Robert Hamel

The Tricarboxylic Acid Cycle, an Ancient Metabolic Network with a Novel Twist

The tricarboxylic acid (TCA) cycle is an essential metabolic network in all oxidative organisms and provides precursors for anabolic processes and reducing factors (NADH and FADH2) that drive the generation of energy. Here, we show that this metabolic network is also an integral part of the oxidative defence machinery in living organisms and α-ketoglutarate (KG) is a key participant in the detoxification of reactive oxygen species (ROS). Its utilization as an anti-oxidant can effectively diminish ROS and curtail the formation of NADH, a situation that further impedes the release of ROS via oxidative phosphorylation. Thus, the increased production of KG mediated by NADP-dependent isocitrate dehydrogenase (NADP-ICDH) and its decreased utilization via the TCA cycle confer a unique strategy to modulate the cellular redox environment. Activities of α-ketoglutarate dehydrogenase (KGDH), NAD-dependent isocitrate dehydrogenase (NAD-ICDH), and succinate dehydrogenase (SDH) were sharply diminished in the cellular systems exposed to conditions conducive to oxidative stress. These findings uncover an intricate link between TCA cycle and ROS homeostasis and may help explain the ineffective TCA cycle that characterizes various pathological conditions and ageing.

An ATP and Oxalate Generating Variant Tricarboxylic Acid Cycle Counters Aluminum Toxicity in Pseudomonas fluorescens

Although the tricarboxylic acid (TCA) cycle is essential in almost all aerobic organisms, its precise modulation and integration in global cellular metabolism is not fully understood. Here, we report on an alternative TCA cycle uniquely aimed at generating ATP and oxalate, two metabolites critical for the survival of Pseudomonas fluorescens. The upregulation of isocitrate lyase (ICL) and acylating glyoxylate dehydrogenase (AGODH) led to the enhanced synthesis of oxalate, a dicarboxylic acid involved in the immobilization of aluminum (Al). The increased activity of succinyl-CoA synthetase (SCS) and oxalate CoA-transferase (OCT) in the Al-stressed cells afforded an effective route to ATP synthesis from oxalyl-CoA via substrate level phosphorylation. This modified TCA cycle with diminished efficacy in NADH production and decreased CO2-evolving capacity, orchestrates the synthesis of oxalate, NADPH, and ATP, ingredients pivotal to the survival of P. fluorescens in an Al environment. The channeling of succinyl-CoA towards ATP formation may be an important function of the TCA cycle during anaerobiosis, Fe starvation and O2-limited conditions.

Aluminum-tolerant Pseudomonas fluorescens : ROS toxicity and enhanced NADPH production

Aluminum (Al) triggered a marked increase in reactive oxygen species (ROS) such as O2− and H2O2 in Pseudomonas fluorescens. Although the Al-stressed cells were characterized with higher amounts of oxidized lipids and proteins than controls, NADPH production was markedly increased in these cells. Blue native polyacrylamide gel electrophoresis (BN-PAGE) analyses coupled with activity and Coomassie staining revealed that NADP+ -dependent isocitrate dehydrogenase (ICDH, E.C. 1.1.1.42) and glucose-6-phosphate dehydrogenase (G6PDH, E.C. 1.1.1.49) played a pivotal role in diminishing the oxidative environment promoted by Al. These enzymes were overexpressed in the Al-tolerant microbes and were modulated by the presence of either Al or hydrogen peroxide (H2O2) or menadione. The activity of superoxide dismutase (SOD, E.C. 1.15.1.1), an enzyme known to combat ROS stress was also increased in the cells cultured in millimolar amounts of Al. Hence, Al-tolerant P. fluorescens invokes an anti-oxidative defense strategy in order to survive.

Aluminum detoxification in Pseudomonas fluorescens is mediated by oxalate and phosphatidylethanolamine.

13C NMR studies with aluminum (Al)-stressed Pseudomonas fluorescens revealed that the trivalent metal was secreted in association with oxalate and phosphatidylethanolamine (PE). These moieties were observed in the insoluble pellet obtained upon incubation of these resting cells in the presence of either Al-citrate or citrate. This extrusion process was concomitant with the utilization of either of these tricarboxylic acids as a substrate. While only minimal amounts of Al were secreted in the presence of such carbon source as glucose, succinate or oxaloacetate, oxalate did permit the efflux of Al. Neither alpha-ketoglutarate nor ethylenediaminetetraacetic acid (EDTA) was effective in dislocating Al from the cells. The elimination of Al from the cells did not appear to be affected by p-dinitrophenol (DNP) or dicyclohexylcarbodiimide (DCCD) or azide, but was sensitive to temperature, pH and cerulenin, an inhibitor of lipid synthesis. Thus, it appears that P. fluorescens detoxifies Al via its extrusion in association with oxalate and PE in a process that apparently does not necessitate the direct utilization of energy.

