria. Here, we describe the role of clpC operon in Bacillus anthracis. We generated knockout strains of the clpC operon genes to investigate the impact of CtsR, McsA, McsB and ClpC deletion on essential processes of B. anthracis. We observed that growth, cell division, sporulation and germination were severely affected in mcsB and clpC deleted strains, while none of deletions affected toxin secretion. Growth defect in these strains was pronounced at elevated temperature. The growth pattern gets restored on complementation of mcsB and clpC in respective mutants. Electron microscopic examination revealed that mcsB and clpC deletion also causes defect in septum formation leading to cell elongation. These vegetative cell deformities were accompanied by inability of mutant strains to generate morphologically intact spores. Higher levels of polyhydroxybutyrate granules accumulation were also observed in these deletion strains, indicating a defect in sporulation process. Our results demonstrate, for the first time, the vital role played by McsB and ClpC in physiology of B. anthracis and open up further interest on this operon, which might be of importance to success of B. anthracis as pathogen.
Assuntos
Antígenos de Bactérias/metabolismo , Bacillus anthracis/citologia , Bacillus anthracis/fisiologia , Proteínas de Bactérias/fisiologia , Toxinas Bacterianas/metabolismo , Proteínas de Choque Térmico/fisiologia , Óperon/fisiologia , Antígenos de Bactérias/biossíntese , Antígenos de Bactérias/genética , Bacillus anthracis/genética , Proteínas de Bactérias/genética , Toxinas Bacterianas/biossíntese , Toxinas Bacterianas/genética , Proteínas de Choque Térmico/genética , Óperon/genética , Esporos Bacterianos/citologia , Esporos Bacterianos/genética , Esporos Bacterianos/fisiologiaRESUMO
Reversible protein phosphorylation is an important and ubiquitous protein modification in all living cells. Here we report that protein phosphorylation on arginine residues plays a physiologically significant role. We detected 121 arginine phosphorylation sites in 87 proteins in the gram-positive model organism Bacillus subtilis in vivo. Moreover, we provide evidence that protein arginine phosphorylation has a functional role and is involved in the regulation of many critical cellular processes, such as protein degradation, motility, competence, and stringent and stress responses. Our results suggest that in B. subtilis the combined activity of a protein arginine kinase and phosphatase allows a rapid and reversible regulation of protein activity and that protein arginine phosphorylation can play a physiologically important and regulatory role in bacteria.
Assuntos
Arginina/metabolismo , Bacillus subtilis/metabolismo , Bacillus subtilis/fisiologia , Proteínas de Bactérias/metabolismo , Sequência de Aminoácidos , Arginina/genética , Bacillus subtilis/genética , Proteínas de Bactérias/genética , Sítios de Ligação/genética , Eletroforese em Gel de Poliacrilamida , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Espectrometria de Massas , Fosfopeptídeos/metabolismo , Fosforilação/fisiologia , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , ProteóliseRESUMO
Protein quality networks are required for the maintenance of proper protein homeostasis and essential for viability and growth of all living organisms. Hence, regulation and coordination of these networks are critical for survival during stress as well as for virulence of pathogenic species. In low GC, Gram-positive bacteria central protein quality networks are under the control of the global repressor CtsR. Here, we provide evidence that CtsR activity during heat stress is mediated by intrinsic heat sensing through a glycine-rich loop, probably in all Gram-positive species. Moreover, a function for the recently identified arginine kinase McsB is confirmed, however, not for initial inactivation and dissociation of CtsR from the DNA, but for heat-dependent auto-activation of McsB as an adaptor for ClpCP-mediated degradation of CtsR.
