Fumarate Reductase Activity Maintains an Energized Membrane in Anaerobic Mycobacterium tuberculosis

Abstract

Oxygen depletion of Mycobacterium tuberculosis engages the DosR regulon that coordinates an overall down-regulation of metabolism while up-regulating specific genes involved in respiration and central metabolism. We have developed a chemostat model of M. tuberculosis where growth rate was a function of dissolved oxygen concentration to analyze metabolic adaptation to hypoxia. A drop in dissolved oxygen concentration from 50 mmHg to 0.42 mmHg led to a 2.3 fold decrease in intracellular ATP levels with an almost 70-fold increase in the ratio of NADH/NAD+. This suggests that re-oxidation of this co-factor becomes limiting in the absence of a terminal electron acceptor. Upon oxygen limitation genes involved in the reverse TCA cycle were upregulated and this upregulation was associated with a significant accumulation of succinate in the extracellular milieu. We confirmed that this succinate was produced by a reversal of the TCA cycle towards the non-oxidative direction with net CO2 incorporation by analysis of the isotopomers of secreted succinate after feeding stable isotope (13C) labeled precursors. This showed that the resulting succinate retained both carbons lost during oxidative operation of the TCA cycle. Metabolomic analyses of all glycolytic and TCA cycle intermediates from 13C-glucose fed cells under aerobic and anaerobic conditions showed a clear reversal of isotope labeling patterns accompanying the switch from normoxic to anoxic conditions. M. tuberculosis encodes three potential succinate-producing enzymes including a canonical fumarate reductase which was highly upregulated under hypoxia. Knockout of frd, however, failed to reduce succinate accumulation and gene expression studies revealed a compensatory upregulation of two homologous enzymes. These major realignments of central metabolism are consistent with a model of oxygen-induced stasis in which an energized membrane is maintained by coupling the reductive branch of the TCA cycle to succinate secretion. This fermentative process may offer unique targets for the treatment of latent tuberculosis.

Rifamycin action on RNA polymerase in antibiotic-tolerant Mycobacterium tuberculosis results in differentially detectable populations

“…Inclusion of charcoal in the cfu plates to absorb RIF carried over from the original culture (23) did not increase the number of cfus and thereby affect the estimation of DD Mtb, nor did addition of hemin, an alternate source of iron (24), or fumarate, an alternate electron acceptor (25) (Fig. S2A).…”

An obligately aerobic soil bacterium activates fermentative hydrogen production to survive reductive stress during hypoxia

“…These data provide evidence that hydrogen oxidation can be coupled to the reduction of the traditionally anaerobic acceptor fumarate when oxygen is depleted. Succinate production has previously been shown to have an essential role in adaptation of mycobacteria to hypoxia; the multifunctional compound is capable of being oxidized for ATP synthesis, used for anaplerosis, or excreted as a potential fermentative end product (9,12). Hence, transferring electrons from H 2 to fumarate may provide a mechanism to sustain both the proton-motive force and ATP synthesis through anaerobic respiration while maintaining redox balance in the cell.…”

“…M. tuberculosis uses anaerobic electron sinks such as nitrate and possibly fumarate (9,10). In contrast to the pathogen, M. smegmatis lacks a functional nitrate reductase (10,11), and it is unclear if one of the putative succinate dehydrogenase/fumarate reductase operons (9, 12) acts as a fumarate reductase.…”

“…This genus is generally modeled to be strictly respiratory, given their F 1 F o -ATP synthase is required for growth even on fermentable carbon sources (16,17). However, a recent observation of succinate secretion during hypoxia suggests that some mycobacteria may in fact have the capacity to excrete fermentative end products (9,12). It has also been suggested, but never proven, that mycobacteria dissipate excess reductant by coupling the reoxidation of NAD(P)H to the production of H 2 (14,15,18,19).…”

Mycobacterium tuberculosis Phosphoenolpyruvate Carboxykinase Is Regulated by Redox Mechanisms and Interaction with Thioredoxin

“…In fact, hypoxia induces widespread changes in MTb metabolism due to the induction of the DosR/S two-component system (6) and the KstR repressor (7). During the transition to the hypoxia-induced non-replicating state of the bacteria, the intracellular ATP level decreases, and the absence of oxygen as a terminal electron acceptor leads to the accumulation of reduced cofactors (8,9). Transcriptional and metabolomics studies of MTb under hypoxic conditions have indicated a rearrangement of central carbon metabolism, leading to the synthesis of oxaloacetate (OAA) by an anaplerotic CO 2 -fixing reaction and to the secretion of succinic acid (9,10).…”