pH-dependent catabolic protein expression during anaerobic growth of Escherichia coli K-12

During aerobic growth of Escherichia coli, expression of catabolic enzymes and envelope and periplasmic proteins is regulated by pH. Additional modes of pH regulation were revealed under anaerobiosis. E. coli K-12 strain W3110 was cultured anaerobically in broth medium buffered at pH 5.5 or 8.5 for protein identification on proteomic two-dimensional gels. A total of 32 proteins from anaerobic cultures show pH-dependent expression, and only four of these proteins (DsbA, TnaA, GatY, and HdeA) showed pH regulation in aerated cultures. The levels of 19 proteins were elevated at the high pH; these proteins included metabolic enzymes (DhaKLM, GapA, TnaA, HisC, and HisD), periplasmic proteins (ProX, OppA, DegQ, MalB, and MglB), and stress proteins (DsbA, Tig, and UspA). High-pH induction of the glycolytic enzymes DhaKLM and GapA suggested that there was increased fermentation to acids, which helped neutralize alkalinity. Reporter lac fusion constructs showed base induction of sdaA encoding serine deaminase under anaerobiosis; in addition, the glutamate decarboxylase genes gadA and gadB were induced at the high pH anaerobically but not with aeration. This result is consistent with the hypothesis that there is a connection between the gad system and GabT metabolism of 4-aminobutanoate. On the other hand, 13 other proteins were induced by acid; these proteins included metabolic enzymes (GatY and AckA), periplasmic proteins (TolC, HdeA, and OmpA), and redox enzymes (GuaB, HmpA, and Lpd). The acid induction of NikA (nickel transporter) is of interest because E. coli requires nickel for anaerobic fermentation. The position of the NikA spot coincided with the position of a small unidentified spot whose induction in aerobic cultures was reported previously; thus, NikA appeared to be induced slightly by acid during aeration but showed stronger induction under anaerobic conditions. Overall, anaerobic growth revealed several more pH-regulated proteins; in particular, anaerobiosis enabled induction of several additional catabolic enzymes and sugar transporters at the high pH, at which production of fermentation acids may be advantageous for the cell.     

pH-dependent expression of periplasmic proteins and amino acid catabolism in Escherichia coli

Escherichia coli grows over a wide range of pHs (pH 4.4 to 9.2), and its own metabolism shifts the external pH toward either extreme, depending on available nutrients and electron acceptors. Responses to pH values across the growth range were examined through two-dimensional electrophoresis (2-D gels) of the proteome and through lac gene fusions. Strain W3110 was grown to early log phase in complex broth buffered at pH 4.9, 6.0, 8.0, or 9.1. 2-D gel analysis revealed the pH dependence of 19 proteins not previously known to be pH dependent. At low pH, several acetate-induced proteins were elevated (LuxS, Tpx, and YfiD), whereas acetate-repressed proteins were lowered (Pta, TnaA, DksA, AroK, and MalE). These responses could be mediated by the reuptake of acetate driven by changes in pH. The amplified proton gradient could also be responsible for the acid induction of the tricarboxylic acid (TCA) enzymes SucB and SucC. In addition to the autoinducer LuxS, low pH induced another potential autoinducer component, the LuxH homolog RibB. pH modulated the expression of several periplasmic and outer membrane proteins: acid induced YcdO and YdiY; base induced OmpA, MalE, and YceI; and either acid or base induced OmpX relative to pH 7. Two pH-dependent periplasmic proteins were redox modulators: Tpx (acid-induced) and DsbA (base-induced). The locus alx, induced in extreme base, was identified as ygjT, whose product is a putative membrane-bound redox modulator. The cytoplasmic superoxide stress protein SodB was induced by acid, possibly in response to increased iron solubility. High pH induced amino acid metabolic enzymes (TnaA and CysK) as well as lac fusions to the genes encoding AstD and GabT. These enzymes participate in arginine and glutamate catabolic pathways that channel carbon into acids instead of producing alkaline amines. Overall, these data are consistent with a model in which E. coli modulates multiple transporters and pathways of amino acid consumption so as to minimize the shift of its external pH toward either acidic or alkaline extreme.     

