Reserve Flux Capacity in the Pentose Phosphate Pathway Enables Escherichia coli's Rapid Response to Oxidative Stress

Abstract

To counteract oxidative stress and reactive oxygen species (ROS), bacteria evolved various mechanisms, primarily reducing ROS through antioxidant systems that utilize cofactor NADPH. Cells must stabilize NADPH levels by increasing flux through replenishing metabolic pathways like pentose phosphate (PP) pathway. Here, we investigate the mechanism enabling the rapid increase in NADPH supply by exposing Escherichia coli to hydrogen peroxide and quantifying the immediate metabolite dynamics. To systematically infer active regulatory interactions governing this response, we evaluated ensembles of kinetic models of glycolysis and PP pathway, each with different regulation mechanisms. Besides the known inactivation of glyceraldehyde 3-phosphate dehydrogenase by ROS, we reveal the important allosteric inhibition of the first PP pathway enzyme by NADPH. This NADPH feedback inhibition maintains a below maximum-capacity PP pathway flux under non-stress conditions. Relieving this inhibition instantly increases PP pathway flux upon oxidative stress. We demonstrate that reducing cells' capacity to rapidly reroute their flux through the PP pathway increases their oxidative stress sensitivity.

Modulated efficacy CRISPRi reveals evolutionary conservation of essential gene expression-fitness relationships in bacteria

“…Second, we examined the expression-fitness relationships of murA/murAA and folA/dfrA using comprehensive mismatched sgRNA libraries. Consistent with previous studies (22)(23)(24), we find that CRISPRi targeting of murA/murAA bimodally affects fitness ( Fig. 2B-C), while CRISPRi targeting of folA/dfrA linearly affects growth rate above an initial threshold of activity ( Fig.…”

“…This observation is consistent with the small-colony but non-culturable phenotype of essential cofactor biosynthesis gene deletions (21) and suggests that these cofactors and/or the enzymes producing them are present in excess of what is required for exponential growth. This buffer may be required to enable rapid shifts in metabolism in response to changing environmental conditions, similar to what has been proposed for the pentose-phosphate pathway (22).…”

Investigating the deregulation of metabolic tasks via Minimum Network Enrichment Analysis (MiNEA) as applied to nonalcoholic fatty liver disease using mouse and human omics data

“…Thus, the 290 different patterns of regulation observed for these reactions can unbalance both NADPH concentration and H 2 O 2 synthesis. Interestingly it has been reported that the unbalance in NADPH production through PPP by either an under or over-production of NADPH may induce oxidative stress [25,26].…”

Model balancing: in search of consistent metabolic states and in-vivo kinetic constants

“…The tests show what information about kinetic constants can be extracted from omics data and reveal practical limits of estimating kinetic constants in vivo.Large models have been parameterised [21,22], and pipelines for model parameterization have been developed [23,24]. Finally, even if parameters are unknown, methods for parameter sampling and ensemble modelling allow to find plausible parameter sets [25] and to draw conclusions about possible dynamic behaviour [26].The key problem here is to obtain realistic, consistent values of kinetic constants. In-vivo values are hard to measure, and in-vitro values as proxies may be unreliable -or at least, this is hard to assess unless in-vivo values are known.…”

“…In theory, parameter fitting and optimisation can be performed by random screening or by Monte-Carlo methods for optimisation, such as genetic algorithms or simulated annealing. For example, one may generate a large ensemble of possible parameter sets, compute for each of them the likelihood or posterior density values, and choose the one that performs best (see [25] for an example).…”