RESUMO
Two-component systems (TCSs) in bacteria are molecular circuits that allow the perception of and response to diverse stimuli. These signaling circuits rely on phosphoryl-group transfers between transmitter and receiver domains of sensor kinase and response regulator proteins, and regulate several cellular processes in response to internal or external cues. Phosphorylation, and thereby activation, of response regulators has been demonstrated to occur by their cognate histidine kinases but also by low molecular weight phosphodonors such as acetyl phosphate and carbamoyl phosphate. Here, we present data indicating that the intermediates of the de novo syntheses of purines and histidine, 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranosyl 5'-monophosphate (ZMP) and/or 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranosyl 5'-triphosphate (ZTP), activate the response regulator UvrY, by promoting its autophosphorylation at the conserved aspartate at position 54. Moreover, these Z nucleotides are shown to also activate the nonrelated response regulators ArcA, CpxR, RcsB, and PhoQ. We propose that ZMP and/or ZTP act as alarmones for a wide range of response regulators in vivo, providing a novel mechanism by which they could impact gene expression in response to metabolic cues.
Assuntos
Escherichia coli/metabolismo , Nucleotídeos/farmacologia , Transdução de Sinais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Genes Bacterianos , Mutação/genética , Fosfatos/farmacologia , Fosforilação/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacosRESUMO
The Arc (anoxic redox control) two-component system of Escherichia coli, comprising ArcA as the response regulator and ArcB as the sensor histidine kinase, modulates the expression of numerous genes in response to respiratory growth conditions. Under reducing growth conditions, ArcB autophosphorylates at the expense of ATP, and transphosphorylates ArcA via a His292 â Asp576 â His717 â Asp54 phosphorelay, whereas under oxidizing growth conditions, ArcB catalyzes the dephosphorylation of ArcA-P by a reverse Asp54 â His717 â Asp576 â Pi phosphorelay. However, the exact phosphoryl group transfer routes and the molecular mechanisms determining their directions are unclear. Here, we show that, during signal propagation, the His292 â Asp576 and Asp576 â His717 phosphoryl group transfers within ArcB dimers occur intra- and intermolecularly, respectively. Moreover, we report that, during signal decay, the phosphoryl group transfer from His717 to Asp576 takes place intramolecularly. In conclusion, we present a mechanism that dictates the direction of the phosphoryl group transfer within ArcB dimers and that enables the discrimination of the kinase and phosphatase activities of ArcB.
Assuntos
Ácido Aspártico/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Histidina/metabolismo , Proteínas de Membrana/metabolismo , Mutação , Proteínas Quinases/metabolismo , Ácido Aspártico/química , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Histidina/química , Proteínas de Membrana/química , Proteínas de Membrana/genética , Fosforilação , Proteínas Quinases/química , Proteínas Quinases/genética , Transdução de SinaisRESUMO
Lipid rafts or membrane microdomains have been proposed to compartmentalize cellular processes by spatially organizing diverse molecules/proteins in eukaryotic cells. Such membrane microdomains were recently reported to also exist in a few bacterial species. In this work, we report the development of a procedure for membrane microdomain isolation from Escherichia coli plasma membranes as well as a method to purify the latter. The method here reported could easily be adapted to other gram-negative bacteria, wherein the isolation of this kind of sub-membrane preparation imposes special difficulties. The analysis of isolated membrane microdomains might provide important information on the nature and function of these bacterial structures and permit their comparison with the ones of eukaryotic cells.
Assuntos
Escherichia coli/química , Microdomínios da Membrana/química , Escherichia coli/metabolismo , Microdomínios da Membrana/metabolismoRESUMO
The Arc two-component system, comprising the ArcB sensor kinase and the ArcA response regulator, modulates the expression of numerous genes in response to respiratory growth conditions. Under aerobic growth conditions, the ubiquinone electron carriers were proposed to silence the kinase activity of ArcB by oxidizing two cytosol-located redox-active cysteine residues that participate in intermolecular disulfide bond formation. Here, we confirm the role of the ubiquinone electron carriers as the silencing signal of ArcB in vivo, we show that the redox potential of ArcB is about -41 mV, and we demonstrate that the menaquinols are required for proper ArcB activation upon a shift from aerobic to anaerobic growth conditions. Thus, an essential link in the Arc signal transduction pathway connecting the redox state of the quinone pool to the transcriptional apparatus is elucidated.
