RESUMEN
The production of MG (methylglyoxal) in bacterial cells must be maintained in balance with the capacity for detoxification and protection against this electrophile. Excessive production of MG leads to cell death. Survival of exposure to MG is best understood in the Gram-negative bacteria. The major mechanism of protection is the spontaneous reaction of MG with GSH to form hemithiolacetal, followed by detoxification by the glyoxalase system leading to the production of D-lactate. The KefB and KefC glutathione-gated K(+) efflux systems are integrated with the activity of the glyoxalase system to regulate the cytoplasmic pH in response to exposure to electrophiles. Bacteria only produce MG when an imbalance occurs in metabolism. Operation of the MG bypass enables cells to adapt, such that balance is restored to metabolism. Excessive production of MG is an adaptive ploy, which, if it fails, has fatal consequences. On this basis one might define MG-induced loss of life as "death by misadventure" rather than suicide!
Asunto(s)
Bacterias/metabolismo , Piruvaldehído/metabolismoRESUMEN
Long-term antibiotic treatment offers a rare opportunity to study the evolution of bacteria within the same individual. The appearance of new variants has been suggested to take place via the selection of enhanced resistance in compartments of the body in which the antibiotic concentration is low. Laboratory models of protected compartments have elegantly demonstrated their potential in selecting novel variants. However, comparable data from patients have been rare. In this study, extended antibiotic therapy in a single patient suffering from multiple infected liver cysts has provided the opportunity to observe and analyse the molecular evolution of antibiotic resistance. Each isolate has the same basic ompC gene sequence that is distinct from other Escherichia coli isolates, which suggests that they derive from the same founder population. However, the isolates differ in their auxotrophic markers, in the pI values of their dominant beta-lactamase activities and in the mutations in the promoter region of the ampC gene leading to increased expression of the AmpC enzyme. The data provide strong evidence for a single focal infection expanding via parallel pathways of evolution to give a range of antibiotic-resistant isolates. These data suggest that the infected cysts provide numerous protected environments that are the foci for the separate development of distinct variants.
Asunto(s)
Antibacterianos/farmacología , Proteínas Bacterianas , Enfermedad de Caroli/complicaciones , Farmacorresistencia Bacteriana Múltiple/genética , Infecciones por Escherichia coli/tratamiento farmacológico , Escherichia coli/efectos de los fármacos , Evolución Molecular , Adulto , Antibacterianos/uso terapéutico , Secuencia de Bases , Enfermedad de Caroli/tratamiento farmacológico , Enfermedad de Caroli/microbiología , Escherichia coli/enzimología , Escherichia coli/genética , Infecciones por Escherichia coli/complicaciones , Infecciones por Escherichia coli/microbiología , Humanos , Recién Nacido , Masculino , Pruebas de Sensibilidad Microbiana , Datos de Secuencia Molecular , Porinas/genética , Porinas/metabolismo , Análisis de Secuencia de ADN , Factores de Tiempo , beta-Lactamasas/genética , beta-Lactamasas/metabolismoRESUMEN
A new subunit, YabF, for the KefC K(+) efflux system in Escherichia coli has been identified. The subunit is required for maximum activity of KefC. Deletion of yabF reduces KefC activity 10-fold, and supply of YabF in trans restores activity. IS2 and IS10R insertions in yabF can be isolated as suppressors of KefC activity consequent upon the V427A and D264A KefC mutations.
Asunto(s)
Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteínas de Escherichia coli , Escherichia coli/genética , Canales de Potasio/genética , Canales de Potasio/metabolismo , Escherichia coli/química , Escherichia coli/metabolismo , Eliminación de Gen , Genes Bacterianos , Genes Supresores , Glutatión/metabolismo , MutaciónRESUMEN
The presence of Escherichia coli O157 in the faeces of farm animals appears to provide a primary route for human infection, either through physical contact or by contamination of the food chain. Controlling the survival and proliferation of this pathogen in the ruminant gut could offer a measure of protection in the short term, and ultimately complement alternative biotechnological based solutions. Normally, E. coli is greatly outnumbered in the ruminant gut by anaerobic bacteria, producers of weak acids inhibitory to the growth of this species. Withdrawal of feed prior to animal slaughter reduces the concentration of these acids in the gut and may be accompanied by the proliferation of E. coli. There are conflicting reports concerning the effects of changes in the ruminant diet upon faecal shedding of E. coli O157. It is contended that it is important to identify animal husbandry methods or feed additives that may be accompanied by an increased risk of proliferation of this pathogen. Greater understanding of the mechanisms involved in bacterial survival in the presence of weak acids, in the interactions between E. coli and other gut bacteria, and of the effects of some antibacterial plant secondary plant compounds on E. coli, could lead to the development of novel control methods.
