RESUMO
The glucose-phosphotransferase system (PTS) in Escherichia coli K-12 is a complex sensory and regulatory system. In addition to its central role in glucose uptake, it informs other global regulatory networks about carbohydrate availability and the physiological status of the cell. The expression of the ptsG gene encoding the glucose-PTS transporter EIICB(Glc) is primarily regulated via the repressor Mlc, whose inactivation is glucose dependent. During transport of glucose and dephosphorylation of EIICB(Glc), Mlc binds to the B domain of the transporter, resulting in derepression of several Mlc-regulated genes. In addition, Mlc can also be inactivated by the cytoplasmic protein MtfA in a direct protein-protein interaction. In this study, we identified the binding site for Mlc in the carboxy-terminal region of MtfA by measuring the effect of mutated MtfAs on ptsG expression. In addition, we demonstrated the ability of MtfA to inactivate an Mlc super-repressor, which cannot be inactivated by EIICB(Glc), by using in vivo titration and gel shift assays. Finally, we characterized the proteolytic activity of purified MtfA by monitoring cleavage of amino 4-nitroanilide substrates and show Mlc's ability to enhance this activity. Based on our findings, we propose a model of MtfA as a glucose-regulated peptidase activated by cytoplasmic Mlc. Its activity may be necessary during the growth of cultures as they enter the stationary phase. This proteolytic activity of MtfA modulated by Mlc constitutes a newly identified PTS output signal that responds to changes in environmental conditions.
Assuntos
Escherichia coli K12/metabolismo , Proteínas de Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Peptídeo Hidrolases/metabolismo , Sistema Fosfotransferase de Açúcar do Fosfoenolpiruvato/biossíntese , Mapeamento de Interação de Proteínas , Proteínas Repressoras/metabolismo , Sítios de Ligação , Ensaio de Desvio de Mobilidade Eletroforética , Escherichia coli K12/genética , Peptídeo Hidrolases/genética , Ligação Proteica , Domínios e Motivos de Interação entre ProteínasRESUMO
MtfA of Escherichia coli (formerly YeeI) was previously identified as a regulator of the phosphoenolpyruvate (PEP)-dependent:glucose phosphotransferase system. MtfA homolog proteins are highly conserved, especially among beta- and gammaproteobacteria. We determined the crystal structures of the full-length MtfA apoenzyme from Klebsiella pneumoniae and its complex with zinc (holoenzyme) at 2.2 and 1.95 Å, respectively. MtfA contains a conserved H(149)E(150)XXH(153)+E(212)+Y(205) metallopeptidase motif. The presence of zinc in the active site induces significant conformational changes in the region around Tyr205 compared to the conformation of the apoenzyme. Additionally, the zinc-bound MtfA structure is in a self-inhibitory conformation where a region that was disordered in the unliganded structure is now observed in the active site and a nonproductive state of the enzyme is formed. MtfA is related to the catalytic domain of the anthrax lethal factor and the Mop protein involved in the virulence of Vibrio cholerae, with conservation in both overall structure and in the residues around the active site. These results clearly provide support for MtfA as a prototypical zinc metallopeptidase (gluzincin clan).
Assuntos
Antígenos de Bactérias/química , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/química , Klebsiella pneumoniae/enzimologia , Metaloproteases/química , Metaloproteases/metabolismo , Zinco/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Antígenos de Bactérias/genética , Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/genética , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Domínio Catalítico , Cristalografia por Raios X , Klebsiella pneumoniae/química , Klebsiella pneumoniae/genética , Metaloendopeptidases/química , Metaloendopeptidases/genética , Metaloendopeptidases/metabolismo , Metaloproteases/genética , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Alinhamento de SequênciaRESUMO
Glucose is the classical carbon source that is used to investigate the transport, metabolism, and regulation of nutrients in bacteria. Many physiological phenomena like nutrient limitation, stress responses, production of antibiotics, and differentiation are inextricably linked to nutrition. Over the years glucose transport systems have been characterized at the molecular level in more than 20 bacterial species. This review aims to provide an overview of glucose uptake systems found in the eubacterial kingdom. In addition, it will highlight the diverse and sophisticated regulatory features of glucose transport systems.
