The Modulation of NADPH, NADH and A-Ketoglutarate in P Flourescens Exposed to Oxidative Stress

The Modulation of NADPH, NADH and A-Ketoglutarate in P Flourescens Exposed to Oxidative Stress
Introduction

Oxygen and Life

The metabolic processes such as respiration and photosynthesis have now been proven to unavoidably lead to the production of reactive oxygen species (ROS).1 The common feature of all known ROS species is their inherent capacity to cause oxidative damage to proteins, DNA, lipids and carbohydrates in-vivo. In that there is such widespread cytotoxicity due to the by-products of energy metabolism within the aerobe, elaborate and redundant non-enzymatic and enzymatic ROS detoxification mechanisms have evolved to enable aerobic organisms to combat this danger.

The inherent paradox of aerobic life is exemplified in the statement of French chemist and oxidative theory pioneer Lavoisier in 1777: “while we cannot live without (it), we are constantly traumatized by the consequences of oxidative degradation.”The knowledge of oxygen’s pertinence to present biology dates back some 300 years B.C., hidden within the texts of Sumerian creationist myths. Since then this bi-radical molecule has found dual roles as both the giver and taker of aerobic life. The logic of using oxygen as the terminal electron acceptor in the Electron Transport Chain (ETC) is evident in its relative abundance in air and its capacity to readily reduce to harmless H2O. The biology, physiology and metabolism of an organism are now understood to be highly integrated and allows the organism to function effectively. However, problems that arise from the main ETC reaction tend to affect all aspects of life. In fact, over the past four hundred years, knowledge has accumulated at a seemingly exponential rate regarding the properties, function and hazards of oxygen in aerobic organisms. Signs of oxidative stress as either inactivated antioxidant defence or increased production of ROS themselves have been implicated in the majority of prominent diseases, and may potentially be involved in the aging process.Hence, a better understanding of the biological and exogenous sources of ROS, their properties and their detoxification is of paramount importance if one is to understand life.

The high susceptibility of aerobes to form ROS is intricately linked to the ETC, the molecular nature of oxygen, and oxidative phosphorylation. ROS are free radical species with a propensity for a short half-life. The term free radical refers to any chemical species capable of independent existence while having one or more unpaired electrons circulating its vicinity. In comparison, a pro-oxidant species is any species that satisfies the above criteria or is instrumental in the formation of such species in a biological setting. One should note that radical species are often employed within the biological setting in tightly regulated fashion in biopolymer formation, in the phagocytotic immune response, and in cell signalling cascades. Thus, ROS formation is not a biological abnormality, when generated in a controlled manner like in enzymatic reactions involving peroxidases, cytochrome p450, and xanthine oxidases. The phagocytic response to invading pathogens involves a respiratory burst comprised of low level ROS production. However, unregulated production of ROS creates an oxidative environment and inactivates essential biomolecules.

Generation of ROS
Ground state molecular oxygen is a bi-radical with its two outermost valence electrons occupying separate orbitals with parallel spins. Thus, to oxidize a nonradical atom or molecule, triplet oxygen would require a partner that provides a pair of electrons with opposite parallel spins to fit into its free orbitals. Herein lies the core of the oxidative stress theory. Pairs of electrons typically travel with opposing spins, and thus this favourably restricts the reactions of oxygen with most organic molecules. However O2 can readily accept electrons. The reduction of oxygen for covalent bond formation becomes step-wise and sometimes partial. In fact, literature boasts of some 2-5% of all oxygen processed by ETC components has been shown to be siphoned off towards incomplete reduction and thus the formation of such ROS as O2.-, H2O2, etc.

Despite having relatively short biological half-lives, the deleterious effects of ROS to proximal targets, such as ETC components, lipid, protein and nucleic acid structure, has been well documented and proven to be very deleterious. Oxidative stress itself is a description of a biological imbalance between ROS generation and ROS detoxification by the various defence mechanisms of the organism. Oxidative stress can initiate the peroxidation of polyunsaturated fatty acids, breaks in nucleic acid polymers, induce improper mitochondrial permeability pore opening for apoptotic events, and allow for alteration of protein carbonyl content and thiol groups.