Assuntos
Bacillus subtilis/metabolismo , Proteínas de Bactérias/metabolismo , Bactérias Gram-Positivas/metabolismo , Resposta ao Choque Térmico/fisiologia , Temperatura Alta , Proteínas Repressoras/metabolismo , Bacillus subtilis/genética , Bacillus subtilis/crescimento & desenvolvimento , Proteínas de Bactérias/genética , Northern Blotting , Eletroforese em Gel Bidimensional , Ensaio de Desvio de Mobilidade Eletroforética , Regulação Bacteriana da Expressão Gênica , Bactérias Gram-Positivas/genética , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Immunoblotting , Mutação Puntual/genética , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , RNA Mensageiro/genética , Proteínas Repressoras/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Intracellular proteolysis carried out by energy-dependent proteases is one of the most conserved biological processes. In all cells proteolysis maintains and shapes the cellular proteome by ridding the cell of damaged proteins and by regulating abundance of functional proteins such as regulatory proteins. The ATP-dependent ClpP protease is highly conserved among eubacteria and in the chloroplasts and mitochondria of eukaryotic cells. In the serious human pathogen, Staphylococcus aureus inactivation of clpP rendered the bacterium avirulent emphasizing the central role of proteolysis in virulence. The contribution of the Clp proteins to virulence is likely to occur at multiple levels. First of all, both Clp ATPases and the Clp protease are central players in stress responses required to cope with the adverse conditions met in the host. The ClpP protease has a dual role herein, as it both eliminates stress-damaged proteins as well as ensures the timely degradation of major stress regulators such as Spx, LexA and CtsR. Additionally, as we will summarize in this review, Clp proteases and Clp chaperones impact on such central processes as virulence gene expression, cell wall metabolism, survival in stationary phase, and cell division. These observations together with recent findings that Clp proteins contribute to adaptation to antibiotics highlights the importance of this interesting proteolytic machinery both for understanding pathogenicity of the organism and for treating staphylococcal infections.
Assuntos
Farmacorresistência Bacteriana , Endopeptidase Clp/metabolismo , Chaperonas Moleculares/metabolismo , Staphylococcus aureus/enzimologia , Staphylococcus aureus/fisiologia , Estresse Fisiológico , Animais , Calmodulina , Humanos , Viabilidade Microbiana , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/patogenicidade , VirulênciaRESUMO
The cellular amount of proteins not only depends on synthesis but also on degradation. Here, we expand the understanding of differential protein levels by complementing synthesis data with a proteome-wide, mass spectrometry-based stable isotope labeling with amino acids in cell culture analysis of protein degradation in the human pathogen Staphylococcus aureus during glucose starvation. Monitoring protein stability profiles in a wild type and an isogenic clpP protease mutant revealed that 1) proteolysis mainly affected proteins with vegetative functions, anabolic and selected catabolic enzymes, whereas the expression of TCA cycle and gluconeogenesis enzymes increased; 2) most proteins were prone to aggregation in the clpP mutant; 3) the absence of ClpP correlated with protein denaturation and oxidative stress responses, deregulation of virulence factors and a CodY repression. We suggest that degradation of redundant, inactive proteins disintegrated from functional complexes and thereby amenable to proteolytic attack is a fundamental cellular process in all organisms to regain nutrients and guarantee protein homeostasis.
Assuntos
Proteínas de Bactérias/metabolismo , Glucose/metabolismo , Staphylococcus aureus/metabolismo , Proteínas de Bactérias/antagonistas & inibidores , Ciclo do Ácido Cítrico , Endopeptidase Clp/genética , Endopeptidase Clp/metabolismo , Regulação Bacteriana da Expressão Gênica , Gluconeogênese , Mutação , Estresse Oxidativo , Biossíntese de Proteínas , Proteólise , Proteínas Repressoras/antagonistas & inibidores , Staphylococcus aureus/enzimologia , Staphylococcus aureus/crescimento & desenvolvimentoRESUMO
In the important human pathogen Staphylococcus aureus the cytoplasmic ClpP protease is essential for mounting cellular stress responses and for virulence. To directly identify substrates of the ClpP protease, we expressed in vivo a proteolytic inactive form of ClpP (ClpP(trap)) that will retain but not degrade substrates translocated into its proteolytic chamber. Substrates captured inside the proteolytic barrel were co-purified along with the His-tagged ClpP complex and identified by mass spectrometry. In total, approximately 70 proteins were trapped in both of the two S. aureus strains NCTC8325-4 and Newman. About one-third of the trapped proteins are previously shown to be unstable or to be substrates of ClpP in other bacteria, supporting the validity of the ClpP-TRAP. This group of proteins encompassed the transcriptional regulators CtsR and Spx, the ClpC adaptor proteins McsB and MecA, and the cell division protein FtsZ. Newly identified ClpP substrates include the global transcriptional regulators PerR and HrcA, proteins involved in DNA damage repair (RecA, UvrA, UvrB), and proteins essential for protein synthesis (RpoB and Tuf). Our study hence underscores the central role of Clp-proteolysis in a number of pathways that contribute to the success of S. aureus as a human pathogen.