Assuntos
Bactérias/metabolismo , Glucose/metabolismo , Bactérias/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Transporte Biológico , Regulação Bacteriana da Expressão Gênica , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismoRESUMO
The PEP-dependent carbohydrate:phosphotransferase systems (PTSs) of enteric bacteria constitute a complex sensory system which involves as its central element a PEP-dependent His-protein kinase (Enzyme I). As a unit, the PTS comprises up to 20 different transporters per cell which correspond to its chemoreceptors for PTS carbohydrates, and several targeting subunits, which include in the low [G+C] Gram-positive bacteria an ancillary Ser/Thr-protein kinase. The PTS senses the presence of carbohydrates, in particular glucose, in the medium and the energy state of the cell, in the form of either the intracellular PEP-to-pyruvate ratio or the D-fructose-bisphosphate levels. This information is subsequently communicated to cellular targets, in particular those involved in the chemotactic response of the cell towards PTS carbohydrates, and in sensing glucose in the medium, using cAMP and several targeting subunits as intermediates. Peptide targeting subunits ensure the fast, transient, and yet accurate communication of the PTS with its more than hundred different targets, avoiding at the same time unwanted cross-talk. Many elements of this sensory system are simultaneously elements of specific and global regulatory networks. Thus, the PTS controls, besides the immediate (in the ms to s range) chemotactic responses, the activity of the various carbohydrate transporters and enzymes involved in carbon and energy metabolism through inducer exclusion, and in a delayed response (in the min to h range) the synthesis of these transporters and catabolic enzymes through catabolite repression. Indirect consequences of this program are phenomena related to cell surface rearrangements, which include flagella synthesis, as well as memory, adaptation, and learning effects. The analogy between the PTS and other prokaryotic systems, and more complex sensory systems from eukaryotic organisms which share elements with regulatory systems is obvious.
Assuntos
Fenômenos Fisiológicos Bacterianos , Metabolismo dos Carboidratos , Sistema Fosfotransferase de Açúcar do Fosfoenolpiruvato/fisiologia , Percepção de Quorum/fisiologia , Transdução de Sinais , Transporte Biológico , Quimiotaxia , Simulação por Computador , Glucose/metabolismo , Fosfoenolpiruvato/metabolismo , Ácido Pirúvico/metabolismoRESUMO
An automated flow-injection system with an integrated biosensor array using bacterial cells for the selective and simultaneous determination various mono- and disaccharides is described. The selectivity of the individually addressable sensors of the array was achieved by the combination of the metabolic response, measured as the O(2) consumption, of bacterial mutants of Escherichia coli K12 lacking different transport systems for individual carbohydrates. Kappa-carrageenan was used as immobilization matrix for entrapment of the bacterial cells in front of 6 individually addressable working electrodes of a screen-printed sensor array. The local consumption of molecular oxygen caused by the metabolic activity of the immobilized cells was amperometrically determined at the underlying screen-printed gold electrodes at a working potential of -600 mV vs. Ag/AgCl. Addition of mono- or disaccharides for which functional transport systems exist in the used transport mutant strains of E. coli K12 leads to an enhanced metabolic activity of the immobilized bacterial cells and to a concomitant depletion of oxygen at the electrode. Parallel determination of fructose, glucose, and sucrose was performed demonstrating the high selectivity of the proposed analytical system.