B: Protein Oxidation8

C: Sulfhydryl Oxidation and Repair

The products of such reactions are often dysfunctional macromolecules that can be highly toxic as well, possibly containing further radical moieties. The inactivation of key metabolic proteins, the destabilization of the lipid membrane integrity, the severing of the nucleic acid code, and the production of toxic aldehydes are but a few of the potential negative impact of an oxidative stress. One can liken the relationship of ROS and aerobic metabolism to the damage of sparks that fly from a roaring fire. For the majority of times, the fire will burn optimally, but occasionally one will find that a cinder shoots out and burns the carpet. Given time, the carpet will degrade, revealing the vulnerable layers of the floor. Fire is the harvesting of energy through strategic and step wise oxidation of fuel molecules from various carbon sources. The sparks are ROS formed from electron leakage of ETC components, particularly the hydroxyl radical, and one can see the carpeting and the underlying floor as the various levels of organic biology in the aerobe. Such damage to the human body may be exemplified in brain disorders such as Parkinson’s or Alzheimer’s disease, or metabolic conditions like diabetes, atherosclerosis and heart disease.

Oxidative Phosphorylation and the Superoxide Anion (O2._ )
By the transfer of a single free electron to oxygen at the level of Complex I and 3, the superoxide anion, O2._ is generated. In the grand hierarchy of ROS species, superoxide has both a short half-life and is excessively polar preventing its crossing of membranes in the cell.10

The superoxide anion (O2.-), however, is readily scavenged by the SOD to produce the more stable hydrogen peroxide, H2O2. An Mn-containing SOD, localized close to ETC components, provides immediate quenching of superoxide from electron leakage, while any superoxide produced in the cytosol, possibly though excessive pooling of labile transition-metal ions and subsequent Fenton/Haber-Weiss type chemistry, can be effectively scavenged by Cu, Zn, containing soluble SOD.

Even though both O2.- and H2O2 have relatively longer biological half-lives, it is the hydroxyl anion that is considered most dangerous, and mechanisms for its production are linked directly to the presence of both ROS species. Fenton and Haber-Weiss both independently proposed the potential for O2.- and H2O2, in the presence of redox-active metals such as Cu and Fe, to readily produce the hydroxyl radical. The dual effect would be to inactivate metabolically pertinent metalloenzymes while perpetuating further oxidative stress.
1. Fe(II) + O2↔Fe(III) + O2.-, producing the superoxide anion.
2. Fe(II) + H2O2 → Fe(III) + ∙OH + OH-, producing the hydroxyl radical.
Another potential mechanism proposed by Fenton:
3. Fe(II) + H2O2 → Fe(III)OH + OH-, whereby both the iron/hydroxyl complex and the hydroxyl radical are highly reactive to lipids, proteins and nucleic acids. Another potential free iron reaction under oxidative stress is the Haber-Weiss reaction, a coupling of the above reactions, 1 and 2, into a single mechanism, whereby iron involvement is implied:
Fe(III) + O2.- → Fe(II) + O2,
Fe(II) + H2O2 → Fe(III) + ∙OH + OH- ,
O2.- + H2O2 → O2 + ∙OH + OH-
Note how the labile iron is returned to its original valency, primed for further reactions with O2.-. Thus, in theory, any mechanism that will increase the amount of free iron within a cell will contribute to an overall increase in ROS, particularly the hydroxyl radical, leading to the endogenous damage of the biological macromolecules.

Iron sulphur integrity of such metalloproteins as aconitase have been assessed under oxidative stress and found to be highly susceptible to denaturation. 13 Conceivably, ROS could liberate labile iron from aconitase, hindering the primary step of the TCA cycle.
Although the main function of ETC is ATP synthesis, there are two main side-reactions to consider beyond the simple passing electrons from reduced substrates to oxygen. One reaction describes the faulty leakage of protons back across the membrane to divert conserved energy and produce heat. This is an adaptive measure by the cell to restrict excess ATP formation. The other possible reaction, however, involves the major natural source of oxidative stress and mitochondrial dysfunction: electron leakage. Several studies of the ETC component and O2.- production in various organisms pinpoint Complex I as the main agent of electron leakage, while implicating Complex III are a minor contributor. The susceptibilities of the metal components of these two proteins to an oxidative stress are highly suspect. Interestingly, mild uncouplers, which slightly reduce both membrane potential and pH gradients of eukaryotic systems effectively, decreased the high O2.-production of Complex I. Complex III of the ETC is also suspect in that ubiquinone and superoxide production are well correlated, but its overall contribution is small in comparison.