Asunto(s)
Animales Domésticos/microbiología , Reservorios de Enfermedades/veterinaria , Escherichia coli O157/aislamiento & purificación , Alimentación Animal , Crianza de Animales Domésticos/normas , Animales , Ácidos Grasos Volátiles/análisis , Cadena Alimentaria , Concentración de Iones de Hidrógeno , Estiércol/microbiología , Rumen/química , Rumen/microbiología , Rumiantes/microbiologíaRESUMEN
The electrophile N-ethylmaleimide (NEM) elicits rapid K(+) efflux from Escherichia coli cells consequent upon reaction with cytoplasmic glutathione to form an adduct, N-ethylsuccinimido-S-glutathione (ESG) that is a strong activator of the KefB and KefC glutathione-gated K(+) efflux systems. The fate of the ESG has not previously been investigated. In this report we demonstrate that NEM and N-phenylmaleimide (NPM) are rapidly detoxified by E. coli. The detoxification occurs through the formation of the glutathione adduct of NEM or NPM, followed by the hydrolysis of the imide bond after which N-substituted maleamic acids are released. N-ethylmaleamic acid is not toxic to E. coli cells even at high concentrations. The glutathione adducts are not released from cells, and this allows glutathione to be recycled in the cytoplasm. The detoxification is independent of new protein synthesis and NAD(+)-dependent dehydrogenase activity and entirely dependent upon glutathione. The time course of the detoxification of low concentrations of NEM parallels the transient activation of the KefB and KefC glutathione-gated K(+) efflux systems.
Asunto(s)
Escherichia coli/metabolismo , Etilmaleimida/metabolismo , Glutatión/metabolismo , Maleatos/metabolismo , Escherichia coli/crecimiento & desarrollo , Glutatión/análogos & derivados , Espectroscopía de Resonancia Magnética , Maleimidas/metabolismo , NAD/metabolismo , Succinimidas/metabolismoRESUMEN
The Kdp K+ uptake system of Escherichia coli is induced by limitation for K+ and/or high osmolarity. In the present study, the regulation of the activity of the Kdp system has been investigated in E. coli mutants possessing only the Kdp system as the mechanism of K+ accumulation. Cells grown in the presence of low K+ (0.1-1 mM) exhibit normal growth. However, growth inhibition results from exposure of cells to moderate levels of external K+ (> 5 mM). Measurement of the cytoplasmic pH, of K+ pools and of transport via the Kdp system demonstrates that the Kdp system is rapidly and irreversibly inhibited by moderate external K+. Concentrations of K+ greater than 2 mM are sufficient to cause inhibition of Kdp. At pH 6, this results in rapid lowering of the capacity for pH homeostasis, but at pH 7 the intracellular pH is unaffected. Parallel analysis of the expression of the Kdp system in a Kdp+/kdpFABC-lacZ strain shows that levels of K+ that are sufficient to inhibit Kdp activity also repress expression. As a result, growth inhibition of strains solely possessing Kdp arises jointly from inhibition of Kdp activity and repression of Kdp gene expression. These data identify an important aspect of the regulation of potassium transport via the Kdp system and also provide support for a model of regulation of Kdp expression via at least two mechanisms: sensing of both turgor and external K+ concentration.