Assuntos
Proteínas de Bactérias/genética , Reparo do DNA/genética , DNA Bacteriano , Endopeptidase Clp/genética , Regulação Bacteriana da Expressão Gênica , Proteoma/genética , Staphylococcus aureus/genética , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Farmacorresistência Bacteriana/genética , Endopeptidase Clp/metabolismo , Resposta ao Choque Térmico/genética , Proteínas de Ligação às Penicilinas , Fator Tu de Elongação de Peptídeos/genética , Fator Tu de Elongação de Peptídeos/metabolismo , Ligação Proteica , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Proteólise , Proteoma/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Staphylococcus aureus/metabolismoRESUMO
The general stress regulon of Bacillus subtilis comprises approximately 200 genes and is under the control of the alternative sigma factor σ(B). The activation of σ(B) occurs in response to multiple physical stress stimuli as well as energy starvation conditions. The expression of the general stress proteins provides growing and stationary nonsporulating vegetative cells with nonspecific and broad stress resistance. A previous comprehensive phenotype screening analysis of 94 general stress gene mutants in response to severe growth-inhibiting stress stimuli, including ethanol, NaCl, heat, and cold, indicated that secondary oxidative stress may be a common component of severe physical stress. Here we tested the individual contributions of the same set of 94 mutants to the development of resistance against exposure to the superoxide-generating agent paraquat and hydrogen peroxide (H(2)O(2)). In fact, 62 mutants displayed significantly decreased survival rates in response to paraquat and/or H(2)O(2) stress compared to the wild type at a confidence level of an α value of ≤ 0.01. Thus, we were able to assign 47 general stress genes to survival against superoxide, 6 genes to protection from H(2)O(2) stress, and 9 genes to the survival against both. Furthermore, we show that a considerable overlap exists between the phenotype clusters previously assumed to be involved in oxidative stress management and the actual group of oxidative-stress-sensitive mutants. Our data provide information that many general stress proteins with still unknown functions are implicated in oxidative stress resistance and further support the notion that different severe physical stress stimuli elicit a common secondary oxidative stress.
Assuntos
Bacillus subtilis/fisiologia , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica/fisiologia , Estresse Oxidativo/fisiologia , Proteínas de Bactérias/genética , Perfilação da Expressão Gênica , Genômica , Peróxido de Hidrogênio , Viabilidade Microbiana/efeitos dos fármacos , Oxidantes/farmacologia , Paraquat/farmacologiaRESUMO
Spo0Aâ¼P is the master regulator of sporulation in Bacillus subtilis. Activity of Spo0A is regulated by a phosphorelay integrating multiple positive and negative signals by the action of kinases and phosphatases. The phosphatase Spo0E specifically inactivates the response regulator Spo0Aâ¼P by dephosphorylation. We identified a σ(B)-type promoter adjacent to spo0E that is activated by the general stress response sigma factor σ(B) and is responsible for spo0E induction in vivo. Ectopic expression of σ(B) and subsequent induction of spo0E cause a σ(B)-dependent block of sporulation-specific transcription of the spo0A and spoIIE genes and produces a sporulation-deficient phenotype. This effect could be erased by a deletion of the σ(B) promoter of spo0E and thus solely addresses σ(B) activity. Here, a molecular mechanism is shown that integrates σ(B) activity into the decision-making process of sporulation and provides a link to interconnect these two dominant and probably mutually exclusive adaptive responses in the regulatory network of B. subtilis.
Assuntos
Bacillus subtilis/crescimento & desenvolvimento , Bacillus subtilis/genética , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Fator sigma/metabolismo , Esporos Bacterianos/crescimento & desenvolvimento , Esporos Bacterianos/genética , Proteínas de Bactérias/genética , Sítios de Ligação , Perfilação da Expressão Gênica , Regiões Promotoras Genéticas , Ligação Proteica , Fator sigma/genéticaRESUMO
CtsR, the global heat shock repressor in low GC, Gram+ bacteria, regulates a crucial subset of genes involved in protein quality control. CtsR de-repression occurs not only during heat stress but also during a variety of other environmental stresses, most notably thiol-specific oxidative stress. Here we report that McsA acts as a molecular redox switch that regulates CtsR de-repression via the activation of McsB. Once critical thiols of McsA become oxidized, the strong interaction between McsA and McsB is interrupted and free McsB is no longer inhibited by McsA, resulting in the inactivation of CtsR. This mechanism differs significantly from inactivation of CtsR during heat stress demonstrating a dual activity control of CtsR. Moreover, we show that in those low GC, Gram+ bacteria, which lack the McsA/McsB complex, the Zn finger protein ClpE is able to sense and respond to oxidative stress, also resulting in CtsR inactivation.
