Acid- and base-induced proteins during aerobic and anaerobic growth of Escherichia coli revealed by two-dimensional gel electrophoresis

Proteins induced by acid or base, during long-term aerobic or anaerobic growth in complex medium, were identified in Escherichia coli. Two-dimensional gel electrophoresis revealed pH-dependent induction of 18 proteins, nine of which were identified by N-terminal sequencing. At pH 9, tryptophan deaminase (TnaA) was induced to a high level, becoming one of the most abundant proteins observed. TnaA may reverse alkalinization by metabolizing amino acids to produce acidic products. Also induced at high pH, but only in anaerobiosis, was glutamate decarboxylase (GadA). The gad system (GadA/GadBC) neutralizes acidity and enhances survival in extreme acid; its induction during anaerobic growth may help protect alkaline-grown cells from the acidification resulting from anaerobic fermentation. To investigate possible responses to internal acidification, cultures were grown in propionate, a membrane-permeant weak acid which acidifies the cytoplasm. YfiD, a homologue of pyruvate formate lyase, was induced to high levels at pH 4.4 and induced twofold more by propionate at pH 6; both of these conditions cause internal acidification. At neutral or alkaline pH, YfiD was virtually absent. YfiD is therefore a strong candidate for response to internal acidification. Acid or propionate also increased the expression of alkyl hydroperoxide reductase (AhpC) but only during aerobic growth. At neutral or high pH, AhpC showed no significant difference between aerobic and anaerobic growth. The increase of AhpC in acid may help protect the cell from the greater concentrations of oxidizing intermediates at low pH. Isocitrate lyase (AceA) was induced by oxygen across the pH range but showed substantially greater induction in acid or in base than at pH 7. Additional responses observed included the induction of MalE at high pH and induction of several enzymes of sugar metabolism at low pH: the phosphotransferase system components ManX and PtsH and the galactitol fermentation enzyme GatY. Overall, our results indicate complex relationships between pH and oxygen and a novel permeant acid-inducible gene, YfiD.     

Chromosomally-determined induced tolerance to copper in Escherichia coli

Pre-exposure of Echerichia coli to sub-lethal concentrations of cupric sulphate induced copper tolerance: pre-exposed (habituated) organisms were essentially unaffected by concentrations of Cu²⁺ which completely prevented colony formation by non-habituated ones. The observed copper tolerance was not dependent on the selection of copper-resistant mutants but resulted from a phenotypic change in the organisms during the pre-exposure period.     

Divergent roles of RpoS in Escherichia coli under aerobic and anaerobic conditions

Escherichia coli exhibited different levels of rpoS expression and general stress resistance under aerobiosis and anaerobiosis. Expression measured using reporter gene fusions and protein levels was lower under anaerobic conditions. Consistent with earlier findings, rpoS mutants were selected in aerobic nutrient-limited cultures but rpoS mutants were not enriched under anaerobiosis. This result suggested that, despite its decreased level, RpoS had a function under anaerobic conditions not essential under aerobiosis. Competition experiments between rpoS(+) and rpoS bacteria confirmed the advantage conferred by RpoS under anaerobiosis. In contrast, stress resistance assays suggested RpoS made a greater contribution to general stress resistance under aerobiosis than anaerobiosis. These results indicate a significant, but different role of RpoS in aerobic and anaerobic environments.     

Are the aerobic and anaerobic phosphofructokinases of Escherichia coli different?

Phosphofructokinase has been purified from Escherichia coli strain K-12 grown in a glucose-limited chemostat, both aerobically and anaerobically. The enzymes migrated together in polyacrylamide gel electrophoresis, had the same subunit size in denaturing (dodecylsulfate) gels (Mr approx. 34000) and the same kinetic characteristics as described earlier for E. coli phosphofructokinase [e.g. Blangy et al. (1968) J. Mol. Biol. 31, 13-35]: a sigmoid curve of velocity vs. fructose 6-phosphate concentration, activation by ADP, and inhibition by phosphoenolpyruvate. Findings [e.g. Doelle (1975) Eur. J. Biochem, 50, 335-342] of quite different enzymes in aerobic and anaerobic cells were not confirmed.     