Assuntos
Biofilmes , Técnicas Biossensoriais/instrumentação , Dissacarídeos/análise , Eletrodos , Escherichia coli/metabolismo , Monossacarídeos/análise , Transporte Biológico Ativo/genética , Técnicas Biossensoriais/métodos , Células Cultivadas , Células Imobilizadas , Eletroquímica/instrumentação , Eletroquímica/métodos , Escherichia coli/genética , Análise de Injeção de Fluxo/instrumentação , Análise de Injeção de Fluxo/métodos , Frutose/análise , Glucose/análise , Mutação , Oxigênio/metabolismo , Sensibilidade e Especificidade , Especificidade da Espécie , Sacarose/análiseRESUMO
The monocarboxylate pyruvate is an important metabolite and can serve as sole carbon source for Escherichia coli. Although specific pyruvate transporters have been identified in two bacterial species, pyruvate transport is not well understood in E. coli. In the present study, pyruvate transport was investigated under different growth conditions. The transport of pyruvate shows specific activities depending on the growth substrate used as sole carbon source, suggesting the existence of at least two systems for pyruvate uptake: i) one inducible system and probably highly specific for pyruvate and ii) one system active under non-induced conditions. Using the toxic pyruvate analog 3-fluoropyruvate, a mutant was isolated unable to grow on and transport pyruvate. Further investigation revealed that a revertant selected for growth on pyruvate regained the inducible pyruvate transport activity. Characterization of pyruvate excretion showed that the pyruvate transport negative mutant accumulated pyruvate in the growth medium suggesting an additional transport system for pyruvate excretion. The here presented data give valuable insight into the pyruvate metabolism and transport of E. coli suggesting the presence of at least two uptake systems and one excretion system to balance the intracellular level of pyruvate.
Assuntos
Escherichia coli/genética , Escherichia coli/metabolismo , Mutação , Ácido Pirúvico/farmacocinética , Transporte Biológico/efeitos dos fármacos , Transporte Biológico/genética , Escherichia coli/crescimento & desenvolvimento , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Cinética , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Potássio/farmacologia , Piruvatos/metabolismo , Piruvatos/farmacocinética , Ácido Pirúvico/metabolismoRESUMO
Escherichia coli is a widely used microorganism in biotechnological processes. An obvious goal for current scientific and technical research in this field is the search for new tools to optimize productivity. Usually glucose is the preferred carbon source in biotechnological applications. In E. coli, glucose is taken up by the phosphoenolpyruvate-dependent glucose phosphotransferase system (PTS). The regulation of the ptsG gene for the glucose transporter is very complex and involves several regulatory proteins. Recently, a novel posttranscriptional regulation system has been identified which consists of a small regulatory RNA SgrS and a small regulatory polypeptide called SgrT. During the accumulation of glucose-6-phosphate or fructose-6-phosphate, SgrS is involved in downregulation of ptsG mRNA stability, whereas SgrT inhibits glucose transport activity by a yet unknown mechanism. The function of SgrS has been studied intensively. In contrast, the knowledge about the function of SgrT is still limited. Therefore, in this paper, we focused our interest on the regulation of glucose transport activity by SgrT. We identified the SgrT target sequence within the glucose transporter and characterized the interaction in great detail. Finally, we suggest a novel experimental approach to regulate artificially carbohydrate uptake in E. coli to minimize metabolic overflow in biotechnological applications.
RESUMO
Metabolism and signalling are tightly coupled in bacteria. Combining several theoretical approaches, a core model is presented that describes transcriptional and allosteric control of glycolysis in Escherichia coli. Experimental data based on microarrays, signalling components and extracellular metabolites are used to estimate kinetic parameters. A newly designed strain was used that adjusts the incoming glucose flux into the system and allows a kinetic analysis. Based on the results, prediction for intracelluar metabolite concentrations over a broad range of the growth rate could be performed and compared with data from literature.
RESUMO
The phosphoenolpyruvate-(PEP)-dependent-carbohydrate:phosphotransferase systems (PTSs) of enteric bacteria constitute a complex transport and sensory system. Such a PTS usually consists of two cytoplasmic energy-coupling proteins, Enzyme I (EI) and HPr, and one of more than 20 different carbohydrate-specific membrane proteins named Enzyme II (EII), which catalyze the uptake and concomitant phosphorylation of numerous carbohydrates. The most prominent representative is the glucose-PTS, which uses a PTS-typical phosphorylation cascade to transport and phosphorylate glucose. All components of the glucose-PTS interact with a large number of non-PTS proteins to regulate the carbohydrate flux in the bacterial cell. Several aspects of the glucose-PTS have been intensively investigated in various research projects of many groups. In this article we will review our recent findings on a Glc-PTS-dependent metalloprotease, on the interaction of EIICB(Glc) with the regulatory peptide SgrT, on the structure of the membrane spanning C-domain of the glucose transporter and on the modeling approaches of ptsG regulation, respectively, and discuss them in context of general PTS research.