Salmonella typhimurium poultry isolate growth response to propionic acid and sodium propionate under aerobic and anaerobic conditions

Propionic acid and its sodium salt have long been used as additives in poultry feed to reduce microbial populations, including Salmonella spp. Propionic acids in poultry feed may have a potential role in inhibiting growth of Salmonella in the chicken intestine. In this study, we determined growth response of a Salmonella typhimurium poultry isolate to propionic acid and sodium propionate under aerobic and anaerobic conditions. Growth rate consistently decreased with the addition of greater concentrations of either propionic acid or sodium propionate. The extent of growth inhibition was much greater with propionic acid than the sodium form. Media pH decreased only with addition of propionic acid. Growth inhibition was more effective under anaerobic growth conditions with either propionic acid or sodium propionate. When determined at the same pH level, growth rate was significantly lowered by addition of 25 mM of either propionate or sodium propionate alone, and also by the decrease in pH levels (P<0.05). These results showed that growth inhibition of S. typhimurium by propionic acid or sodium propionate is greatly enhanced by pH decrease, and to lesser extent by anaerobiosis. We also found that sodium propionate was more inhibitory for growth of S. typhimurium than propionic acid when compared at the same pH levels.     

Batch cultures of Escherichia coli with added substrate under various aeration conditions

The growth parameters (Xmax, Y, mu) of Escherichia coli batch cultures were studied when a substrate was added under different aeration conditions. If the pH was not adjusted, the bacterial growth stopped at pH 4.5 under the aerobic conditions or at pH 5.8 under the anaerobic conditions. When the pH 7.0 was constantly maintained, the accumulation of acid products in the aerobic culture was 3 times as high as in the anaerobic culture by the time the growth ceased. The anaerobic culture resumed its growth upon aeration. A hypothesis is proposed to explain the data.     

Energy-dependent inactivation of citrate lyase in Enterobacter aerogenes.

Enterobacter aerogenes was grown in continous culture with ammonia as the growth-limiting substrate, and changes in citrate lyase and citrate synthase activities were monitored after growth shifts from anaerobic growth on citrate to aerobic growth on citrate, aerobic growth on glucose, anaerobic growth on glucose, and anaerobic growth on glucose plus nitrate. Citrate lyase was inactivated during aerobic growth on glucose and during anaerobic growth with glucose plus nitrate. Inactivation did not occur during anaerobic growth on glucose, and as a result of the simultaneous presence of citrate lyase and citrate synthase, growth difficulties were observed. Citrate lyase inactivation consisted of deacetylation of the enzyme. The corresponding deacetylase could not be demonstrated in cell extracts, and it is concluded that, as in a number of other inactivations, electron transport to oxygen or nitrate was required for inactivation.     

Induction of citrate lyase in Enterobacter cloacae grown under aerated conditions and its effect on citrate metabolism.

Growth of Enterobacter cloacae on K+ citrate under aerated conditions (no detectable oxygen tension in the medium even though it was aerated) was slower (mean generation time, 130 min) than under aerobic conditions (mean generation time, 72 min), but with a faster utilization of citrate, resulting in a molar growth yield of 10.6 g (dry weight) of cells per mol of citrate utilized versus 40 g (dry weight) of cells per mol of citrate utilized for aerobic growth. The rapid utilization of citrate under aerated conditions was apparently due to the induction of citrate lyase and was supported by the finding that cells excreted acetate and a small amount of oxalacetate under aerated conditions, but not under aerobic conditions when the cells were devoid of citrate lyase activity. The activity of oxalacetate decarboxylase in aerated cells was slightly lower than in aerobic cells, indicating that little of the oxalacetate produced by the citrate lyase was metabolized by the decarboxylase. Oxalacetate was probably metabolized by malate dehydrogenase, previously shown to be present in anaerobic and aerobic cells. Thus, about 70% of the citrate was cleaved by the citrate lyase, resulting in little or no production of energy for growth. The remaining citrate was metabolized via the citric acid cycle under aerated conditions, since the cells contained alpha-ketoglutarate dehydrogenase at the same level as in aerobically grown cells. The presence of the other enzymes of the cycle was shown in earlier studies.     

