RESUMO
Respiratory NADH dehydrogenase-2 (NDH-2) of Escherichia coli is a peripheral membrane-bound flavoprotein. By eliminating its C-terminal region, a water soluble truncated version was obtained in our laboratory. Overall conformation of the mutant version resembles the wild-type protein. Considering these data and the fact that the mutant was obtained as an apo-protein, the truncated version is an ideal model to study the interaction between the enzyme and its cofactor. Here, the FAD binding properties of this version were characterized using far-UV circular dichroism (CD), differential scanning calorimetry (DSC), limited proteolysis, and steady-state and dynamic fluorescence spectroscopy. CD spectra, thermal unfolding and DSC profiles did not reveal any major difference in secondary structure between apo- and holo-protein. In addition, digestion site accessibility and tertiary conformation were similar for both proteins, as seen by comparable chymotryptic cleavage patterns. FAD binding to the apo-protein produced a parallel increment of both FAD fluorescence quantum yield and steady-state emission anisotropy. On the other hand, addition of FAD quenched the intrinsic fluorescence emission of the truncated protein, indicating that the flavin cofactor should be closely located to the protein Trp residues. Analysis of the steady-state and dynamic fluorescence data confirms the formation of the holo-protein with a 1:1 binding stoichiometry and an association constant KA=7.0(±0.8)×10(4)M(-1). Taken together, the FAD-protein interaction is energetically favorable and the addition of FAD is not necessary to induce the enzyme folded state. For the first time, a detailed characterization of the flavin:protein interaction was performed among alternative NADH dehydrogenases.
Assuntos
Citosol/enzimologia , Escherichia coli/enzimologia , Flavina-Adenina Dinucleotídeo/metabolismo , NADH Desidrogenase/metabolismo , Varredura Diferencial de Calorimetria , Dicroísmo Circular , Espectrometria de Fluorescência , Espectrofotometria UltravioletaRESUMO
In most natural environments, association with a surface in a structure known as biofilm is the prevailing microbial life-style of bacteria. Polyphosphate (polyP), an ubiquitous linear polymer of hundreds of orthophosphate residues, has a crucial role in stress responses, stationary-phase survival, and it was associated to bacterial biofilm formation and production of virulence factors. In previous work, we have shown that Escherichia coli cells grown in media containing a critical phosphate concentration >37 mM maintained an unusual high polyP level in stationary phase. The aim of the present work was to analyze if fluctuations in polyP levels in stationary phase affect biofilm formation capacity in E. coli. Polymer levels were modulated by the media phosphate concentration or using mutant strains in polyP metabolism. Cells grown in media containing phosphate concentrations higher than 25 mM were defective in biofilm formation. Besides, there was a disassembly of 24 h preformed biofilm by the addition of high phosphate concentration to the medium. These phenotypes were related to the maintenance or re-synthesis of polyP in stationary phase in static conditions. No biofilm formation was observed in ppk(-)ppx(-) or ppk(-)ppx(-)/ppk(+) strains, deficient in polyP synthesis and hydrolysis, respectively. luxS and lsrK mutants, impaired in autoinducer-2 quorum sensing signal metabolism, were unable to form biofilm unless conditioned media from stationary phase wild type cells grown in low phosphate were used. We conclude that polyP degradation is required for biofilm formation in sufficient phosphate media, activating or triggering the production of autoinducer-2. According to our results, phosphate concentration of the culture media should be carefully considered in bacterial adhesion and virulence studies.
Assuntos
Proteínas de Bactérias/genética , Liases de Carbono-Enxofre/genética , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Polifosfatos/metabolismo , Percepção de Quorum/genética , Proteínas de Bactérias/metabolismo , Biofilmes/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Liases de Carbono-Enxofre/metabolismo , Meios de Cultura/metabolismo , Meios de Cultivo Condicionados/farmacologia , Escherichia coli/efeitos dos fármacos , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/metabolismo , Homosserina/análogos & derivados , Homosserina/biossíntese , Lactonas , Polifosfatos/farmacologia , Percepção de Quorum/efeitos dos fármacos , Fatores de Virulência/biossíntese , Fatores de Virulência/genéticaRESUMO
Respiratory NADH dehydrogenase-2 (NDH-2) of Escherichia coli is a membrane-bound flavoprotein. Bioinformatics approaches suggested the involvement of NDH-2 C-terminal region in membrane anchorage. Here, we demonstrated that NDH-2 is a peripheral membrane protein and that its predicted C-terminal amphipathic Arg390-Ala406 helix is sufficient to bind the protein to lipid membranes. Additionally, a cytosolic NDH-2 protein (Trun-3), lacking the last 43 aminoacids, was purified and characterized. FAD cofactor was absent in purified Trun-3. Upon the addition of FAD, Trun-3 maximum velocity was similar to native NDH-2 rate with ferricyanide and MTT acceptors. However, Trun-3 activity was around 5-fold lower with quinones. No significant difference in K(m) values was observed for both enzymes. For the first time, an active and water soluble NDH-2 was obtained, representing a major improvement for structural/functional characterizations.