Oxalic acid production and aluminum tolerance in Pseudomonas fluorescens

13C NMR studies on intact cells from Al-stressed Pseudomonas fluorescens incubated with citric acid or Al-citrate yielded peaks at 158 and 166 ppm that were attributable to free and complexed oxalic acid, respectively. The presence of oxalic acid was further confirmed with the aid of oxalate oxidase. These peaks were not discernable in experiments performed with cells taken from control cultures. Enzymatic analyses of cell fractions showed the highest production of oxalic acid in the inner membrane fraction of Al-stressed cells incubated with glyoxylate. There was an eight-fold increase in the synthesis of oxalic acid in the inner membrane fraction from the Al-stressed cells compared to the control cells. Although oxalic acid production was observed when citrate, Al-citrate and isocitrate were utilized as substrates, the inner membrane fraction did not mediate the formation of oxalic acid from glycine/pyruvate, glycolic acid, oxaloacetate or ascorbate. These data suggest that the increased oxalic acid production in response to Al stress is effected via the oxidation of glyoxylate.

Adaptation of Pseudomonas fluorescens to Al-citrate: involvement of tricarboxylic acid and glyoxylate cycle enzymes and the influence of phosphate.

The degradation of Aluminum-citrate by Pseudomonas fluorescens necessitated a major restructuring of the various enzymatic activities involved in the TCA and glyoxylate cycles. While a six-fold increase in fumarase (FUM EC 4.2.1.2) activity was observed in cells subjected to Al-citrate compared to control cells, citrate synthase (CS EC 4.1.3.7) activity experienced a two-fold increase. On the other hand, in the Al-stressed cells malate synthase (MS EC 4.1.3.2) activity underwent a five-fold decrease in activity. This modulation of enzymatic activities appeared to be evoked by Al stress, as the incubation of Al-stressed cells in control media led to the complete reversal of these enzymatic profiles. These observations were further confirmed by 1H NMR and 13C NMR spectroscopy. No significant variations were observed in the activities of other glyoxylate and TCA cycle enzymes, like isocitrate lyase (ICL EC 4.1.3.1), malate dehydrogenase (MDH EC 1.1.1.37), and succinate dehydrogenase (SDH EC 1.3.99.1). This reconfiguration of the metabolic pathway appears to favour the production of a citrate-rich aluminophore that is involved in the sequestration of Al.

The Metabolism of Aluminum Citrate and Biosynthesis of Oxalic Acid in Pseudomonas fluorescens

Abstract13CNMR and 1HNMR studies revealed that aluminum citrate (Al-citrate) was metabolized intracellularly and that oxalic acid was an important product in the Al-stressed cells. This dicarboxylic acid was produced via the oxidation of glyoxylate, a precursor generated through the cleavage of isocitrate. In the control cells, citrate was biotransformed essentially with the aid of regular tricarboxylic cycle (TCA) enzymes. However, these control cells were able neither to uptake nor to metabolize Al-citrate. Al-stressed cells obtained at 38–40 h of growth showed maximal Al-citrate uptake and biotransforming activities. At least a fourfold increase in the activity of the enzyme isocitrate lyase (ICL, E. C. 4.1.3.1) has been observed in the Al-stressed cells compared with the control cells. The transport of Al-citrate was sensitive to p-dinitrophenol and sodium azide, but not to dicyclohexylcarbodiimide. Experiments with the dye 9-aminoacridine revealed that the translocation of Al-citrate led to an increase in intracellular pH. Thus, it appears that after the uptake of Al-citrate, this complex is metabolized intracellularly.

Phosphatidylethanolamine production and iron homeostasis in Pseudomonas fluorescens

Abstract Pseudomonas fluorescens was found to grow in a minimal mineral medium containing citrate, the sole carbon source, complexed to 5 mM iron(III). As the tricarboxylic acid was utilized, dialysis and ultracentrifigation analysis of the spent fluid revealed that iron (III) was associated with phosphatidylethanolamine (PE). Atomic absorption studies revealed that the iron concentration in the soluble cellular fraction was maximal at 35 h of incubation and accounted for 1.3% of the total iron. However, at stationary phase of growth, most of the iron was deposited as a PE containing residue. A transmission electron microscope, equipped with an electron energy loss spectrometer (EELS), allowed the visualization of iron bodies within the bacterial cells. However, such metal inclusions were absent in cells isolated at stationary phase of growth.

Bioaccumulation of Yttrium: A Microbial Model for the Management of Nuclear Wastes

Pseudomonas fluorescens was found to multiply readily in a minimal mineral medium supplemented with millimolar amounts of yttrium complexed to citrate, the sole carbon source. At stationary phase of growth, the microbe accumulated 65% of the trivalent metal originally found in the growth medium. The examination of cell fractions revealed that most of the metal was associated with the outer membranes. Subsequent exposure of these membranes to yttrium pointed to their ability to further accumulate the metal. Electrophoresis of the membranes isolated from the yttrium stressed cells revealed the presence of numerous polypeptide bands that were absent in the membranes from the control cells. Transmission electron microscopy aided in the identification of yttrium in the membrane components. This model system has the potential of removing yttrium from contaminated sites.