Cell yields of Escherichia coli during anaerobic growth on fumarate and molecular hydrogen

Escherichia coli was grown anaerobically on sodium fumarate and molecular hydrogen or sodium formate in continuous culture. The maximal growth yield and the maintenance coefficient were determined. In a mineral medium a Y _fum ^max value of 6.6 g dry weight per mol fumarate was found. This value increased to 7.5 when casamino acids were present in the medium. From these data and the corresponding Y _ATP ^max values it could be calculated that per mol of fumarate reduced, 0.4 mol of ATP became available for growth. In batch culture a Y_fum value of 4.8 g dry weight per mol fumarate was determined.     

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.     

Differential effect of YidC depletion on the membrane proteome of Escherichia coli under aerobic and anaerobic growth conditions

YidC of Escherichia coli belongs to the evolutionarily conserved Oxa1/Alb3/YidC family. Members of the family have all been implicated in membrane protein biogenesis of respiratory and energy transducing proteins. The number of proteins identified thus far to require YidC for their membrane biogenesis remains limited and the identification of new substrates may allow the elucidation of properties that define the YidC specificity. To this end we investigated changes in the membrane proteome of E. coli upon YidC depletion using metabolic labeling of proteins with ¹⁵N/¹⁴N combined with a MS‐centered proteomics approach and compared the effects of YidC depletion under aerobic and anaerobic growth conditions. We found that YidC depletion resulted in protein aggregation/misfolding in the cytoplasm as well as in the inner membrane of E. coli. A dramatic increase was observed in the chaperone‐mediated stress response upon YidC depletion and this response was limited to aerobically grown cells. A number of transporter proteins were identified as possible candidates for the YidC‐dependent insertion and/or folding pathway. These included the small metal ion transporter CorA, numerous ABC transporters, as well as the MFS transporters KgtP and ProP, providing a new subset of proteins potentially requiring YidC for membrane biogenesis.     

Alternative hydroxylases for the aerobic and anaerobic biosynthesis of ubiquinone in Escherichia coli.

The synthesis of ubiquinone under anaerobic conditions was examined in a variety of strains of Escherichia coli K12. All were shown to synthesize appreciable quantities of ubiquinone 8 when grown anaerobically on glycerol in the presence of fumarate. Under these conditions, ubiquinone 8 was in most cases the principal quinone formed, and levels in the range 50--70% of those obtained aerobically were observed. Studies with mutants blocked in the various reactions of the aerobic pathway for ubiquinone 8 synthesis established that under anaerobic conditions three alternative hydroxylation reactions not involving molecular oxygen are used to derive the C-4, -5, and -6 oxygens of ubiquinone 8. Thus, mutants blocked in either of the three hydroxylation reactions of the aerobic pathway (ubiB, ubiH, or ubiF) are each able to synthesize ubiquinone 8 anaerobically, whereas mutants lacking the octaprenyltransferase (ubiA), carboxy-lyase (ubiD), or methyltransferases (ubiE or ubiG) of the aerobic pathway remain blocked anaerobically. The demonstration that E. coli possesses a special mechanism for the anaerobic biosynthesis of ubiquinone suggests that this quinone may play an important role in anaerobic metabolism.     

Stoichiometry of the H+-ATPase of Escherichia coli cells during anaerobic growth

The H+/ATP stoichiometry of the H+-ATPase was investigated in Escherichia coli cells growing under anaerobic conditions at pH 6 and 7. The protonmotive force was determined from the intracellular accumulation of benzoate and tetraphenylphosphonium ions, as well as the accumulation of lactose in this lac operon inducible, but beta-galactosidase negative strain. The phosphorylation potential was calculated from the cellular concentrations of ATP, ADP and inorganic phosphate. By comparing the phosphorylation potential and the proton motive force under these steady state conditions, the H+/ATP stoichiometry was determined to be 3, similar to the value previously found in the same cells growing under aerobic conditions.     

Osmotic repression of anaerobic metabolic systems in Escherichia coli.

The influence of the osmolarity of the growth medium on anaerobic fermentation and nitrate respiratory pathways was analyzed. The levels of several enzymes, including formate dehydrogenase, hydrogenase, and nitrate reductase, plus a nickel uptake system were examined, as was the expression of the corresponding structural and regulatory genes. While some functions appear to be only moderately affected by an increase in osmolarity, others were found to vary considerably. An increase in the osmolarity of the medium inhibits both fermentation and anaerobic respiratory pathways, though in a more dramatic fashion for the former. fnr expression is affected by osmolarity, but the repression of anaerobic gene expression was shown to be independent of FNR regulatory protein, at least for hyd-17 and fdhF. This repression could be mediated by the intracellular concentration of potassium and is reversed by glycine betaine.     