Thioredoxin-dependent hydroperoxide peroxidase activity of bacterioferritin comigratory protein (BCP) as a new member of the thiol-specific antioxidant protein (TSA)/Alkyl hydroperoxide peroxidase C (AhpC) family

Escherichia coli bacterioferritin comigratory protein (BCP), a putative bacterial member of the TSA/AhpC family, was characterized as a thiol peroxidase. BCP showed a thioredoxin-dependent thiol peroxidase activity. BCP preferentially reduced linoleic acid hydroperoxide rather than H(2)O(2) and t-butyl hydroperoxide with the use of thioredoxin as an in vivo immediate electron donor. The value of V(max)/K(m) of BCP for linoleic acid hydroperoxide was calculated to be 5-fold higher than that for H(2)O(2), implying that BCP has a selective capability to reduce linoleic acid hydroperoxide. Replacement of Cys-45 with serine resulted in the complete loss of thiol peroxidase activity, suggesting that BCP is a new bacterial member of TSA/AhpC family having a conserved cysteine as the primary site of catalysis. BCP exists as a monomer, and its functional Cys-45 appeared to exist as cysteine sulfenic acid. The expression level of BCP gradually elevated during exponential growth until mid-log phase growth, beyond which the expression level was decreased. BCP was induced 3-fold by the oxidative stress given by changing the growth conditions from the anaerobic to aerobic culture. Bcp null mutant grew more slowly than its wild type in aerobic culture and showed the hypersensitivity toward various oxidants such as H(2)O(2), t-butyl hydroperoxide, and linoleic acid hydroperoxide. The peroxide hypersensitivity of the null mutant could be complemented by the expression of bcp gene. Taken together, these data suggest that BCP is a new member of thioredoxin-dependent TSA/AhpC family, acting as a general hydroperoxide peroxidase.     

Bioenergetics of lactic acid bacteria: cytoplasmic pH and osmotolerance

Abstract Lactic acid bacteria maintain a cytoplasm that is more alkaline than the medium, but whose pH decreases as the medium is acidified during growth and fermentation. Streptococci generally acidify the cytoplasm from approximately pH 7.6 to 5.7 (external pH 4.5) before growth and then fermentation cease. The internal enzyme machinery of these anaerobic fermenters thus tolerates a fairly wide range in internal proton concentration. Lactobacilli tolerate a significantly more acidic cytoplasmic pH of 4.4 (external pH 3.5). However, when the cytoplasmic pH decreases below a threshold pH, which depends on the organism cellular functions are inhibited. Fermentation end-products, such as organic acids or alcohols, exert their deleterious effects by bringing about acidification of the cytoplasm below the permissible pH. Organic acids, which act as protonophores, or solvents, which perturb membrane phospholipids, at high concentrations increase the inward leak of H⁺ so that H⁺ efflux is not rapid enough to alkalinize the cytoplasm. The membrane pH gradient is thus dissipated.
A specific strain of Lactobacillus acidophilus has been found to be unusually osmotolerant. The osmoresistance is due to the cells' capacity to accumulate glycine betaine by a transport carrier that is activated, but not induced, by high medium osmotic pressure.     

Role of pH, lactate, and anaerobiosis in controlling the growth of some fermentative Gram-negative bacteria on beef.

At 5 degrees C four strains of fermentative, gram-negative bacteria (Serratia liquefaciens, Yersinia enterocolitica, Enterobacter cloacae, and Aeromonas hydrophila) grew aerobically and anaerobically on adipose tissue removed from beef muscle of low pH (5.4 to 5.6). All four strains also grew aerobically and anaerobically on muscle tissue of high pH (6.0 to 6.3). However, none of the four grew anaerobically on beef muscle of low pH, and the aeromonad strain also failed to grow aerobically on such muscle. Growth of S. liquefaciens and E.cloacae on vacuum-packaged beef muscle was dependent on the pH of the tissue and the oxygen transmission rate of the packaging film. Although the four strains grew in broth buffered at pH 5.55, L-lactate, at the concentration found in muscle of low pH (ca. 100 mM), prevented anaerobic growth of all four isolates and prevented the aerobic growth of th aeromonad. At pH 6.1 in buffered broth, the concentration of L-lactate occurring in muscle of high pH did not prevent aerobic or anaerobic growth of any of the strains.     