Assuntos
Escherichia coli K12/metabolismo , Sistema Fosfotransferase de Açúcar do Fosfoenolpiruvato/metabolismo , Metabolismo dos Carboidratos , Escherichia coli K12/enzimologia , Fosforilação , Transdução de SinaisRESUMO
BACKGROUND: The pyruvate dehydrogenase regulator protein (PdhR) of Escherichia coli acts as a transcriptional regulator in a pyruvate dependent manner to control central metabolic fluxes. However, the complete PdhR regulon has not yet been uncovered. To achieve an extended understanding of its gene regulatory network, we combined large-scale network inference and experimental verification of results obtained by a systems biology approach. RESULTS: 22 new genes contained in two operons controlled by PdhR (previously only 20 regulatory targets in eight operons were known) were identified by analysing a large-scale dataset of E. coli from the Many Microbes Microarray Database and novel expression data from a pdhR knockout strain, as well as a PdhR overproducing strain. We identified a regulation of the glycolate utilization operon glcDEFGBA using chromatin immunoprecipitation and gel shift assays. We show that this regulation could be part of a cross-induction between genes necessary for acetate and pyruvate utilisation controlled through PdhR. Moreover, a link of PdhR regulation to the replication machinery of the cell via control of the transcription of the dcw-cluster was verified in experiments. This augments our knowledge of the functions of the PdhR-regulon and demonstrates its central importance for further cellular processes in E. coli. CONCLUSIONS: We extended the PdhR regulon by 22 new genes contained in two operons and validated the regulation of the glcDEFGBA operon for glycolate utilisation and the dcw-cluster for cell division proteins experimentally. Our results provide, for the first time, a plausible regulatory link between the nutritional status of the cell and cell replication mediated by PdhR.
Assuntos
Proteínas de Escherichia coli/fisiologia , Escherichia coli/genética , Proteínas Repressoras/fisiologia , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Redes Reguladoras de Genes , Genes Bacterianos , Redes e Vias Metabólicas , Regulon/fisiologia , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Biologia de Sistemas/métodosRESUMO
BACKGROUND: Although Escherichia coli is one of the best studied model organisms, a comprehensive understanding of its gene regulation is not yet achieved. There exist many approaches to reconstruct regulatory interaction networks from gene expression experiments. Mutual information based approaches are most useful for large-scale network inference. RESULTS: We used a three-step approach in which we combined gene regulatory network inference based on directed information (DTI) and sequence analysis. DTI values were calculated on a set of gene expression profiles from 19 time course experiments extracted from the Many Microbes Microarray Database. Focusing on influences between pairs of genes in which one partner encodes a transcription factor (TF) we derived a network which contains 878 TF - gene interactions of which 166 are known according to RegulonDB. Afterward, we selected a subset of 109 interactions that could be confirmed by the presence of a phylogenetically conserved binding site of the respective regulator. By this second step, the fraction of known interactions increased from 19% to 60%. In the last step, we checked the 44 of the 109 interactions not yet included in RegulonDB for functional relationships between the regulator and the target and, thus, obtained ten TF - target gene interactions. Five of them concern the regulator LexA and have already been reported in the literature. The remaining five influences describe regulations by Fis (with two novel targets), PhdR, PhoP, and KdgR. For the validation of our approach, one of them, the regulation of lipoate synthase (LipA) by the pyruvate-sensing pyruvate dehydrogenate repressor (PdhR), was experimentally checked and confirmed. CONCLUSIONS: We predicted a set of five novel TF - target gene interactions in E. coli. One of them, the regulation of lipA by the transcriptional regulator PdhR was validated experimentally. Furthermore, we developed DTInfer, a new R-package for the inference of gene-regulatory networks from microarrays using directed information.
