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Uncoupling of substrate-level phosphorylation in Escherichia coli during glucose-limited growth

Abstract (Expand)

The respiratory chain of Escherichia coli contains three different cytochrome oxidases. Whereas the cytochrome bo oxidase and the cytochrome bd-I oxidase are well characterized and have been shown to … contribute to proton translocation, physiological data suggested a nonelectrogenic functioning of the cytochrome bd-II oxidase. Recently, however, this view was challenged by an in vitro biochemical analysis that showed that the activity of cytochrome bd-II oxidase does contribute to proton translocation with an H(+)/e(-) stoichiometry of 1. Here, we propose that this apparent discrepancy is due to the activities of two alternative catabolic pathways: the pyruvate oxidase pathway for acetate production and a pathway with methylglyoxal as an intermediate for the production of lactate. The ATP yields of these pathways are lower than those of the pathways that have so far always been assumed to catalyze the main catabolic flux under energy-limited growth conditions (i.e., pyruvate dehydrogenase and lactate dehydrogenase). Inclusion of these alternative pathways in the flux analysis of growing E. coli strains for the calculation of the catabolic ATP synthesis rate indicates an electrogenic function of the cytochrome bd-II oxidase, compatible with an H(+)/e(-) ratio of 1. This analysis shows for the first time the extent of bypassing of substrate-level phosphorylation in E. coli under energy-limited growth conditions.

Authors: , Klaas J Hellingwerf, Maarten J Teixeira de Mattos,

Date Published: 27th Jul 2012

Publication Type: Not specified

Using a Genome-Scale Metabolic Model of Enterococcus faecalis V583 To Assess Amino Acid Uptake and Its Impact on Central Metabolism

Abstract (Expand)

Increasing antibiotic resistance in pathogenic bacteria necessitates the development of new medication strategies. Interfering with the metabolic network of the pathogen can provide novel drug targets … but simultaneously requires a deeper and more detailed organism-specific understanding of the metabolism, which is often surprisingly sparse. In light of this, we reconstructed a genome-scale metabolic model of the pathogen Enterococcus faecalis V583. The manually curated metabolic network comprises 642 metabolites and 706 reactions. We experimentally determined metabolic profiles of E. faecalis grown in chemically defined medium in an anaerobic chemostat setup at different dilution rates and calculated the net uptake and product fluxes to constrain the model. We computed growth-associated energy and maintenance parameters and studied flux distributions through the metabolic network. Amino acid auxotrophies were identified experimentally for model validation and revealed seven essential amino acids. In addition, the important metabolic hub of glutamine/glutamate was altered by constructing a glutamine synthetase knockout mutant. The metabolic profile showed a slight shift in the fermentation pattern toward ethanol production and increased uptake rates of multiple amino acids, especially l-glutamine and l-glutamate. The model was used to understand the altered flux distributions in the mutant and provided an explanation for the experimentally observed redirection of the metabolic flux. We further highlighted the importance of gene-regulatory effects on the redirection of the metabolic fluxes upon perturbation. The genome-scale metabolic model presented here includes gene-protein-reaction associations, allowing a further use for biotechnological applications, for studying essential genes, proteins, or reactions, and the search for novel drug targets.

Authors: N. Veith, M. Solheim, K. W. van Grinsven, B. G. Olivier, J. Levering, R. Grosseholz, J. Hugenholtz, H. Holo, I. Nes, B. Teusink, U. Kummer

Date Published: 19th Dec 2014

Publication Type: Not specified

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