Asunto(s)
Adenosina Trifosfatasas/antagonistas & inhibidores , Proteínas Portadoras/antagonistas & inhibidores , Proteínas de Transporte de Catión , Proteínas de Escherichia coli , Escherichia coli/enzimología , Potasio/metabolismo , Adenosina Trifosfatasas/genética , Proteínas Portadoras/genética , Escherichia coli/crecimiento & desarrollo , Regulación Bacteriana de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Genes Bacterianos , Homeostasis , Concentración de Iones de Hidrógeno , Cinética , Concentración Osmolar , Potasio/químicaRESUMEN
The isolation of rhizobial strains which exhibit an intrinsic tolerance to acidic conditions has been reported and has facilitated studies on the basic mechanisms underlying acid tolerance. Rhizobium tropici strain CIAT899 displays a high intrinsic tolerance to acidity and therefore was used in this work to study the molecular basis of bacterial responses to acid conditions and other environmental stresses. We generated a collection of R. tropici CIAT899 mutants affected in acid tolerance using Tn5-luxAB mutagenesis, and one mutant strain (CIAT899-13T2), which fails to grow under acid conditions, was characterized in detail. Strain CIAT899-13T2 was found to contain a single Tn5-luxAB insertion in a gene showing a high degree of similarity with the Escherichia coli gshB gene, encoding the enzyme glutathione synthetase. Intracellular potassium pools and intracellular pH levels were found to be lower in the mutant than in the parent. The glutathione-deficient mutant was shown to be sensitive to weak organic acids, osmotic and oxidative stresses, and the presence of methylglyoxal. Glutathione restores responses to these stresses almost to wild-type levels. Our data show that in R. tropici the production of glutathione is essential for growth in extreme environmental conditions. The mutant strain CIAT899-13T2 induced effective nodules; however, it was found to be outcompeted by the wild-type strain in coinoculation experiments.
Asunto(s)
Glutatión/metabolismo , Rhizobium/fisiología , Elementos Transponibles de ADN , Fabaceae/microbiología , Concentración de Iones de Hidrógeno , Mutagénesis Insercional , Presión Osmótica , Plantas Medicinales , Plásmidos/genética , Potasio/metabolismo , Piruvaldehído/toxicidad , Rhizobium/genéticaRESUMEN
The effect of the toxic metabolite methylglyoxal on the DNA of Escherichia coli cells has been investigated. Exposure of E. coli cells to methylglyoxal reduces the transformability of plasmid DNA and results in the degradation of genomic DNA. The activity of the KefB and KefC potassium channels protects E. coli cells against methylglyoxal and limits the amount of DNA damage. In mutants lacking KefB and KefC, methylglyoxal-induced DNA damage was reduced by incubation with a weak acid that lowers the pHi to the same extent as through KefB and KefC activation. This provides evidence that acidification of the cytoplasm protects E. coli DNA against methylglyoxal. By the analysis of cells lacking UvrA, we demonstrate that this repair protein is required for the degradation of the DNA upon methylglyoxal exposure. However, protection by KefB and KefC occurred independently of UvrA. Although we present evidence that exposure of E. coli cells to methylglyoxal results in DNA degradation, our results suggest this event is not essential for methylglyoxal-induced death. The implications of these findings will be discussed.
Asunto(s)
Antiportadores/fisiología , Proteínas Bacterianas/fisiología , Proteínas de Escherichia coli , Escherichia coli/efectos de los fármacos , Canales de Potasio/fisiología , Piruvaldehído/toxicidad , ADN Bacteriano , Escherichia coli/fisiología , Plásmidos , Antiportadores de Potasio-HidrógenoRESUMEN
The effect of meat peptone type I (Sigma) on the growth of Escherichia coli cells under hyperosmotic stress has been investigated. Peptone is a complex mixture of peptides with a small content of free amino acids, which resembles nutrients found in natural environments. Our data showed that peptone enhances the growth of E. coli cells in high-osmolarity medium to levels higher than those achieved with the main compatible solute in bacteria, glycine betaine. The mechanism of osmoprotection by peptone comprises the uptake and accumulation of the compatible solute, proline. The main role of the peptides contained in peptone is the provision of nutrients rather than the intracellular accumulation of osmolytes. In contrast to Listeria monocytogenes (M. R. Amezaga, I. Davidson, D. McLaggan, A. Verheul, T. Abee, and I. R. Booth, Microbiology 141:41-49, 1995), E. coli does not accumulate exogenous peptides for osmoprotection and peptides containing proline do not lead to the accumulation of proline as a compatible solute. In late-logarithmic-phase cultures of E. coli growing at high osmolarity plus peptone, proline becomes the limiting factor for growth, and the intracellular pools of proline are not maintained. This is a consequence of the low concentration of free proline in peptone, the catabolism of proline by E. coli, and the inability of E. coli to utilize proline-containing peptides as a source of compatible solutes. Our data highlight the role that natural components in food such as peptides play in undermining food preservation regimes, such as high osmolarity, and also that the specific mechanisms of osmoprotection by these compounds differ according to the organism.
