Deletion of Genes Encoding Cytochrome Oxidases and Quinol Monooxygenase Blocks the Aerobic-Anaerobic Shift in Escherichia coli K-12 MG1655

Abstract

The constitutive activation of the anoxic redox control transcriptional regulator (ArcA) in Escherichia coli during aerobic growth, with the consequent production of a strain that exhibits anaerobic physiology even in the presence of air, is reported in this work. Removal of three terminal cytochrome oxidase genes (cydAB, cyoABCD, and cbdAB) and a quinol monooxygenase gene (ygiN) from the E. coli K-12 MG1655 genome resulted in the activation of ArcA aerobically. These mutations resulted in reduction of the oxygen uptake rate by nearly 98% and production of D-lactate as a sole by-product under oxic and anoxic conditions. The knockout strain exhibited nearly identical physiological behaviors under both conditions, suggesting that the mutations resulted in significant metabolic and regulatory perturbations. In order to fully understand the physiology of this mutant and to identify underlying metabolic and regulatory reasons that prevent the transition from an aerobic to an anaerobic phenotype, we utilized whole-genome transcriptome analysis, 13 C tracing experiments, and physiological characterization. Our analysis showed that the deletions resulted in the activation of anaerobic respiration under oxic conditions and a consequential shift in the content of the quinone pool from ubiquinones to menaquinones. An increase in menaquinone concentration resulted in the activation of ArcA. The activation of the ArcB/ArcA regulatory system led to a major shift in the metabolic flux distribution through the central metabolism of the mutant strain. Flux analysis indicated that the mutant strain had undetectable fluxes around the tricarboxylic acid (TCA) cycle and elevated flux through glycolysis and anaplerotic input to oxaloacetate. Flux and transcriptomics data were highly correlated and showed similar patterns.Escherichia coli has been studied extensively with respect to its physiology, genetics, and metabolism. One of the unique features of its metabolism is the ability to support robust growth under both oxic and anoxic conditions (38). During aerobic growth, when oxygen is used as a terminal electron acceptor, E. coli divides rapidly and produces carbon dioxide and acetate as major growth by-products (38), representing an efficient form of energy metabolism. In the absence of oxygen, E. coli and other microorganisms rely on anaerobic respiration and fermentation in order to oxidize substrates, recycle electron carriers, and produce ATP (38). This metabolic versatility of E. coli allows it to survive and thrive over a wide range of conditions.Since the ability to produce a number of reduced by-products, such as organic acids and ethanol, is of importance in the field of metabolic engineering, the majority of metabolic engineering designs rely on anaerobic conditions (15,30,31). It has also been shown that E. coli strains developed for overproduction of commodity chemicals can further be improved using adaptive evolution strategies (15). Adaptive evolution is often performed through a series of dilutions allowing cells to...

Ubiquinone and Menaquinone Electron Carriers Represent the Yin and Yang in the Redox Regulation of the ArcB Sensor Kinase

“…Data represent the averages from four experiments, and the standard deviation values are indicated. and coworkers (41). Our results are also in accordance with the previous findings that despite the aerobic growth conditions, an ArcA-P expression profile was observed in mutant strains lacking both cytochrome bo and cytochrome bd terminal oxidases (42), the ubiG gene (43), or the three terminal cytochrome oxidase genes (cydAB, cyoABCD, and cbdAB) and a quinol monooxygenase gene (ygiN) (41).…”

“…Therefore, the authors concluded that inhibition of ArcB phosphatase activity mediated by fermentation products, such as acetate, may be the dominant mechanism for regulating ArcB and, thereby, ArcA activity instead of the redox state of the ubiquinone pool (26). However, the 2-fold difference in total ubiquinone between anaerobiosis and full aerobiosis contrasts the ϳ5-fold increase reported earlier (25,39,41,44,45) and weakens the above-mentioned conclusion. Moreover, this mode of ArcB regulation has been discarded in earlier in vivo and in vitro studies that demonstrate that fermentation products, such as acetate and lactate, have no effect on the ArcB phosphatase activity (12) and do not act as direct signals for activation of ArcB E=°ϭ ϩ100 mV) constitute the major quinones in the respiratory chain of E. coli.…”

“…and coworkers (41). Our results are also in accordance with the previous findings that despite the aerobic growth conditions, an ArcA-P expression profile was observed in mutant strains lacking both cytochrome bo and cytochrome bd terminal oxidases (42), the ubiG gene (43), or the three terminal cytochrome oxidase genes (cydAB, cyoABCD, and cbdAB) and a quinol monooxygenase gene (ygiN) (41). These observations are promptly explained by the demonstration that these mutations result in a shift in the content of the quinone pool from the ubiquinones to menaquinones (41).…”

“…A central component in these networks is the quinone pool that acts as a redox mediator. During aerobic growth, the major quinone is ubiquinone (E=°ϭ ϩ100 mV), which is replaced by menaquinone (E=°ϭ Ϫ74 mV) in anaerobic growth (25,39,41,44,45). Thus, during aerobic growth conditions or upon a shift from anaerobic to aerobic growth, the predominant ubiquinone pool acts to oxidize the cysteine residues of ArcB (E=°Ϸ Ϫ41 mV), resulting in disulfide bond formation and immediate silencing of the ArcB kinase activity, leading to inactivation of ArcA.…”

Insights into the Mode of Action of Benzyl Isothiocyanate on Campylobacter jejuni

“…Similar shifts in metabolism were observed for an E. coli mutant strain deleted for the three terminal cytochrome oxidase genes and therefore unable to use oxygen for oxidative phosphorylation and ATP synthesis. In this mutant, genes for anaerobic respiration were induced even under oxic conditions, as were glycolysis-related genes, probably to sustain ATP generation through substrate-level phosphorylation (88).…”