Assuntos
Escherichia coli/genética , Redes Reguladoras de Genes , Análise de Sequência de DNA , Biologia de Sistemas/métodos , Sequência de Bases , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Genômica , Reprodutibilidade dos TestesRESUMO
We report here the molecular identification of a glucose permease from Mycobacterium smegmatis,a model organism for our understanding of the life patterns of the major pathogens Mycobacterium tuberculosis and Mycobacterium leprae. A computer-based search of the available genome of M. smegmatis mc(2) 155 with the sequences of well-characterized glucose transporters revealed the gene msmeg4187 as a possible candidate. The deduced protein belongs to the major facilitator superfamily of proton symporters and facilitators and exhibits up to 53% of amino acid identity to other members of this family. Heterologous expression of msmeg4187 in an Escherichia coli glucose-negative mutant led to the restoration of growth on glucose. The determination of the biochemical features characterize MSMEG4187 (GlcP) as a high affinity (K(m) of 19 microM), glucose-specific permease. The results represent the first molecular characterization of a sugar permease in mycobacteria, and thus supply fundamental data for further in-depth analysis on the nutritional lifestyle of these bacteria.
Assuntos
Transporte Biológico , Glucose/metabolismo , Proteínas de Membrana Transportadoras/genética , Mycobacterium smegmatis/enzimologia , Mycobacterium smegmatis/genética , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Glucose/genética , Proteínas de Membrana Transportadoras/metabolismo , Dados de Sequência Molecular , Mycobacterium smegmatis/fisiologia , FilogeniaRESUMO
In Escherichia coli K-12, components of the phosphoenolpyruvate-dependent phosphotransferase systems (PTSs) represent a signal transduction system involved in the global control of carbon catabolism through inducer exclusion mediated by phosphoenolpyruvate-dependent protein kinase enzyme IIA(Crr) (EIIA(Crr)) (= EIIA(Glc)) and catabolite repression mediated by the global regulator cyclic AMP (cAMP)-cAMP receptor protein (CRP). We measured in a systematic way the relation between cellular growth rates and the key parameters of catabolite repression, i.e., the phosphorylated EIIA(Crr) (EIIA(Crr) approximately P) level and the cAMP level, using in vitro and in vivo assays. Different growth rates were obtained by using either various carbon sources or by growing the cells with limited concentrations of glucose, sucrose, and mannitol in continuous bioreactor experiments. The ratio of EIIA(Crr) to EIIA(Crr) approximately P and the intracellular cAMP concentrations, deduced from the activity of a cAMP-CRP-dependent promoter, correlated well with specific growth rates between 0.3 h(-1) and 0.7 h(-1), corresponding to generation times of about 138 and 60 min, respectively. Below and above this range, these parameters were increasingly uncoupled from the growth rate, which perhaps indicates an increasing role executed by other global control systems, in particular the stringent-relaxed response system.
Assuntos
Proteína Receptora de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Reatores Biológicos/microbiologia , Proteína Receptora de AMP Cíclico/genética , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Proteínas de Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Glucose/metabolismo , Manitol/metabolismo , Sistema Fosfotransferase de Açúcar do Fosfoenolpiruvato/genética , Sistema Fosfotransferase de Açúcar do Fosfoenolpiruvato/metabolismo , Fosforilação , Regiões Promotoras Genéticas/genética , Sacarose/metabolismoRESUMO
Carbon metabolism and regulation is poorly understood in mycobacteria, a genus that includes some major pathogenic species like Mycobacterium tuberculosis and Mycobacterium leprae. Here, we report the identification of a glucose kinase from Mycobacterium smegmatis. This enzyme serves in glucose metabolism and global carbon catabolite repression in the related actinomycete Streptomyces coelicolor. The gene, msmeg1356 (glkA), was found by means of in silico screening. It was shown that it occurs in the same genetic context in all so far sequenced mycobacterial species, where it is located in a putative tricistronic operon together with a glycosyl hydrolase and a putative malonyl-CoA transacylase. Heterologous expression of glkA in an Escherichia coli glucose kinase mutant led to the restoration of glucose growth, which provided in vivo evidence for glucose kinase function. GlkA(Msm) was subsequently overproduced in order to study its enzymatic features. We found that it can form a dimer and that it efficiently phosphorylates glucose at the expense of ATP. The affinity constant for glucose was with 9 mM about eight times higher and the velocity was about tenfold slower when compared to the parallel measured glucose kinase of S. coelicolor. Both enzymes showed similar substrate specificity, which consists in an ATP-dependent phosphorylation of glucose and no, or very inefficient, phosphorylation of the glucose analogues 2-deoxyglucose and methyl alpha-glucoside. Hence, our data provide a basis for studying the role of mycobacterial glucose kinase in vivo to unravel possible catalytic and regulatory functions.