Asunto(s)
Aminoácidos/metabolismo , Escherichia coli/fisiología , Péptidos/metabolismo , Peptonas/metabolismo , Prolina/metabolismo , Transporte Biológico , Escherichia coli/efectos de los fármacos , Escherichia coli/genética , Genotipo , Cinética , Listeria monocytogenes/fisiología , Concentración Osmolar , Peptonas/farmacologíaRESUMEN
When Escherichia coli cells are subject to hypoosmotic shock they are subject to substantial flows of water that can be equivalent to a 4-5-fold increase in the pressure exerted from the cytoplasm on the membrane and peptidoglycan wall. The recently described aquaporin that facilitates rapid water movement across the cytoplasmic membrane is repressed during growth at high osmolarity. This may enable the cell to reduce the rate of pressure build up during transitions from high to low osmolarity. The presence of multiple mechanosensitive channels in the E. coli cell membrane is well documented. The recent identification of genes that inactivate the MscL and MscS channels has established their role in releasing the pressure built up by hypoosmotic shock. The isolation of specific mutations and the structural studies on MscL now pave the way to a molecular understanding of the mechanism of activation of mechanosensitive channels.
Asunto(s)
Acuaporinas/metabolismo , Escherichia coli/fisiología , Canales Iónicos/metabolismo , Presión Osmótica , Agua/metabolismo , Acuaporinas/genética , Regulación Bacteriana de la Expresión Génica , Canales Iónicos/genética , Estimulación Física , Transducción de SeñalRESUMEN
Mechanosensitive channels are ubiquitous amongst bacterial cells and have been proposed to have major roles in the adaptation to osmotic stress, in particular in the management of transitions from high to low osmolarity environments. Electrophysiological measurements have identified multiple channels in Escherichia coli cells. One gene, mscL, encoding a large conductance channel has previously been described, but null mutants were without well-defined phenotypes. Here, we report the characterization of a new gene family required for MscS function, YggB and KefA, which has enabled a rigorous test of the role of the channels. The channel determined by KefA does not appear to have a major role in managing the transition from high to low osmolarity. In contrast, analysis of mutants of E.coli lacking YggB and MscL shows that mechanosensitive channels are designed to open at a pressure change just below that which would cause cell disruption leading to death.
Asunto(s)
Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteínas de Escherichia coli , Escherichia coli/genética , Escherichia coli/metabolismo , Genes Bacterianos , Canales Iónicos/genética , Canales Iónicos/metabolismo , Secuencia de Bases , Cartilla de ADN/genética , Mutación , Presión Osmótica , Protoplastos/metabolismoRESUMEN
The regulation of intracellular pH (pHi) in bacterial cells is achieved through control over cation (and anion) permeability. In addition to the active components of homeostasis there are contributions from essentially passive elements, such as the lipid composition of the membrane and the buffering capacity of the cytoplasm. Active homeostasis involves control over the movement of K+, Na+ and H+. Alterations in the membrane permeability for any of these cations may cause perturbation of homeostasis. In Escherichia coli this is exemplified by the controlled activation of K+ efflux systems by glutathione adducts leading to temporary acidification of the cytoplasm. This is achieved by sophisticated control over the KefB and KefC systems, and is tightly integrated with glutathione-dependent detoxification mechanisms. Such control over pHi facilitates survival of the cell following exposure to toxic electrophiles. The components of pH homeostasis will be reviewed and the molecular mechanisms, and role of, the KefB and KefC systems will be discussed.