The effect of cyclic AMP on anaerobic growth of Escherichia coli

Adenosine 3′,5′-cyclic phosphate (cyclic AMP) stimulated a cyclic AMP-deficient mutant strain of $\text{Escherichia coli}$ to grow anaerobically on glucose in a minimal medium and in media supplemented with nitrate or casein hydrolysate. Cyclic AMP was found to stimulate the production of the formic hydrogenlyase system in this mutant strain, but had no effect on its ability to carry out anaerobic reductions of nitrate or nitrite. It was also observed that CO₂ stimulated the anaerobic growth of the mutant in the absence of cyclic AMP.     

Crystalline index change in cellulose during aerobic and anaerobic Cellulomonas uda growth

Summary Cultures of Cellulomonas uda were monitored under both aerobic and anaerobic conditions using three commercially available celluloses with varying degrees of crystallinity. In all cases, a high level of cellulose was metabolized and the same maximum carboxymethylcellulase activity (2.6 IU/mg of cellular protein) was observed. Measurement of the crystalline index of celluloses during cellulose growth revealed that the amorphous and crystalline regions were solubilized simultaneously. Investigation of the solubilization rate showed that a decline occurred when a considerable amount of cellulose still remained in the medium. Hypotheses were suggested to explain the biphasic pattern of the kinetics obtained.     

Microcalorimetric study of aerobic growth of Escherichia coli in batch culture

Thermograms of an aerobic batch culture of Escherichia coli K-12 in synthetic medium were obtained by using a newly designed mecrocalorimeter. The thermograms reflected sharp changes of metabolic activity in glucose-, nitrogen-, or oxygen-limited cultures. The thermo chemical analysis for each culture condition was done using the data of the heat evolved, the head of combustion of cells, and the elementary analysis of the cells. The efficiency of energy conversion determined experimentally revealed nutritional differences.     

CueO is a multi-copper oxidase that confers copper tolerance in Escherichia coli

The putative multi-copper oxidase CueO had previously been implicated in intrinsic copper resistance in Escherichia coli. In this report we showed that the presence of CueO in the periplasm protected alkaline phosphatase from copper-induced damage. CueO contained four copper atoms per molecule and displayed spectroscopic properties typical of blue copper oxidases. CueO catalyzed the oxidation of p-phenylenediamine (pPD), 2,6-dimethoxyphenol (DMP) and exhibited ferroxidase activity in vitro.     

Mutations permitting the anaerobic growth of Escherichia coli on trehalose

Wild-type strains of Escherichia coli K-12 do not grow anaerobically on trehalose or galactose. We isolated two operon fusion mutants of E. coli which gained the ability to grow on trehalose anaerobically (tan). The tanA-lac mutation was located at 41 min on the E. coli genetic map and also abolished growth on glucuronic acid both aerobically and anaerobically. The tanB-lac mutation was mapped to 68 min and permitted anaerobic growth on galactose as well as trehalose. The tanB-lac fusion was induced anaerobically whereas tanA-lac showed more or less constitutive beta-galactosidase expression.     

Differential sensitivities of the growth of Escherichia coli to acrylate under aerobic and anaerobic conditions and its effect on product formation

The effect of acrylate on the growth of Escherichia coli was determined under aerobic and anaerobic conditions in glucose-defined medium. Growth occurred with up to 35 mM acrylate under aerobic conditions but ceased at 5 mM acrylate under anaerobic conditions. This differential sensitivity can be attributed to inhibition of pyruvate formate lyase and/or pflB gene repression, as this enzyme is necessary for anaerobic growth of E. coli. The effect of acrylate on end-product distribution was also determined by growing E. coli first aerobically, then switching to anaerobic conditions. In the absence of acrylate, E. coli generated the typical distribution of mixed-acid products, with about 12 % of pyruvate being metabolically converted to lactate. In contrast, in the presence of 5 mM acrylate, E. coli converted 83 % of pyruvate to lactate, consistent with a reduction in pyruvate formate lyase activity.