Assuntos
Proteínas de Bactérias/metabolismo , Genes Bacterianos , Glucoquinase/metabolismo , Glucose/metabolismo , Mycobacterium smegmatis/enzimologia , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Dimerização , Escherichia coli/genética , Escherichia coli/metabolismo , Glucoquinase/genética , Dados de Sequência Molecular , Mutação , Mycobacterium smegmatis/genética , Óperon , Fosforilação , Filogenia , Especificidade por SubstratoRESUMO
A dynamic mathematical model was developed to describe the uptake of various carbohydrates (glucose, lactose, glycerol, sucrose, and galactose) in Escherichia coli. For validation a number of isogenic strains with defined mutations were used. By considering metabolic reactions as well as signal transduction processes influencing the relevant pathways, we were able to describe quantitatively the phenomenon of catabolite repression in E. coli. We verified model predictions by measuring time courses of several extra- and intracellular components such as glycolytic intermediates, EII-ACrr phosphorylation level, both LacZ and PtsG concentrations, and total cAMP concentrations under various growth conditions. The entire data base consists of 18 experiments performed with nine different strains. The model describes the expression of 17 key enzymes, 38 enzymatic reactions, and the dynamic behavior of more than 50 metabolites. The different phenomena affecting the phosphorylation level of EIIACrr, the key regulation molecule for inducer exclusion and catabolite repression in enteric bacteria, can now be explained quantitatively.
Assuntos
Metabolismo dos Carboidratos , Escherichia coli/metabolismo , Modelos Biológicos , Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Cinética , Mutação , Sistema Fosfotransferase de Açúcar do Fosfoenolpiruvato/metabolismo , Fosforilação , Transdução de SinaisRESUMO
The membrane-bound protein EIICB(Glc) encoded by the ptsG gene is the major glucose transporter in Escherichia coli. This protein is part of the phosphoenolpyruvate:glucose-phosphotransferase system, a very important transport and signal transduction system in bacteria. The regulation of ptsG expression is very complex. Among others, two major regulators, the repressor Mlc and the cyclic AMP-cyclic AMP receptor protein activator complex, have been identified. Here we report identification of a novel protein, YeeI, that is involved in the regulation of ptsG by interacting with Mlc. Mutants with reduced activity of the glucose-phosphotransferase system were isolated by transposon mutagenesis. One class of mutations was located in the open reading frame yeeI at 44.1 min on the E. coli K-12 chromosome. The yeeI mutants exhibited increased generation times during growth on glucose, reduced transport of methyl-alpha-d-glucopyranoside, a substrate of EIICB(Glc), reduced induction of a ptsG-lacZ operon fusion, and reduced catabolite repression in lactose/glucose diauxic growth experiments. These observations were the result of decreased ptsG expression and a decrease in the amount of EIICB(Glc). In contrast, overexpression of yeeI resulted in higher expression of ptsG, of a ptsG-lacZ operon fusion, and of the autoregulated dgsA gene. The effect of a yeeI mutation could be suppressed by introducing a dgsA deletion, implying that the two proteins belong to the same signal transduction pathway and that Mlc is epistatic to YeeI. By measuring the surface plasmon resonance, we found that YeeI (proposed gene designation, mtfA) directly interacts with Mlc with high affinity.