Assuntos
Bacillus subtilis/metabolismo , Proteínas de Bactérias/metabolismo , Composição de Bases/genética , Proteínas Repressoras/metabolismo , Estresse Fisiológico , Compostos de Sulfidrila/metabolismo , Adenosina Trifosfatases/metabolismo , Bacillus subtilis/efeitos dos fármacos , Bacillus subtilis/genética , Proteínas de Bactérias/genética , Dissulfetos/farmacologia , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Proteínas de Choque Térmico/metabolismo , Resposta ao Choque Térmico/efeitos dos fármacos , Cinética , Modelos Biológicos , Oxirredução/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Proteínas Quinases/metabolismo , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteínas Repressoras/genética , Estresse Fisiológico/efeitos dos fármacos , Dedos de ZincoRESUMO
The general stress response and the decision-making processes of sporulation initiation are interconnected pathways in the regulatory network of Bacillus subtilis. In a previous study we provided evidence for a mechanism capable of impairing sporulation by σ(B) -dependent induction of spo0E, encoding a phosphatase specifically inactivating the sporulation master regulator Spo0A~P. Here we show that the σ(B) promoter (Pσ(B)) of spo0E is responsive to sub-inhibitory levels of ethanol stress, producing a σ(B) -dependent sporulation deficient phenotype. In addition to positive regulation by σ(B) , we identified Rok, the repressor of comK, to be a direct repressor of spo0E expression from Pσ(B) . This constellation provides the possibility to integrate signals negatively acting on sporulation initiation through the σ(B) branch as well as a positive feedback loop acting on Pσ(B) by Rok that is most likely a direct consequence of Spo0A~P activity. Thus, the molecular mechanism described here offers the opportunity for cross-talk between the general stress response and sporulation initiation in the adaptational gene expression network of B. subtilis.
Assuntos
Bacillus subtilis/fisiologia , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Estresse Fisiológico/fisiologia , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Proteínas de Bactérias/genética , Etanol/farmacologia , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Monoéster Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/metabolismo , Regiões Promotoras Genéticas/genética , Esporos Bacterianos/genética , Esporos Bacterianos/metabolismo , Esporos Bacterianos/fisiologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
The alternative sigma factor σ(B) is the master regulator of the general stress regulon that comprises approximately 200 genes whose products confer a comprehensive stress resistance to Bacillus subtilis. The characterization of MgsR (modulator of the general stress response) revealed that the activation and induction of σ(B) are a prerequisite but not sufficient for a full expression of all general stress genes. MgsR is a paralogue of the global regulator of the diamide stress response, Spx, and controls a subregulon of the general stress response. Here we demonstrate that MgsR activity is controlled at multiple levels. These mechanisms include a positive autoregulatory loop on mgsR transcription, a post-translational redox-sensitive activation step by an intramolecular disulfide bond formation in response to ethanol stress in vivo, as well as rapid proteolytic degradation of MgsR by the ClpXP and ClpCP proteases. Our results indicate an elaborate regulatory network integrating secondary oxidative stress signals into a σ(B) -mediated regulatory cascade that is aimed at rapid and finely tuned target gene expression to coordinately fulfil the physiological needs of the cell in the face of multiple environmental changes.
Assuntos
Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Fator sigma/metabolismo , Estresse Fisiológico/genética , Homeostase/genética , Oxirredução , Estabilidade Proteica , Fator sigma/genéticaRESUMO
ATP-dependent intracellular proteolysis is essential for all living organisms. ClpP, the proteolytic subunit of the ATP-dependent Clp proteases, shares 56% protein identity between B. subtilis and man. The aim of this study was to verify, whether human ClpP (HClpP) is able to substitute the bacterial pendant, BClpP, irrespectively of the huge evolutionary distance. For this reason hclpP was expressed from the natural B. subtilis promoters at the original chromosomal site. Growth at 37 °C as well as sporulation in the presence of hclpP depict an intermediate phenotype between wild type and clpP mutant suggesting a partial functional substitution of BClpP by HClpP. Northern as well as Western blot analyses show a similar induction pattern of both, bclpP and hclpP during heat stress on the mRNA as well as on the protein levels. Co-immunoprecipitation experiments imply specific interaction of HClpP with bacterial ClpC, ClpX and ClpE during control as well as heat stress conditions. Radioactive pulse-chase labeling and immunoprecipitation revealed that a ClpXP substrate, the short-living regulatory protein MgsR, is degraded by HClpP, although with an extremely slower rate in comparison to BClpP. The occurrence of an exceptional thickened cell wall of a clpP mutant can be almost fully reversed by the complementation with HClpP. The utilization of the HClpP expressing strain as a test system for new biological or synthetic active substances targeting BClpP is discussed.