Asunto(s)
Bacterias/metabolismo , Concentración de Iones de HidrógenoRESUMEN
KefB and KefC are glutathione-gated K+ efflux systems in Escherichia coli, and the proteins exhibit strong similarity at the level of both primary sequence and domain organization. The proteins are maintained closed by glutathione and are activated by binding of adducts formed between glutathione and electrophiles. By construction of equivalent mutations in each protein, this study has analyzed the control over inactive state of the proteins. A UV-induced mutation in KefB, L75S, causes rapid spontaneous K+ efflux but has only a minor effect on K+ efflux via KefC. Similarly amino acid substitutions that cause increased spontaneous activity in KefC have only small effects in KefB. Exchange of an eight amino acid region from KefC (HALESDIE) with the equivalent sequence from KefB (HELETAID) has identified a role for a group of acidic residues in controlling KefC activity. The mutations HELETAID and L74S in KefC act synergistically, and the activity of the resultant protein resembles that of KefB. We conclude that, despite the high degree of sequence similarity, KefB and KefC exhibit different sensitivities to the same site-specific mutations.
Asunto(s)
Antiportadores/fisiología , Proteínas Bacterianas/fisiología , Proteínas de Escherichia coli , Glutatión/fisiología , Activación del Canal Iónico/fisiología , Canales de Potasio/fisiología , Secuencia de Aminoácidos , Antiportadores/genética , Antiportadores/efectos de la radiación , Proteínas Bacterianas/genética , Proteínas Bacterianas/efectos de la radiación , Clonación Molecular , Secuencia Conservada , Escherichia coli/genética , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Canales de Potasio/genética , Canales de Potasio/efectos de la radiación , Antiportadores de Potasio-Hidrógeno , Piruvaldehído/farmacología , Alineación de Secuencia , Relación Estructura-Actividad , Rayos UltravioletaRESUMEN
The acid tolerance of Escherichia coli O157:H7 strains can be overcome by addition of lactate, ethanol, or a combination of the two agents. Killing can be increased by as much as 4 log units in the first 5 min of incubation at pH 3 even for the most acid-tolerant isolates. Exponential-phase, habituated, and stationary-phase cells are all sensitive to incubation with lactate and ethanol. Killing correlates with disruption of the capacity for pH homeostasis. Habituated and stationary-phase cells can partially offset the effects of the lowering of cytoplasmic pH.
Asunto(s)
Infecciones por Escherichia coli/microbiología , Escherichia coli O157/crecimiento & desarrollo , Etanol/farmacología , Lactatos/farmacología , Productos de la Carne/microbiología , Animales , Pollos , Recuento de Colonia Microbiana , Medios de Cultivo , Escherichia coli/efectos de los fármacos , Escherichia coli/crecimiento & desarrollo , Escherichia coli/aislamiento & purificación , Escherichia coli O157/aislamiento & purificación , Humanos , Concentración de Iones de Hidrógeno , Carne/microbiología , PorcinosRESUMEN
Methylglyoxal is a toxic electrophile. In Escherichia coli cells, the principal route of methylglyoxal production is from dihydroxyacetone phosphate by the action of methylglyoxal synthase. The toxicity of methylglyoxal is believed to be due to its ability to interact with the nucleophilic centres of macromolecules such as DNA. Bacteria possess an array of detoxification pathways for methylglyoxal. In E. coli, glutathione-based detoxification is central to survival of exposure to methylglyoxal. The glutathione-dependent glyoxalase I-II pathway is the primary route of methylglyoxal detoxification, and the glutathione conjugates formed can activate the KefB and KefC potassium channels. The activation of these channels leads to a lowering of the intracellular pH of the bacterial cell, which protects against the toxic effects of electrophiles. In addition to the KefB and KefC systems, E. coli cells are equipped with a number of independent protective mechanisms whose purpose appears to be directed at ensuring the integrity of the DNA. A model of how these protective mechanisms function will be presented. The production of methylglyoxal by cells is a paradox that can be resolved by assigning an important role in adaptation to conditions of nutrient imbalance. Analysis of a methylglyoxal synthase-deficient mutant provides evidence that methylglyoxal production is required to allow growth under certain environmental conditions. The production of methylglyoxal may represent a high-risk strategy that facilitates adaptation, but which on failure leads to cell death. New strategies for antibacterial therapy may be based on undermining the detoxification and defence mechanisms coupled with deregulation of methylglyoxal synthesis.
