Characterization of E. coli MG1655 and frdA and sdhC mutants at various aerobiosis levels

Depending on the availability of oxygen, Escherichia coli is able to switch between aerobic respiratory metabolism and anaerobic mixed acid fermentation. An important, yet understudied, metabolic mode is the micro-aerobic metabolism at intermediate oxygen availabilities. The relationship between oxygen input, physiology and gene expression of E. coli MG1655 and two isogenic mutants lacking succinate dehydrogenase (SDH) and fumarate reductase (FRD) activities was analyzed at different aerobiosis levels. Growth rate and cell yield were very similar to the parent strain. By-product formation was altered in the sdhC mutant to higher acetic acid and glutamate production in batch cultures. In continuous cultures with defined oxygen input gene expression analysis revealed a dependency of many catabolic genes to aerobiosis. Acetate excretion was still detectable under aerobic conditions in the sdhC mutant; the frdA mutant lacked anaerobic succinate excretion. Anaerobic repression of the sdh operon was diminished in the frdA strain, possibly to allow SDH to partially replace FRD. The experiments illustrate the remarkable adaptability of E. coli physiology—to compensate for the absence of important metabolic genes by altering carbon flux and/or gene expression such that there are only minor changes in growth capability across the aerobiosis range.     

Two global regulators repress the anaerobic expression of MnSOD in Escherichia coli: Fur (ferric uptake regulation) and Arc (aerobic respiration control)

The expression of sodA, the Escherichia coli gene encoding manganese superoxide dismutase (MnSOD) is induced by aerobiosis and superoxide generators such as paraquat. Analysis of variants expressing sodA in the absence of oxygen has revealed that mutations in genes for two global regulatory systems, Fur (ferric uptake regulation) and Arc (aerobic respiration control), are simultaneously required for the expression of sodA in anaerobiosis. The Fur protein still represses sodA in an iron-dependent fashion in aerobiosis. Moreover, all mutants remain inducible by paraquat, indicating that the positive control of SoxR, which mediates the response to superoxide in E. coli, is still operative. Thus, in addition to the response to the superoxide-mediated oxidative stress which depends on SoxR, two global controls regulate MnSOD expression: ArcA couples MnSOD expression to respiration, and Fur couples it to the intracellular concentration of iron.     

Quantitative assessment of oxygen availability: perceived aerobiosis and its effect on flux distribution in the respiratory chain of Escherichia coli

Despite a large number of studies on the role of oxygen in cellular processes, there is no consensus as to how oxygen availability to the cell should be defined, let alone how it should be quantified. Here, a quantitative definition for oxygen availability (perceived aerobiosis) is presented; the definition is based on a calibration with reference to the minimal oxygen supply rate needed for fully oxidative catabolism (i.e., complete conversion of the energy source to CO(2) and water for glucose-limited conditions). This quantitative method is used to show how steady-state electron fluxes through the alternative cytochrome oxidases of Escherichia coli are distributed as a function of the extent of aerobiosis of glucose-limited chemostat cultures. At low oxygen availability the electron flux is mainly via the high-affinity cytochrome bd oxidase, and, at higher oxygen availability, a similar phenomenon occurs but now via the low-affinity cytochrome bo oxidase. The main finding is that the catabolic activities of E. coli (and specifically its respiratory activity) are affected by the actual oxygen availability per unit of biomass rather than by the residual dissolved oxygen concentration of the culture.     

Effect of oxygen on the Escherichia coli ArcA and FNR regulation systems and metabolic responses

Escherichia coli has several elaborate sensing mechanisms for response to the availability of oxygen and the presence of other electron acceptors. The adaptive responses are coordinated by a group of global regulators, which include the one-component Fnr protein, and the two-component Arc system. To quantitate the contribution of Arc and Fnr-dependent regulation in catabolism, arcA and fnr mutant strains were constructed using the recently developed lambda derived recombination system. The metabolic activity of wildtype E. coli, an arcA mutant, an fnr mutant, and a double arcA-fnr mutant, via the fermentative pathways in glucose-limited cultures and different oxygen concentrations was studied in chemostat cultures at steady state. It was found that the most significant role of ArcA is under microaerobic conditions, while that of FNR is under more strictly anaerobic conditions. The FNR protein is normally inactive during microaerobic conditions. However, our results indicate that in the arcA mutant strain the cells behave as if a higher level of the FNR regulator is in the activated form compared to the wildtype strain during the transition from aerobic to microanaerobic growth. The results show a significant increase in the flux through pyruvate formate lyase (PFL) in the presence of oxygen. The activity of FNR-regulated pathways in the arcA mutant strain is correlated with the high redox potential obtained under microaerobic growth.     