The roles of cell-wall acidification and proton-pump stimulation in auxin-induced growth: studies using monensin

The carboxylic ionophore, monensin, rapidly induced cell-wall acidification and a decrease in cytosolic pH when added to maize coleoptiles at low external pH and Na⁺ concentration. Elongation growth at rates equivalent to those obtained with indole-3-acetic acid was induced for about 1 h. Stimulation of the outwardly directed proton pump apparently occurred, since under the same conditions monensin induced membrane hyperpolarization of maize root rhizodermis cells. When the external pH was high (>8) and Na⁺ present, monensin treatment caused only minimal changes in membrane potential and cytosolic pH. Although the ionophore transported protons out of the cell, resulting in cell-wall acidification, no elongation growth occurred. However, under identical conditions, indole-3-acetic acid dit induce growth. The data indicates that stimulation of the outwardly directed electrogenic proton pump rather than the subsequent acidification of the cell wall is vital for the induction of elongation growth.Abbreviations CFA₂ 6-carboxyfluorescein diacetate - FA₂ fluorescein diacetate - Hepes 4-(2-hydroxyethyl-1-piperazinepropanesulfonic acid - IAA indole-3-acetic acid - Mes 2-(N-morpholino) ethanesulfonic acid - Tris 2-amino-2-(hydroxymethyl)-1,3-propanediol     

Anoxia tolerance in the aquatic monocot Potamogeton pectinatus absence of oxygen stimulates elongation in association with an unusually large pasteur effect

Elongation by stems of overwintered tubers of Potamogeton pectinatus (L.) is strongly promoted over several days by oxygen-free conditions. Characteristics of the respiration underpinning this unusual response were examined. Anaerobic plants produced ethanol and CO(2) in approximately equimolar amounts, indicating that glycolysis coupled to alcoholic fermentation was the principal CO(2)-producing respiratory pathway. Rates of CO(2) evolution by aerobic and anaerobic whole plants (shoot and tuber) were similar, suggesting a rate of glycolysis three times that of aerobic plants, i.e. a strong Pasteur effect. In the shoot alone, anaerobic CO(2) production was twice the aerobic rate indicating a 6-fold increase in the rate of glycolysis in this tissue. Anoxic stems contained more sucrose at a stronger concentration than slower-growing aerobic stems or anaerobic leaves, demonstrating that sugar supply to the site of most rapid growth exceeded demand in the absence of oxygen. Concentrations of potentially toxic acetaldehyde in the external medium were small (approximately 0.2 mol m(-3)) during anoxia and on return to aerated conditions. Lactic acid was undetectable under anaerobic conditions and in vivo (31)P-NMR analysis of shoots revealed a cytoplasmic acidification of only 相似文献   

Induction of Acid Resistance of Salmonella typhimurium by Exposure to Short-Chain Fatty Acids

Exposure to short-chain fatty acids (SCFA) is one of the stress conditions Salmonella typhimurium encounters during its life cycle, because SCFA have been widely used as food preservatives and SCFA are also present at high concentrations in the gastrointestinal tracts of host animals. The effects of SCFA on the acid resistance of the organism were examined in an attempt to understand the potential role of SCFA in the pathogenesis of S. typhimurium. The percent survival of S. typhimurium at pH 3.0 was determined after exposure to SCFA for 1 h at pH 7.0. The percent acid survival, which varied depending on the SCFA species and the concentration used, was 42 after exposure to 100 mM propionate at pH 7.0 under aerobic incubation conditions, while less than 1% could survive without exposure. The SCFA-induced acid resistance was markedly enhanced by anaerobiosis (64%), lowering pH conditions (138% at pH 5.0), or increasing incubation time (165% with 4 h) during exposure to propionic acid. When protein synthesis during exposure to propionate was blocked by chloramphenicol, the percent acid survival was less than 1, indicating that the protein synthesis induced by exposure to propionate is required for the induction of the acid resistance. The percent acid survival determined with the isogenic mutant strains defective in acid tolerance response revealed that AtrB protein is necessary for the full induction of acid resistance by exposure to propionate, while unexpectedly, inactivation of PhoP significantly increased acid resistance over that of the wild type (P < 0.05). The results suggest that the virulence of S. typhimurium may be enhanced by increasing acid resistance upon exposure to SCFA during its life cycle and further enhanced by anaerobiosis, low pH, and prolonged exposure time.     