Asunto(s)
Bacterias/metabolismo , Proteínas de Escherichia coli , Piruvaldehído/metabolismo , Antiportadores/metabolismo , Bacterias/enzimología , Proteínas Bacterianas/metabolismo , ADN Bacteriano/metabolismo , Escherichia coli/química , Escherichia coli/enzimología , Escherichia coli/fisiología , Glutatión/metabolismo , Glucólisis/fisiología , Canales de Potasio/metabolismo , Antiportadores de Potasio-Hidrógeno , Factor sigma/metabolismoRESUMEN
The role of the tripeptide glutathione in the growth and survival of Escherichia coli cells has been investigated. Glutathione-deficient mutants leak potassium and have a reduced cytoplasmic pH. These mutants are more sensitive to methylglyoxal than the parent strain, indicating that in the absence of glutathione-dependent detoxification, acidification of the cytoplasm cannot fully protect cells. However, increasing the intracellular pH of the glutathione-deficient strain resulted in enhanced sensitivity to methylglyoxal. This suggests that acidification of the cytoplasm can provide some protection to E. coli cells in the absence of glutathione. In the presence of the Kdp system, glutathione-deficient mutants are highly sensitive to methylglyoxal. This is due to the higher intracellular pH in these cells. In the absence of methylglyoxal, the presence of the Kdp system in a glutathione-deficient strain also leads to an extended lag upon dilution into fresh medium. These data highlight the importance of glutathione for the regulation of the K+ pool and survival of exposure to methylglyoxal.
Asunto(s)
Escherichia coli/metabolismo , Glutatión/metabolismo , Potasio/metabolismo , Piruvaldehído/farmacología , División Celular/efectos de los fármacos , Citoplasma/metabolismo , Escherichia coli/efectos de los fármacos , Concentración de Iones de Hidrógeno , Piruvaldehído/metabolismoRESUMEN
The mechanisms by which Escherichia coli cells survive exposure to the toxic electrophile N-ethylmaleimide (NEM) have been investigated. Stationary-phase E. coli cells were more resistant to NEM than exponential-phase cells. The KefB and KefC systems were found to play an important role in protecting both exponential- and stationary-phase cells against NEM. Additionally, RpoS and the DNA-binding protein Dps aided the survival of both exponential- and stationary-phase cells against NEM. Double mutants lacking both RpoS and Dps and triple mutants deficient in KefB and KefC and either RpoS or Dps had an increased sensitivity to NEM in both exponential- and stationary-phase cells compared to mutants missing only one of these protective mechanisms. Stationary- and exponential-phase cells of a quadruple mutant lacking all four protective systems displayed even greater sensitivity to NEM. These results indicated that protection by the KefB and KefC systems, RpoS and Dps can each occur independently of the other systems. Alterations in the level of RpoS in exponentially growing cells correlated with the degree of NEM sensitivity. Decreasing the level of RpoS by enriching the growth medium enhanced sensitivity to NEM, whereas a mutant lacking the ClpP protease accumulated RpoS and gained high levels of resistance to NEM. A slower-growing E. coli strain was also found to accumulate RpoS and had enhanced resistance to NEM. These data emphasize the multiplicity of pathways involved in protecting E. coli cells against NEM.