Kinase Activity of ArcB from Escherichia coli Is Subject to Regulation by Both Ubiquinone and Demethylmenaquinone

Expression of the catabolic network in Escherichia coli is predominantly regulated, via oxygen availability, by the two-component system ArcBA. It has been shown that the kinase activity of ArcB is controlled by the redox state of two critical pairs of cysteines in dimers of the ArcB sensory kinase. Among the cellular components that control the redox state of these cysteines of ArcB are the quinones from the cytoplasmic membrane of the cell, which function in ‘respiratory’ electron transfer. This study is an effort to understand how the redox state of the quinone pool(s) is sensed by the cell via the ArcB kinase. We report the relationship between growth, quinone content, ubiquinone redox state, the level of ArcA phosphorylation, and the level of ArcA-dependent gene expression, in a number of mutants of E. coli with specific alterations in their set of quinones, under a range of physiological conditions. Our results provide experimental evidence for a previously formulated hypothesis that not only ubiquinone, but also demethylmenaquinone, can inactivate kinase activity of ArcB. Also, in a mutant strain that only contains demethylmenaquinone, the extent of ArcA phosphorylation can be modulated by the oxygen supply rate, which shows that demethylmenaquinone can also inactivate ArcB in its oxidized form. Furthermore, in batch cultures of a strain that contains ubiquinone as its only quinone species, we observed that the ArcA phosphorylation level closely followed the redox state of the ubiquinone/ubiquinol pool, much more strictly than it does in the wild type strain. Therefore, at low rates of oxygen supply in the wild type strain, the activity of ArcB may be inhibited by demethylmenaquinone, in spite of the fact that the ubiquinones are present in the ubiquinol form.     

Importance of anaerobic superoxide dismutase synthesis in facilitating outgrowth of Escherichia coli upon entry into an aerobic habitat.

The manganese-containing isozyme of superoxide dismutase (MnSOD) is synthesized by Escherichia coli only during aerobiosis, in accordance with the fact that superoxide can be formed only in aerobic environments. In contrast, E. coli continues to synthesize the iron-containing isozyme (FeSOD) even in the absence of oxygen. A strain devoid of FeSOD exhibited no deficits during either anaerobic or continuously aerobic growth, but its growth lagged for 2 h during the transition from anaerobiosis to aerobiosis. Complementation of this defect with heterologous SODs established that anaerobic SOD synthesis per se is necessary to permit a smooth transition to aerobiosis. The growth deficit was eliminated by supplementation of the medium with branched-chain amino acids, indicating that the growth interruption was due to the established sensitivity of dihydroxyacid dehydratase to endogenous superoxide. Components of the anaerobic respiratory chain rapidly generated superoxide when exposed to oxygen in vitro, suggesting that this transition may be a period of acute oxidative stress. These results show that facultative bacteria must preemptively synthesize SOD during anaerobiosis in preparation for reaeration. The data suggest that evolution has chosen FeSOD for this function because of the relative availability of iron, in comparison to manganese, during anaerobiosis.     

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.     

Three hydroxylations incorporating molecular oxygen in the aerobic biosynthesis of ubiquinone in Escherichia coli.

The biosynthetic origin of the oxygen atoms of ubiquinone 8 from aerobically grown Escherichia coli was studied by 18O labeling. An apparatus was developed which allowed the growth of cells under a defined atmosphere. Mass spectral analysis of ubiquinone 8 from cells grown under highly enriched 18O2 showed that three oxygen atoms of the quinone are derived from molecular oxygen. It was established that the molecular oxygen is incorporated into the two methoxyl groups (at C-5 and C-6) and one of the carbonyl positions of the ubiquinone molecule by demonstrating that only one of the incorporated oxygens will exchange with water under acidic conditions that specifically catalyze the exchange of carbonyl, but not methoxyl, oxygens. That the C-4 carbonyl oxygen is derived from molecular oxygen was shown by the incorporation of three atoms of 18O2 into ubiquinone 8 biosynthesized from added 4-hydroxybenzoic acid. Comparison of ubiquinone 8 and menaquinone 8 from E. coli grown under 18O2 confirmed that the labeled carbonyl oxygen of the [18O2]ubiquinone 8 is incorporated biosynthetically and not by chemical exchange in the cell. It is concluded that the three hydroxylation reactions involved in the pathway for the aerobic biosynthesis of ubiquinone are all catalyzed by monooxygenases. The implications of this study for the anaerobic biosynthesis of ubiquinone 8 in E coli are discussed.