Metabolic manipulation by Pseudomonas fluorescens: a powerful stratagem against oxidative and metal stress.

Metabolism is the foundation of all living organisms and is at the core of numerous if not all biological processes. The ability of an organism to modulate its metabolism is a central characteristic needed to proliferate, to be dormant and to survive any assault. Pseudomonas fluorescens is bestowed with a uniquely versatile metabolic framework that enables the microbe to adapt to a wide range of conditions including disparate nutrients and toxins. In this mini-review we elaborate on the various metabolic reconfigurations evoked by this microbial system to combat reactive oxygen/nitrogen species and metal stress. The fine-tuning of the NADH/NADPH homeostasis coupled with the production of α-keto-acids and ATP allows for the maintenance of a reductive intracellular milieu. The metabolic networks propelling the synthesis of metabolites like oxalate and aspartate are critical to keep toxic metals at bay. The biochemical processes resulting from these defensive mechanisms provide molecular clues to thwart infectious microbes and reveal elegant pathways to generate value-added products.

Reactive nitrogen species (RNS)-resistant microbes: adaptation and medical implications.

Nitrosative stress results from an increase in reactive nitrogen species (RNS) within the cell. Though the RNS - nitric oxide (·NO) and peroxynitrite (ONOO-) - play pivotal physiological roles, at elevated concentrations, these moieties can be poisonous to both prokaryotic and eukaryotic cells alike due to their capacity to disrupt a variety of essential biological processes. Numerous microbes are known to adapt to nitrosative stress by elaborating intricate strategies aimed at neutralizing RNS. In this review, we will discuss both the enzymatic systems dedicated to the elimination of RNS as well as the metabolic networks that are tailored to generate RNS-detoxifying metabolites - α-keto-acids. The latter has been demonstrated to nullify RNS via non-enzymatic decarboxylation resulting in the production of a carboxylic acid, many of which are potent signaling molecules. Furthermore, as aerobic energy production is severely impeded during nitrosative stress, alternative ATP-generating modules will be explored. To that end, a holistic understanding of the molecular adaptation to nitrosative stress, reinforces the notion that neutralization of toxicants necessitates significant metabolic reconfiguration to facilitate cell survival. As the alarming rise in antimicrobial resistant pathogens continues unabated, this review will also discuss the potential for developing therapies that target the alternative ATP-generating machinery of bacteria.

Fumarate metabolism and ATP production in Pseudomonas fluorescens exposed to nitrosative stress.

Although nitrosative stress is known to severely impede the ability of living systems to generate adenosine triphosphate (ATP) via oxidative phosphorylation, there is limited information on how microorganisms fulfill their energy needs in order to survive reactive nitrogen species (RNS). In this study we demonstrate an elaborate strategy involving substrate-level phosphorylation that enables the soil microbe Pseudomonas fluorescens to synthesize ATP in a defined medium with fumarate as the sole carbon source. The enhanced activities of such enzymes as phosphoenolpyruvate carboxylase and pyruvate phosphate dikinase coupled with the increased activities of phospho-transfer enzymes like adenylate kinase and nucleoside diphophate kinase provide an effective strategy to produce high energy nucleosides in an O2-independent manner. The alternate ATP producing machinery is fuelled by the precursors derived from fumarate with the aid of fumarase C and fumarate reductase. This metabolic reconfiguration is key to the survival of P. fluorescens and reveals potential targets against RNS-resistant organisms.

Hydrogen peroxide stress provokes a metabolic reprogramming in Pseudomonas fluorescens: enhanced production of pyruvate.

Pseudomonas fluorescens invoked a metabolic reconfiguration that resulted in enhanced production of pyruvate under the challenge of hydrogen peroxide (H2O2). Although this stress led to a sharp reduction in the activities of numerous tricarboxylic acid (TCA) cycle enzymes, there was a marked increase in the activities of catalase and various NADPH-generating enzymes to counter the oxidative burden. The upregulation of phosphoenolpyruvate synthase (PEPS) and pyruvate kinase (PK) coupled with the reduction of pyruvate dehydrogenase (PDH) in the H2O2-challenged cells appear to be important contributors to the elevated levels of pyruvate found in these bacteria. Increased pyruvate synthesis was evident in the presence of a variety of carbon sources including d-glucose. Intact cells rapidly consumed d-glucose with the concomitant formation of this monocarboxylic acid. At least a 12-fold increase in pyruvate production within 1h was observed in the stressed cells. These findings may be exploited in the development of technologies aimed at the conversion of carbohydrates into pyruvate.

How aluminum, an intracellular ROS generator promotes hepatic and neurological diseases: the metabolic tale.

Metal pollutants are a global health risk due to their ability to contribute to a variety of diseases. Aluminum (Al), a ubiquitous environmental contaminant is implicated in anemia, osteomalacia, hepatic disorder, and neurological disorder. In this review, we outline how this intracellular generator of reactive oxygen species (ROS) triggers a metabolic shift towards lipogenesis in astrocytes and hepatocytes. This Al-evoked phenomenon is coupled to diminished mitochondrial activity, anerobiosis, and the channeling of α-ketoacids towards anti-oxidant defense. The resulting metabolic reconfiguration leads to fat accumulation and a reduction in ATP synthesis, characteristics that are common to numerous medical disorders. Hence, the ability of Al toxicity to create an oxidative environment promotes dysfunctional metabolic processes in astrocytes and hepatocytes. These molecular events triggered by Al-induced ROS production are the potential mediators of brain and liver disorders.