Complex II, however, appears to be an evolutionary success story for oxidative metabolism. The enzyme fumarate reductase, catalyzing the reverse reaction in bacteria, has been shown to be a potent superoxide producer in the presence of high oxygen levels. It is theorized that over time, Complex II in higher organisms has perhaps evolved to a point whereby ROS production in the ETC is hindered. Globally speaking, O2.- production has as well been linked to glycerol-3 phosphate dehydrogenase and two enzymes of fatty acid oxidation, electron transfer flavoprotein and electron transfer flavoprotein quinine oxidoreductase.

The potential for O2.- to cause damage in a biological system is fairly high in that it is very reactive. It combines with nitric oxide to form the highly reactive peroxynitrite radical, it is readily dismutated by SOD or spontaneously denatures to H2O2 in vivo, and in the presence of redox active metals, will spawn the hydroxyl radica. Peroxynitrite is known to be 1000 fold more reactive than H2O2.

Living with ROS
It is clear that the evolution of aerobic metabolic processes has led to the production of ROS, but it is clear as well that aerobes are thriving despite it. Thus, a parallel stream of evolution has developed an elaborate antioxidant system, classically defined in two stages. Primary defence is achieved through antioxidant enzyme systems like SOD, CAT, APx, GPx and GR. The secondary defence is achieved through glutathione, vitamin E, α-tocopherol, and thioredoxin, low molecular weight scavengers of ROS and labile redox-active metals such as Cu and Fe. Of key interest to note, however, is that there are no known scavengers of the hydroxyl radical. Thus systems have evolved to control the production of its precursors through enzymatic or non-enzymatic means. This is the ancillary antioxidant system employing pro-oxidant scavengers such as ceruloplasmin and transferrin. Non enzymatic scavengers are comprised of redox buffers like glutathione and ascorbate, along with flavanoids, alkaloids and carotenoids. Mutants showing any impairment of such systems are automatically more susceptible to stress.

It is interesting to note that despite the complexity and redundancy of the antioxidant system, the presence of multiple genes and isoforms for each enzyme and high concentrations of scavengers in vivo, the fuel for the entire process is the NADPH supply. A high ratio of reduced to oxidized ascorbate and GSH is essential for ROS scavenging, thus reduced states must be maintained at sufficient levels by the action of GR, MDAR and DHAR enzymes. Recent studies aimed at analysing redox status vs. antioxidant status show that subjects having mutations of prominent NADPH producing enzymes within the metabolic scheme, such as G6PDH , IDH-NADP+, and ME show more susceptibility to stress than when having an impaired antioxidant defence. The defence system is primed to stop oxidative damage at the source by prevention of electron leakage, interception of ROS prior to hydroxyl radical formation, and repair of the damage caused by ROS on macromolecular structures. Though the dire need for such systems is undisputed in literature, it is rapidly becoming evident that a larger concern of the organism itself under oxidative stress is the maintenance of reducing power to fuel the entire defence scheme and NADPH appears to be a key candidate.

Gluathione
Much research in the past decade has been devoted to the study of the most abundant intracellular small thiol molecule, glutathione. This tri-peptide is composed of glutamate, cysteine and glycine, and it has been shown to reach millimolar concentrations in some tissues such as the brain. The brain, in fact, poses one of the larger problems for higher aerobes in that it has a relative deficit in antioxidant enzymes and an abundance of polyunsaturated fatty acids and oxygen. For the brain, the choice between ATP production and adaptation to the subsequent ROS production is significantly harder to make.16 GSH plays a critical role in detoxification of both electrophilic compounds and peroxides via catalysis with GST and GPx. Its importance is exemplified in its widespread use in plants, mammals, fungi and some prokaryotes. It is also important in the reduction of ribonucleotides to deoxyribonucleotides and regulation of protein/gene expression via thiol:disulfide exchange reactions. The largest GSH pools are maintained in the cytosol. It is then transported into the mitochondria, and even into the nuclear region for redox control of gene expression. Recent literature has shown oxidants such as H2O2 to induce specific Na+-dependent and independent transporters of cysteine.Metabolizing and scavenging systems to remove oxidants and ROS must be tightly regulated. GPx detoxifies in concert with CAT and SOD, using GSH as the electron donor and GSSG as the product, (Figure 10). The reduction of GSSG is mediated by GR, with the aid of NADPH as the essential cofactor. Under conditions of oxidative stress, GR is regulated at both the transcriptional as well as post-translational levels, and mutations are evident in both cancer and aging. The GPx tetramer has been found to adopt five distinct isoforms, highly expressed and tissue specific in higher organisms, while aberrant expression of the gene has been noted during hepatitis, HIV, and many cancers. In fact, the connections of GSH homeostasis and abnormal cellular functions are well documented.