Asunto(s)
Proteínas Bacterianas/fisiología , Proteínas de Unión al ADN/fisiología , Proteínas de Escherichia coli , Escherichia coli/efectos de los fármacos , Etilmaleimida/farmacología , Factor sigma/fisiología , Antiportadores/genética , Antiportadores/fisiología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteínas de Unión al ADN/genética , Farmacorresistencia Microbiana , Escherichia coli/genética , Escherichia coli/crecimiento & desarrollo , Escherichia coli/fisiología , Mutación , Canales de Potasio/genética , Canales de Potasio/fisiología , Antiportadores de Potasio-Hidrógeno , Factor sigma/genética , Factor sigma/metabolismoRESUMEN
The enzyme methylglyoxal synthase (MGS) was partially purified from Escherichia coli extracts, and the amino-terminal sequence of candidate proteins was determined, based on the native protein being a tetramer of about 69 kDa. Database analysis identified an open reading frame in the E. coli genome, YccG, corresponding to a protein of 16.9 kDa. When amplified and expressed from a controlled promoter, it yielded extracts that contained high levels of MGS activity. MGS expressed from the trc promoter accumulated to approximately 20% of total cell protein, representing approximately 900-fold enhanced expression. This caused no detriment during growth on glucose, and the level of methylglyoxal (MG) in the medium rose to only 0.08 mM. High-level expression of MGS severely compromised growth on xylose, arabinose and glycerol. A mutant lacking MGS was constructed, and it grew normally on a range of carbon sources and on low-phosphate medium. However, the mutant failed to produce MG during growth on xylose in the presence of cAMP, and growth was inhibited.
Asunto(s)
Liasas de Carbono-Oxígeno/genética , Liasas de Carbono-Oxígeno/metabolismo , Escherichia coli/enzimología , Escherichia coli/genética , Piruvaldehído/metabolismo , Liasas de Carbono-Oxígeno/aislamiento & purificación , Escherichia coli/química , Escherichia coli/crecimiento & desarrollo , Regulación Bacteriana de la Expresión Génica , Genes Bacterianos , Glucosa/metabolismo , Glicerol/metabolismo , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Fosfatos/metabolismo , Plásmidos/genética , Xilosa/metabolismoRESUMEN
The glyoxalase I gene (gloA) of Escherichia coli has been cloned and used to create a null mutant. Cells overexpressing glyoxalase I exhibit enhanced tolerance of methylglyoxal (MG) and exhibit elevated rates of detoxification, although the increase is not stoichiometric with the change in enzyme activity. Potassium efflux via KefB is also enhanced in the overexpressing strain. Analysis of the physiology of the mutant has revealed that growth and viability are quite normal, unless the cell is challenged with MG either added exogenously or synthesized by the cells. The mutant strain has a low rate of detoxification of MG, and cells rapidly lose viability when exposed to this electrophile. Activation of KefB and KefC is diminished in the absence of functional glyoxalase I. These data suggest that the glutathione-dependent glyoxalase I is the dominant detoxification pathway for MG in E. coli and that the product of glyoxalase I activity, S-lactoylglutathione, is the activator of KefB and KefC.
Asunto(s)
Antiportadores/metabolismo , Proteínas de Escherichia coli , Escherichia coli/enzimología , Lactoilglutatión Liasa/metabolismo , Canales de Potasio/metabolismo , Potasio/metabolismo , Piruvaldehído/metabolismo , Proteínas Bacterianas/metabolismo , Clonación Molecular , Escherichia coli/genética , Escherichia coli/crecimiento & desarrollo , Regulación Bacteriana de la Expresión Génica , Genes Bacterianos , Glutatión/metabolismo , Antiportadores de Potasio-Hidrógeno , Piruvaldehído/farmacologíaRESUMEN
During inhibition of cell growth by weak acids, there is substantial accumulation of the weak acid anions in the cytoplasm. This study was undertaken to determine the impact of anion accumulation on cellular pools. At pH 6, growth in the presence of 8 mM acetate led to an internal pool of greater than 240 mM acetate anion and resulted in reduced levels of glutamate in the cell, but there were no significant changes in K+ and Na+ levels. At low osmolarity, the change in the glutamate pool compensated for only a small fraction of the accumulated acetate anion. However, at high osmolarity, glutamate compensated for over half of the accumulated acetate. Recovery of the normal cytoplasmic pH after the removal of acetate was dependent on the synthesis of glutamate.