Assuntos
Membrana Celular/metabolismo , Escherichia coli/citologia , Escherichia coli/enzimologia , Interações Hidrofóbicas e Hidrofílicas , NADH Desidrogenase/química , NADH Desidrogenase/metabolismo , Sequência de Aminoácidos , Cinética , Dados de Sequência Molecular , NADH Desidrogenase/deficiência , NADH Desidrogenase/genética , Estrutura Secundária de Proteína , Transporte Proteico , Deleção de Sequência , Solubilidade , Água/químicaRESUMO
We found that Escherichia coli grown in media with >37 mM phosphate maintained a high polyphosphate level in late stationary phase, which could account for changes in gene expression and enzyme activities that enhance stationary-phase fitness.
Assuntos
Escherichia coli/crescimento & desenvolvimento , Escherichia coli/metabolismo , Polifosfatos/metabolismo , Escherichia coli/efeitos dos fármacos , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Fosfatos/farmacologiaRESUMO
Escherichia coli gradually decline the capacity to resist oxidative stress during stationary phase. Besides the aerobic electron transport chain components are down-regulated in response to growth arrest. However, we have previously reported that E. coli cells grown in media containing at least 37mM phosphate maintained ndh expression in stationary phase, having high viability and low NADH/NAD(+) ratio. Here we demonstrated that, in the former condition, other aerobic respiratory genes (nuoAB, sdhC, cydA, and ubiC) expression was maintained. In addition, reactive oxygen species production was minimal and consequently the levels of thiobarbituric acid-reactive substances and protein carbonylation were lower than the expected for stationary cells. Interestingly, defense genes (katG and ahpC) expression was also maintained during this phase. Our results indicate that cells grown in high phosphate media exhibit advantages to resist endogenous and exogenous oxidative stress in stationary phase.
Assuntos
Escherichia coli/genética , Escherichia coli/metabolismo , Fosfatos/metabolismo , Meios de Cultura , Transporte de Elétrons/genética , Escherichia coli/efeitos dos fármacos , Escherichia coli/crescimento & desenvolvimento , Expressão Gênica/efeitos dos fármacos , Genes Bacterianos , Peróxido de Hidrogênio/farmacologia , Cinética , Estresse Oxidativo , Substâncias Reativas com Ácido Tiobarbitúrico/metabolismoRESUMO
Escherichia coli NADH dehydrogenase-2 (NDH-2) is a primary dehydrogenase in aerobic respiration that shows cupric-reductase activity. The enzyme is encoded by ndh, which is highly regulated by global transcription factors. It was described that the gene is expressed in the exponential growth phase and repressed in late stationary phase. We report the maintenance of NDH-2 activity and ndh expression in the stationary phase when cells were grown in media containing at least 37 mM phosphate. Gene regulation was independent of RpoS and other transcription factors described to interact with the ndh promoter. At this critical phosphate concentration, cell viability, oxygen consumption rate, and NADH/NAD+ ratio were maintained in the stationary phase. These physiological parameters gradually changed, but NDH-2 activity remained high for up to 94 h. Phosphate seems to trigger an internal signal in the stationary phase mediated by systems not yet described.
Assuntos
Transporte de Elétrons , Escherichia coli/fisiologia , Regulação Bacteriana da Expressão Gênica , NADH Desidrogenase/biossíntese , Fosfatos/metabolismo , Aerobiose , Fusão Gênica Artificial , Proteínas de Bactérias/metabolismo , Escherichia coli/química , Expressão Gênica , Genes Reporter , Viabilidade Microbiana , NAD/metabolismo , Oxigênio/metabolismo , Piridinas/análise , Fator sigma/metabolismo , beta-Galactosidase/biossíntese , beta-Galactosidase/genéticaRESUMO
In this paper we compared the antibacterial activity of native microcin J25, a peptide antibiotic, with the activities of two analogues obtained by chemical modifications. In the first analogue, the negative charge of glutamic carboxyl group was specifically blocked with an L-glycine methyl ester and in the second the histidine imidazole ring was carbethoxylated. Both analogues decreased notably its antibiotic activity against Escherichia coli and Salmonella newport, strains sensible to the native microcin J25. The biological activity of the carbethoxylated analogue was completely recovered after treatment with hydroxylamine. The extreme importance of both polar residues could be interpreted as specific structural features indispensable for the peptide transportation into the cell, extrusion outside the cell or alternatively to inhibit the RNA-polymerase.