Metabolic pathway of Propionibacterium growing with oxygen: enzymes, 13C NMR analysis, and its application for vitamin B12 production with periodic fermentation

The metabolic pathway of Propionibacterium grown under an aerobic condition is still not clear so far. In this work, cell growth, organic acid formation, vitamin B12 synthesis, and enzyme activities were determined in different aerobic cultivation systems. It was found that the propionate, which is accumulated during anaerobic cultivation, was completely decomposed when the cultivation was shifted to an aerobic condition. Moreover, pyruvate was formed in accordance with the decomposition of the propionate. Besides, more acetate was produced and a large amount of malate was formed during the aerobic cultivation. Such phenomena could be repeatedly observed in a periodic cultivation in which the dissolved oxygen concentration was alternatively controlled at 0 or 1 ppm. Enzyme analysis indicates that the regulation of organic acid formation depends on which molecule, i.e., oxygen or fumarate, serves as an electron acceptor in the respiratory chain reactions. No tricarboxylic acid cycle was found to exist in this species grown under an aerobic condition. It is evident that the randomizing pathway worked in a reversed direction in the presence of oxygen, through which the propionate is oxidized to pyruvate. The 13C NMR spectral analysis confirmed this observation.     

Hydrolysis and acidogenic fermentation of a protein,gelatin, in an anaerobic continuous culture

Summary Model studies of anaerobic protein digestion were performed using gelatin dissolved in a mineral medium, which was fed to a mixed population of bacteria in a carbon-substrate limited chemostat culture. The dilution rate and culture pH value were varied progressively in order to determine the optimal conditions for hydrolysis and acidification (i.e., fatty acids formation). The optimum pH value appeared to be in the neutral region (pH>6.3), and the maximal dilution rate allowing steady state growth was 0.23 h^-1. At this dilution rate and at pH 7 hydrolysis of gelatin was 78% complete, and 79% of the protein hydrolysed was fermented to identifiable products. At submaximal dilution rates both these values were higher. The main fermentation products were acetate, propionate, and valerate, and minor amounts of other volatile fatty acids. The product composition was relatively independent of the dilution rate, but varied substantially with the pH value.     

Amino acid uptake and energy coupling dependent on photosynthesis in Anacystis nidulans

Y-7c-s Synechococcus thermophilic strain grew at its maximum rate at pH 8 and above. The growth rate of this strain was inhibited at pH 7.0 and below, and at pH 6.0 there was no sustained growth. At a suboptimal pH, high light intensity further depressed the growth rate. The inhibition of growth resulted neither from pheophytinization nor from a low chlorophyll content. At pH 5.0 a loss of viability preceded the appearance of pheophytin. Cells exposed to low, growth-inhibiting external pH levels continued to maintain a high internal pH (pH 7.1 to 7.3, as determined at moderate light intensities by 31P nuclear magnetic resonance spectroscopy). Even during exposure to pH 4.8, cells retained a relatively high internal pH. Thus, it appeared that the inhibition of growth at low pH was not caused by acidification of the cytoplasm. Darkened cells maintained a slightly lower internal pH than irradiated cells. The ATP/(ATP + ADP) ratio decreased from 0.80 to 0.82 at pH 8.0 to about 0.6 when growth was limited by exposure to pH 6.0 or by low light intensity. It is possible, but not likely, that a limitation of the energy supply may slow or stop growth when the external pH is lowered.     

Acetate metabolism in purple non-sulfur bacteria

Abstract The photosynthetic non-sulfur purple bacterium Rhodobacter capsulatus E1F1 can grow on acetate or dl -malate photoheterotrophically under anerobic conditions or chemoheterotrophically in the dark in the presence of dioxygen. Bacterial cells grown under both anaerobic and aerobic conditions exhibited high amounts of the tricarboxylic acid cycle enzymes especially in dark-aerobic cultures. A high activity of isocitrate lyase was found in cells of R. capsulatus E1F1 and, to a lesser extent, in those of R. capsulatus IP2, Rhodobacter sphaeroides and Rhodospirillum rubrum grown photoheterotrophically on acetate under anaerobic conditions. The second enzyme of the glyoxylate shunt, malate synthase, appears to be constitutive. Itaconate, a powerful inhibitor of isocitrate lyase, severely inhibited growth of R. capsulatus, R. rubrum and R. sphaeroides on acetate, thus corroborating a physiological role of the enzyme in acetate metabolism by Rhodospirillaceae.