Assuntos
Proteínas de Escherichia coli/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Quinases/metabolismo , Ubiquinona/metabolismo , Vitamina K 2/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Membrana/genética , Oxirredução , Proteínas Quinases/genética , Transdução de Sinais/genética , Transdução de Sinais/fisiologiaRESUMO
The Arc (anoxic redox control) two-component system (TCS) is a complex signal transduction system that plays an important role in regulating energy metabolism at the level of transcription in bacteria. This system comprises the ArcB protein, a hybrid membrane-associated sensor kinase, and the ArcA protein, a typical response regulator. Under anoxic growth conditions, ArcB autophosphorylates and transphosphorylates ArcA via a HisâAspâHisâAsp phosphorelay. Under aerobic conditions, the ArcB kinase activity is silenced by the oxidation of two cytosol-located redox-active cysteine residues that participate in intermolecular disulfide bond formation. Under such conditions, ArcB acts as a phosphatase that catalyzes the dephosphorylation of ArcA-P and thereby releasing its transcriptional regulation. This chapter describes general in vitro and in vivo assays and strategies that have been used to characterize the ArcB/A two-component signal transduction system, which could, also, be applied to most other TCS.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas Quinases/metabolismo , Transdução de Sinais/fisiologia , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Bactérias/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Oxirredução , Fosforilação , Proteínas Quinases/genética , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Transdução de Sinais/genéticaRESUMO
The two-component signal transduction system (TCS) BarA/UvrY activates transcription of CsrB and CsrC noncoding RNAs, which act by sequestering the RNA-binding global regulatory protein CsrA. Here, we show that the metabolic end products formate and acetate provide a physiological stimulus for this TCS and thus link posttranscriptional regulation by the Csr system to the metabolic state of the cell.
Assuntos
Proteínas de Escherichia coli/fisiologia , Escherichia coli/fisiologia , Regulação Bacteriana da Expressão Gênica , Proteínas de Membrana/fisiologia , Fosfotransferases/fisiologia , Transdução de Sinais , Ácido Acético/metabolismo , Formiatos/metabolismoRESUMO
The Arc (anoxic redox control) two-component signal transduction system, consisting of the ArcB sensor kinase and the ArcA response regulator, allows adaptive responses of Escherichia coli to changes of O(2) availability. The arcA gene was previously known as the dye gene because null mutants were growth sensitive to the photosensitizer redox dyes toluidine blue and methylene blue, a phenotype whose molecular basis still remains elusive. In this study we report that the toluidine blue O (TBO) effect on the arc mutants is light independent and observed only during aerobic growth conditions. Moreover, 16 suppressor mutants with restored growth were generated and analyzed. Thirteen of those possessed insertion elements upstream of the cydAB operon, rendering its expression ArcA independent. Also, it was found that, in contrast to cythocrome d, cythocrome o was not able to confer toluidine blue resistance to arc mutants, thereby representing an intriguing difference between the two terminal oxidases. Finally, a mechanism for TBO sensitivity and resistance is discussed.
Assuntos
Proteínas da Membrana Bacteriana Externa/genética , Grupo dos Citocromos b/metabolismo , Grupo dos Citocromos d/metabolismo , Citocromos/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/efeitos dos fármacos , Escherichia coli/crescimento & desenvolvimento , Proteínas Repressoras/genética , Cloreto de Tolônio/farmacologia , Anaerobiose , Proteínas da Membrana Bacteriana Externa/metabolismo , Sequência de Bases , Carotenoides/metabolismo , Catalase/metabolismo , Corantes/farmacologia , Grupo dos Citocromos b/genética , Grupo dos Citocromos d/genética , Citocromos/genética , Escuridão , Complexo de Proteínas da Cadeia de Transporte de Elétrons/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Regulação Bacteriana da Expressão Gênica/genética , Regulação Bacteriana da Expressão Gênica/efeitos da radiação , Glucose/farmacologia , Luz , Dados de Sequência Molecular , Mutação/genética , Oxirredutases/genética , Regiões Promotoras Genéticas/genética , Espécies Reativas de Oxigênio/metabolismo , Proteínas Repressoras/metabolismo , Homologia de Sequência do Ácido Nucleico , Superóxido Dismutase/metabolismoRESUMO
BACKGROUND: Members of the genus Rhodococcus are frequently found in soil and other natural environments and are highly resistant to stresses common in those environments. The accumulation of storage compounds permits cells to survive and metabolically adapt during fluctuating environmental conditions. The purpose of this study was to perform a genome-wide bioinformatic analysis of key genes encoding metabolism of diverse storage compounds by Rhodococcus jostii RHA1 and to examine its ability to synthesize and accumulate triacylglycerols (TAG), wax esters, polyhydroxyalkanoates (PHA), glycogen and polyphosphate (PolyP). RESULTS: We identified in the RHA1 genome: 14 genes encoding putative wax ester synthase/acyl-CoA:diacylglycerol acyltransferase enzymes (WS/DGATs) likely involved in TAG and wax esters biosynthesis; a total of 54 genes coding for putative lipase/esterase enzymes possibly involved in TAG and wax ester degradation; 3 sets of genes encoding PHA synthases and PHA depolymerases; 6 genes encoding key enzymes for glycogen metabolism, one gene coding for a putative polyphosphate kinase and 3 putative exopolyphosphatase genes. Where possible, key amino acid residues in the above proteins (generally in active sites, effectors binding sites or substrate binding sites) were identified in order to support gene identification. RHA1 cells grown under N-limiting conditions, accumulated TAG as the main storage compounds plus wax esters, PHA (with 3-hydroxybutyrate and 3-hydroxyvalerate monomers), glycogen and PolyP. Rhodococcus members were previously known to accumulate TAG, wax esters, PHAs and polyP, but this is the first report of glycogen accumulation in this genus. CONCLUSION: RHA1 possess key genes to accumulate diverse storage compounds. Under nitrogen-limiting conditions lipids are the principal storage compounds. An extensive capacity to synthesize and metabolize storage compounds appears to contribute versatility to RHA1 in its responses to environmental stresses.