The unravelling of metabolic dysfunctions linked to metal-associated diseases by blue native polyacrylamide gel electrophoresis.

Gel electrophoresis is routinely used to separate and analyse macromolecules in biological systems. Although many of these electrophoretic techniques necessitate the denaturing of the analytes prior to their analysis, blue native polyacrylamide gel electrophoresis (BN-PAGE) permits the investigation of proteins/enzymes and their supramolecular structures such as the metabolon in native form. This attribute renders this analytical tool conducive to deciphering the metabolic perturbations invoked by metal toxicity. In this review, we elaborate on how BN-PAGE has led to the discovery of the dysfunctional metabolic pathways associated with disorders such as Alzheimer's disease, Parkinson's disease, and obesity that have been observed as a consequence of exposure to various metal toxicants.

Metabolic reengineering invoked by microbial systems to decontaminate aluminum: implications for bioremediation technologies.

As our reliance on aluminum (Al) increases, so too does its presence in the environment and living systems. Although generally recognized as safe, its interactions with most living systems have been nefarious. This review presents an overview of the noxious effects of Al and how a subset of microbes can rework their metabolic pathways in order to survive an Al-contaminated environment. For instance, in order to expulse the metal as an insoluble precipitate, Pseudomonas fluorescens shuttles metabolites toward the production of organic acids and lipids that play key roles in chelating, immobilizing and exuding Al. Further, the reconfiguration of metabolic modules enables the microorganism to combat the dearth of iron (Fe) and the excess of reactive oxygen species (ROS) promoted by Al toxicity. While in Rhizobium spp., exopolysaccharides have been invoked to sequester this metal, an ATPase is known to safeguard Anoxybacillus gonensis against the trivalent metal. Hydroxyl, carboxyl and phosphate moieties have also been exploited by microbes to trap Al. Hence, an understanding of the metabolic networks that are operative in microorganisms residing in polluted environments is critical in devising bioremediation technologies aimed at managing metal wastes. Metabolic engineering is essential in elaborating effective biotechnological processes to decontaminate metal-polluted surroundings.

A blue native polyacrylamide gel electrophoretic technology to probe the functional proteomics mediating nitrogen homeostasis in Pseudomonas fluorescens.

As glutamate and ammonia play a pivotal role in nitrogen homeostasis, their production is mediated by various enzymes that are widespread in living organisms. Here, we report on an effective electrophoretic method to monitor these enzymes. The in gel activity visualization is based on the interaction of the products, glutamate and ammonia, with glutamate dehydrogenase (GDH, EC: 1.4.1.2) in the presence of either phenazine methosulfate (PMS) or 2,6-dichloroindophenol (DCIP) and iodonitrotetrazolium (INT). The intensity of the activity bands was dependent on the amount of proteins loaded, the incubation time and the concentration of the respective substrates. The following enzymes were readily identified: glutaminase (EC: 3.5.1.2), alanine transaminase (EC: 2.6.1.2), aspartate transaminase (EC: 2.6.1.1), glycine transaminase (EC: 2.6.1.4), ornithine oxoacid aminotransferase (EC: 2.6.1.13), and carbamoyl phosphate synthase I (EC: 6.3.4.16). The specificity of the activity band was confirmed by high pressure liquid chromatography (HPLC) following incubation of the excised band with the corresponding substrates. These bands are amenable to further molecular characterization by a variety of analytical methods. This electrophoretic technology provides a powerful tool to screen these enzymes that contribute to nitrogen homeostasis in Pseudomonas fluorescens and possibly in other microbial systems.

Histidine is a source of the antioxidant, alpha-ketoglutarate, in Pseudomonas fluorescens challenged by oxidative stress.

The role of alpha-ketoglutarate (KG) in the detoxification of reactive oxygen species (ROS) has only recently begun to be appreciated. This ketoacid neutralizes ROS in an NADPH-independent manner with the concomitant formation of succinate and CO(2). To further probe this intriguing attribute of KG in living systems, we have evaluated the significance of histidine metabolism in the model organism, Pseudomonas fluorescens, challenged by hydrogen peroxide (H(2)O(2)). Here, we show that this amino acid does contribute to KG homeostasis and appears to be earmarked for the production of KG during oxidative stress. Both the NAD- and the NADP-dependent glutamate dehydrogenases were upregulated in the stressed cells despite the sharp decline in the activities of numerous enzymes mediating the tricarboxylic acid cycle and oxidative phosphorylation. Enzymes such as isocitrate dehydrogenase-NAD dependent, succinate dehydrogenase, alpha-ketoglutarate dehydrogenase, Complex I, and Complex IV were severely affected in the P. fluorescens grown in the presence of H(2)O(2). Studies with fluorocitrate, a potent inhibitor of citrate metabolism, clearly revealed that histidine was preferentially utilized in the production of KG in the H(2)O(2)-challenged cells. Regulation experiments also helped confirm that the metabolic reprogramming, resulting in the enhanced production of KG was induced by H(2)O(2) stress. These data further establish the pivotal role that KG plays in antioxidative defense.