In the case of Parkinson’s disease, the main causative factor stems from destruction of dopaminergic neurons in the substantia nigra pars compacta region of the midbrain. These cells in particular are involved in the metabolism of dopamine, a process notorious for its own ROS production, and thus the disease is labelled a dopamine deficiency. The progression of the disease is marked by reduced GSH levels that once served to protect the neurons from the dangers of their own functions. Clinical trials of supplementation of Parkinson’s patients with both GSH and N-acetyl cysteine show promising results in terms of re-attaining optimal dopamine metabolism. As well, in all cases of HIV infection, reduced levels of GSH have been reported in many affected tissues. The immune response itself is highly dependent on the regulation of the ROS used, and a reduction in GSH levels would severely compromise the actions of T-cells. In alcohol related liver disease, the connections to GSH homeostasis are even more profound. In alcoholics, pools of mitochondrial GSH are depleted with concomitant ROS damage. As well, alcoholism itself has been shown recently to involve partial inactivation of a specific mitochondrial membrane transport protein responsible for the transport of GSH from the cytosol. Thus, while a build-up of cytosolic GSH occurs, inability to transport ensues. Once again, it is important to note that the protective enzyme systems that counteract ROS wherever they may form are all tethered to GSH levels, and ultimately, to the reducing power of the cell. In plants, the situation could be described as markedly more dangerous, as UV radiation becomes a potent source of the hydroxyl radical and NADPH assumes the additional role as an electron acceptor. As light energy is to be converted to chemical energy, plants are routinely exposed to high levels of ROS. In response, plants can readily degrade photosystem II and dissipate excess energy through ATP consumption and NADPH production in photosystem I. Biosynthesis of both ascorbate and glutathione is heavily up-regulated under light stress in the form of increased UDP-glucose dehydrogenase activity for ascorbate and enhanced sulphur and nitrogen fixation levels for GSH. In all aerobic organisms, the presence of water soluble radical scavengers like GSH and ascorbate, or lipid soluble tocopherols, flavanoids, carotenoids, and ubiquinol, coupled to the complex array of SOD, CAT, GR, GPx, and APx isoforms that localize in all regions of a cell, implies a massive evolutionary response to the ROS conundrum. However, this entire array is complemented by the activity and expression of enzymes that maintain a reduced cellular environment in the form of NADPH. The functional redundancy and cooperative interactions of ROS defence accurately illustrates the massive danger of oxidative stress and the importance of maintaining a reductive environment.

Oxidative Stress and Disease
Oxidative stress and free radical damage to tissues and cell systems has been implicated heavily in the progression of cancer, HIV, diabetes, cardiovascular diseases and major forms of neurodegeneration. In the case of Alzheimer’s disease, recent literature aims to implicate the amyloid β protein as both a transition metal scavenger in neurons for Cu, Fe, and for excess Zn, while displaying SOD activity as well. It appears that a threshold is crossed, one that is lowered by age, whereby insoluble amyloid β depositions begin concentrating labile metals very close to neuronal cells in an environment of high oxygen, low antioxidant status and polyunsaturated fatty acids. All the key players for toxicity are present. In the case of Amyolateral Sclerosis (ALS), the rather severe symptoms of the progressive nerve damage all seems to stem from a mutated SOD gene, whereby superoxide can no longer be effectively scavenged, and biological dysfunction ensues. In short, a copper binding site is lacking, and the enzyme, which constitutes 1% of the total protein of some organisms, is rendered inactive.