Assuntos
Bacillus subtilis/fisiologia , Expressão Ectópica do Gene , Endopeptidase Clp/genética , Bacillus subtilis/ultraestrutura , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Teste de Complementação Genética , Resposta ao Choque Térmico , Humanos , Mutação , Fenótipo , Ligação Proteica , Proteólise , RNA Mensageiro/genéticaRESUMO
Regulated ATP-dependent proteolysis is a common feature of developmental processes and plays also a crucial role during environmental perturbations such as stress and starvation. The Bacillus subtilis MgsR regulator controls a subregulon within the stress- and stationary phase σB regulon. After ethanol exposition and a short time-window of activity, MgsR is ClpXP-dependently degraded with a half-life of approximately 6 min. Surprisingly, a protein interaction analysis with MgsR revealed an association with the McsB arginine kinase and an in vivo degradation assay confirmed a strong impact of McsB on MgsR degradation. In vitro phosphorylation experiments with arginine (R) by lysine (K) substitutions in McsB and its activator McsA unraveled all R residues, which are essentially needed for the arginine kinase reaction. Subsequently, site directed mutagenesis of the MgsR substrate was used to substitute all arginine residues with glutamate (R-E) to mimic arginine phosphorylation and to test their influence on MgsR degradation in vivo. It turned out, that especially the R33E and R94/95E residues (RRPI motif), the latter are adjacently located to the two redox-sensitive cysteines in a 3D model, have the potential to accelerate MgsR degradation. These results imply that selective arginine phosphorylation may have favorable effects for Clp dependent degradation of short-living regulatory proteins. We speculate that in addition to its kinase activity and adaptor function for the ClpC ATPase, McsB might also serve as a proteolytic adaptor for the ClpX ATPase in the degradation mechanism of MgsR.
RESUMO
A combination of pulse-chase experiments and 2-D PAGE revealed that protein degradation appears to play a crucial role for the cell physiology of Staphylococcus aureus COL during extended periods of glucose starvation. The synthesis rate of virtually all cytosolic and radioactively labeled proteins from growing cells seemed dramatically reduced in the first 3.5 h of glucose starvation. The stability of proteins synthesized in growing cells was monitored by a pulse-chase approach on a proteome wide scale. Especially, enzymes involved in nucleic acid and amino acid biosyntheses, energy metabolism and biosynthesis of cofactors were found rather rapidly degraded within the onset of the stationary phase, whereas the majority of glycolytic and tricarboxylic acid cycle enzymes remained more stable. Furthermore, single enzymes of biosynthetic pathways were differentially degraded. A metabolite analysis revealed that glucose completely depleted from the medium in the transient phase, and amino acids such as alanine and glycine were taken up by the cells in the stationary phase. We suggest that vegetative proteins no longer required in non-growing cells and thus no longer protected by integration into functional complexes were degraded. Proteolysis of putative non-substrate-bound or "unemployed" proteins appears to be a characteristic feature of S. aureus in order to access nutrients as an important survival strategy under starvation conditions.
Assuntos
Proteínas de Bactérias/metabolismo , Glucose/metabolismo , Proteoma/análise , Staphylococcus aureus/metabolismo , Inanição , Eletroforese em Gel Bidimensional/métodos , Perfilação da Expressão Gênica , Humanos , Hidrólise , Ressonância Magnética Nuclear BiomolecularRESUMO
The alternative sigma factor sigma(B) of Bacillus subtilis is responsible for the induction of the large general stress regulon comprising approximately 150-200 genes. YqgZ, a member of the sigma(B) regulon, resembles the global regulator Spx of the diamide stress regulon in B. subtilis. In this work we conducted a comprehensive transcriptome and proteome analysis of the B. subtilis wild-type 168 and its isogenic DeltasigB and DeltayqgZ mutants following exposure to 4% (v/v) ethanol stress, which led to the characterization of a 'subregulon' within the general stress response that is regulated by YqgZ. Activation and induction of sigma(B) are necessary but not sufficient for a full expression of all general stress genes. Expression of 53 genes was found to be positively regulated and the expression of 18 genes was negatively affected by YqgZ. The identification of the negatively regulated group represents a so far uncharacterized regulatory phenomenon observed in the DeltasigB mutant background that can now be attributed to the function of YqgZ. Due to the strict sigma(B)-dependent expression of YqgZ it was renamed to MgsR (modulator of the general stress response).