Assuntos
Escherichia coli K12/enzimologia , Proteínas de Escherichia coli/metabolismo , Sistema Fosfotransferase de Açúcar do Fosfoenolpiruvato/metabolismo , Cromossomos Bacterianos/genética , Meios de Cultura , Escherichia coli K12/genética , Escherichia coli K12/crescimento & desenvolvimento , Proteínas de Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos , Glucose , Lactose , Mutação , Fases de Leitura Aberta , Sistema Fosfotransferase de Açúcar do Fosfoenolpiruvato/genética , Ligação Proteica , Proteínas Repressoras/metabolismoRESUMO
CheA is a multidomain histidine kinase for chemotaxis in Escherichia coli. CheA autophosphorylates through interaction of its N-terminal phosphorylation site domain (P1) with its central dimerization (P3) and ATP-binding (P4) domains. This activity is modulated through the C-terminal P5 domain, which couples CheA to chemoreceptor control. CheA phosphoryl groups are donated to two response regulators, CheB and CheY, to control swimming behavior. The phosphorylated forms of CheB and CheY turn over rapidly, enabling receptor signaling complexes to elicit fast behavioral responses by regulating the production and transmission of phosphoryl groups from CheA. To promote rapid phosphotransfer reactions, CheA contains a phosphoacceptor-binding domain (P2) that serves to increase CheB and CheY concentrations in the vicinity of the adjacent P1 phosphodonor domain. To determine whether the P2 domain is crucial to CheA's signaling specificity, we constructed CheADeltaP2 deletion mutants and examined their signaling properties in vitro and in vivo. We found that CheADeltaP2 autophosphorylated and responded to receptor control normally but had reduced rates of phosphotransfer to CheB and CheY. This defect lowered the frequency of tumbling episodes during swimming and impaired chemotactic ability. However, expression of additional P1 domains in the CheADeltaP2 mutant raised tumbling frequency, presumably by buffering the irreversible loss of CheADeltaP2-generated phosphoryl groups from CheB and CheY, and greatly improved its chemotactic ability. These findings suggest that P2 is not crucial for CheA signaling specificity and that the principal determinants that favor appropriate phosphoacceptor partners, or exclude inappropriate ones, most likely reside in the P1 domain.
Assuntos
Proteínas de Bactérias/fisiologia , Quimiotaxia , Proteínas de Membrana/fisiologia , Sítios de Ligação , Proteínas de Escherichia coli , Histidina Quinase , Proteínas Quimiotáticas Aceptoras de Metil , Fenótipo , Fosforilação , Transdução de SinaisRESUMO
We have investigated the crr gene of Streptomyces coelicolor that encodes a homologue of enzyme IIAGlucose of Escherichia coli, which, as a component of the phosphoenolpyruvate-dependent sugar phosphotransferase system (PTS) plays a key role in carbon regulation by triggering glucose transport, carbon catabolite repression, and inducer exclusion. As in E. coli, the crr gene of S. coelicolor is genetically associated with the ptsI gene that encodes the general phosphotransferase enzyme I. The gene product IIACrr was overproduced, purified, and polyclonal antibodies were obtained. Western blot analysis revealed that IIACrr is expressed in vivo. The functionality of IIACrr was demonstrated by phosphoenolpyruvate-dependent phosphorylation via enzyme I and the histidine-containing phosphoryl carrier protein HPr. Phosphorylation was abolished when His72, which corresponds to the catalytic histidine of E. coli IIAGlucose, was mutated. The capacity of IIACrr to operate in sugar transport was shown by complementation of the E. coli glucose-PTS. The striking functional resemblance between IIACrr and IIAGlucose was further demonstrated by its ability to confer inducer exclusion of maltose to E. coli. A specific interaction of IIACrr with the maltose permease subunit MalK from Salmonella typhimurium was uncovered by surface plasmon resonance. These data suggest that this IIAGlucose-like protein may be involved in carbon metabolism in S. coelicolor.