It is, however, in the context of aging, that oxidative stress makes its largest mark. Antioxidant defences are not without flaw and it is believed that their gradual weakening over time results in the natural aging process. Although many sources of pro-oxidants can be endogenous, various pollutants and UV radiation add to this situation. Aerobic organisms are under virtually constant bombardment of oxidative stress during their lifetime. The specific dangers of the superoxide anion are best exemplified in the recent studies of Mn-SOD knock-out mice. Though each subject had vastly differing pathological phenotypes, both displayed a severely curtailed life span of 10-21 days. The general concept that oxidative stress is involved in many diseases is well-documented, but the details of toxic interactions are remains unclear. As is the case with aging, the progressive decline in intracellular function over time results in the accumulation of damaged cellular components, the majority of which are found to be overly oxidized. In mammalian mitochondria, the rates of superoxide production from mice were found to be 5 fold higher than in ox, with life spans of 3.5 and 30 years respectively. More telling however, in comparisons of mitochondria from pigeons (life span 35 years) and rats (life span 4 years), superoxide production was significantly lower in pigeons. In fact, the difference was found to be solely at the level Complex I electron leakage. However, the most compelling finding involved the comparison of bats with their longer-lived counterparts known as naked mole rats. In such phenotypically similar species, initial research from a recent study showed significantly lowered superoxide production with longer life, yet again. Caloric restriction in the diets of rats and mice has been shown to lower superoxide production and increase longevity as well, but these processes will only limit what is produced naturally. They do nothing to scavenge what might already be there. One need only examine fragile balance of energy formation and oxidative stress in the mitochondria to realize the massive scope of ROS toxicity.

Our own laboratory has recently demonstrated the susceptibility of other iron containing enzymes (such as aconitase) to ROS. Though ROS production is an integral component of oxidative cellular metabolism any abnormal metal homeostasis may be an important contributor to toxicity. Interference in the uptake, utilization and storage of a metal such as iron will induce single-electron reductions of oxygen species to further produce ROS. It is also hypothesized that the toxic products of lipid peroxidation can possibly act as signals for mitochondrial uncoupler proteins (UCP). Should electron leakage from the complexes reach a toxic threshold, UCPs would conceivably dissipate membrane potential and slow down ETC until ROS is detoxified. Indeed, the inactivation of the entire ETC group of proteins during extreme oxidative stress, slowing potential electron leakage, illustrates the ever-present choice of an organism between adaptation and survival or ATP. Hence, cellular metabolism leading oxidative phosphorylation is an important generator of ROS.

Metabolism and Life
Metabolism is the foundation of the physical and chemical changes that comprise the stress response, and it ultimately facilitates the survival and proliferation of any living organism. Despite the staggering number of reactions occurring in anabolic and catabolic processes, the specific types of reactions are rather few. Thus, this small set of reaction types unifies all living systems as the common means of energy harvesting from exogenous materials and facilitating the stress-response. Thus organisms such as E.coli and human beings share common molecular patterns underlying the major life processes, while displaying vastly different phenotypes. Indeed, the building blocks for all macromolecular assembly remain nearly universal, as is the genetic flow from DNA to RNA and onto functional proteins. What thus accounts for phenotypic variation across this earth remains hidden in the genetic and environmental history of the individual organism and its species. It is in the interplay of individual-specific metabolic processes that are

both borne of and, in turn, regulate the integrity of the genome, the selection of the transcriptome, and the organization of the proteome into the proliferative, adaptive, or apoptotic status of the cell.

Prokaryote Metabolism: Humble Beginnings
On the surface, the prokaryotic world seems rather limited and hardly diversified. In truth, they share common cellular morphologies, exist in clearly defined motile or resting states, and can be rapidly categorized by simple staining procedures. Furthermore, the interior of the cells lack the characteristic compartmentalization and complexity of the eukaryotic scheme of higher organisms. The excessive use of membrane structures within the cell to segregate major biochemical processes has also been bypassed.