Assuntos
Sistema Fosfotransferase de Açúcar do Fosfoenolpiruvato/metabolismo , Fosfotransferases/metabolismo , Streptomyces/enzimologia , Sequência de Aminoácidos , Transporte Biológico , Escherichia coli/enzimologia , Proteínas de Escherichia coli , Dados de Sequência Molecular , Sistema Fosfotransferase de Açúcar do Fosfoenolpiruvato/genética , Fosfotransferases/genética , Análise de Sequência de DNARESUMO
The maltose ATP-binding cassette transporter of Salmonella typhimurium is composed of a membrane-associated complex (MalFGK2) and a periplasmic receptor (MalE). In addition to its role in transport, the complex acts as a repressor of maltose-regulated gene expression and is subject to inhibition in the process of inducer exclusion. These activities are thought to be mediated by interactions of the ATPase subunit, MalK, with the transcriptional activator, MalT, and nonphosphorylated enzyme IIA of the glucose phosphotransferase system, respectively. To gain further insight in protein regions that are critical for these functions, we have generated nine MalK-specific monoclonal antibodies. These bind to four nonoverlapping linear epitopes: 60-LFig-63 (5B5), 113-RVNQVAEVLQL-123 (represented by 4H12), 309-GHETQI-314 (2F9) and 352-LFREDGSACR-361 (represented by 4B3). All mAbs recognize their epitopes in soluble MalK and in the MalFGK2 complex with Kd values ranging from 10-6 to 10-8 m. ATP reduced the affinity of the mAbs for soluble MalK, indicating a conformational change that renders the epitopes less accessible. 4H12 and 5B5 inhibit the ATPase activity of MalK and the MalE/maltose-stimulated ATPase activity of proteoliposomes, while their Fab fragments displayed no significant effect. The results suggest a similar solvent-exposed position of helix 3 in the MalK dimer and in the intact complex and might argue against a direct role in the catalytic process. 4B3 and 2F9 exhibit reduced binding to the MalFGK2 complex in the presence of MalT and enzyme IIAGlc, respectively, thereby providing the first direct evidence for the C-terminal domain of MalK being the site of interaction with the regulatory proteins.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Anticorpos Antibacterianos/imunologia , Anticorpos Monoclonais/imunologia , Proteínas de Bactérias/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Ligação a DNA , Proteínas de Escherichia coli , Salmonella typhimurium/metabolismo , Fatores de Transcrição , Transportadores de Cassetes de Ligação de ATP/imunologia , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Animais , Anticorpos Antibacterianos/isolamento & purificação , Anticorpos Monoclonais/isolamento & purificação , Especificidade de Anticorpos , Antígenos de Bactérias/imunologia , Proteínas de Bactérias/imunologia , Dimerização , Epitopos/imunologia , Feminino , Proteínas Ligantes de Maltose , Camundongos , Camundongos Endogâmicos BALB C , Modelos Moleculares , Dados de Sequência Molecular , Sistema Fosfotransferase de Açúcar do Fosfoenolpiruvato/metabolismo , Conformação Proteica , Estrutura Terciária de Proteína , Proteolipídeos/metabolismo , Salmonella typhimurium/imunologiaRESUMO
Although Escherichia coli strain EC3132 possesses a chromosomally encoded sucrose metabolic pathway, its growth on low sucrose concentrations (5 mM) is unusually slow, with a doubling time of 20 h. In this report we describe the subcloning and further characterization of the corresponding csc genes and adjacent genes. The csc regulon comprises three genes for a sucrose permease, a fructokinase, and a sucrose hydrolase (genes cscB, cscK, and cscA, respectively). The genes are arranged in two operons and are negatively controlled at the transcriptional level by the repressor CscR. Furthermore, csc gene expression was found to be cyclic AMP-CrpA dependent. A comparison of the genomic sequences of the E. coli strains EC3132, K-12, and O157:H7 in addition to Salmonella enterica serovar Typhimurium LT2 revealed that the csc genes are located in a hot spot region for chromosomal rearrangements in enteric bacteria. The comparison further indicated that the csc genes might have been transferred relatively recently to the E. coli wild-type EC3132 at around the time when the different strains of the enteric bacteria diverged. We found evidence that a mobile genetic element, which used the gene argW for site-specific integration into the chromosome, was probably involved in this horizontal gene transfer and that the csc genes are still in the process of optimal adaptation to the new host. Selection for such adaptational mutants growing faster on low sucrose concentrations gave three different classes of mutants. One class comprised cscR(Con) mutations that expressed all csc genes constitutively. The second class constituted a cscKo operator mutation, which became inducible for csc gene expression at low sucrose concentrations. The third class was found to be a mutation in the sucrose permease that caused an increase in transport activity.