In eukaryotes, genetic information is stringently organized in the nucleus, arranged across multiple chromosomes, and thus allows for specific differentiation of cells into complex tissues and organs. By comparison, there appears more phenotypic variation within a single species of eukaryotes than across the entire population of prokaryotic organisms. This is not, however, the case for prokaryotes. It is in the metabolic diversity of prokaryotes, facilitated by the simplicity of their cellular biology, which has allowed them to reach near astronomical populations in seemingly any known environment on earth. The lines drawn between physiology and biochemistry in higher organisms are blurred at this microscopic level. All metabolic processes are in fact highly integrated networks of metabolites and proteins, operating in close communication. Prokaryotes are designed for survival, much like plants, with relative immobility in the environment. Added to this are ancestral lines that span billions of years and countless adaptive progressions per generation. Thus, the prokaryote is able to rapidly alter its metabolism and delineate specific metabolic networks for direct stress response and indirect activation or suppression of the genome. The success of the prokaryote is thus embedded in its fluid metabolic profile, it is capable of producing energy from alcohol and lactic acid fermentation or aerobic respiration, and a host of other processes. Of key note are the uses of NO3 or fumarate as terminal electron acceptors, and the biology of lithotrophy and photoheterotrophy. In fact, the fluidity of microbial metabolism creates significantly more choices for an organism when dealing with stress. It allows for better juggling of both the stress-response and energy production for basic cell functioning, increasing the chances of survival during and after severe environmental stress. Whereas eukaryotic cells are metabolically stereotyped for precise functioning in controlled environmental locations within the organism, prokaryotes are designed to fend for themselves. They have been here longer, they have settled for simplicity over complexity, fluid adaptability over specialization, and they have effectively conducted biological warfare against all eukaryotes since time immemorial.

Metabolism and Disease
Many of today’s diseases affecting higher organisms are metabolic in nature, resulting from arrest or alteration of a major metabolic network. In many prominent forms of neurodegeneration, signs of oxidative stress coupled to abnormal neuronal and glial metabolism are evident, but the precise mechanism of toxicity remains elusive. Such is the case for a plethora of today’s most common diseases, whereby pharmaceuticals can effectively mask symptoms of toxicity, sometimes masking merely the host’s biological response to the stress, while the mechanisms of the toxicity remain much of a mystery. In characterizing and delineating the adaptive molecular mechanisms of the prokaryotic metabolic profile under specific stress, one can begin to elucidate the means by which it survives. Given such knowledge, one will gain significant insight into how therapeutic approaches to similar stress should be handled in higher eukaryotic schemes, like human beings, where adaptive prowess is severely lacking. Much like plant systems, the immobility of microbes demands a dazzling array of flexibility in the response to environmental conditions. Thus, these systems comprise a genotype-by-environment response, capable of producing a specific geno-phenotype relationship that is heavily dependent upon the growth stage. Accordingly, gene function becomes defined in the context of the systems state and environment.

Metabolism is highly integrated. It is by no means segregated into discretely classical components, but rather best understood in a global perspective, as a fluid mosaic. Thus, in characterizing the state of metabolite flux, the control elements of integrated metabolic networks, and the response of such networks to environmental conditions, recent studies have revealed considerable insight into the nature of toxicity. In truth, toxic or genetic perturbation of any part of the integrated metabolic scheme will affect the entire scheme as a whole, with or without affecting phenotype. Rapid analysis of the metabolic profile will allow for specific and readily screened biomarkers for diseased states to be assessed.

Despite the daunting magnitude of metabolites, proteins and transcripts to be characterized, the result of such metabolomic endeavours will be two fold. On one hand, one will gain novel and profound understanding of the regulation, mechanisms and conditions of metabolism. On the other hand, more importantly, one will understand the true scope of the organism’s response to any chemically, biologically, or physically induced abnormal environment. Two more steps towards the concept of individual specific health assessments and therapies, tailor-made for the metabolic profile.

New Perspectives in Metabolism
Metabolism, as a whole, is most efficiently taught in segregated concepts by means of electron source or function, such as carbohydrates and proteins or efflux and growth. It is now evident however, that only in the integration of all metabolic circuits can we begin to elucidate the biochemical processes. Through substrate shuttling and modulation of key reversible and irreversible enzymes in various circuits of the integrated scheme, life has found the means to survive stress, particularly of the oxidative nature. The stress response thus becomes the concerted efforts of newly delineated metabolic circuits that arise from the integrated metabolic scheme. It allows for instant alteration of energy harvesting and consuming processes, and facilitates the removal of toxins, the recruitment of scavenging compounds, repair of damage via stress-response protein activation, or initiation of apoptotic events. It is a highly regulated multitude of reactions as well, interconnected by myriad potentially favourable avenues, depending on the specific needs of the cell. Control is achieved firstly through maintenance of enzyme levels at the transcriptional and translational level. Secondly, the maintenance of catalytic activities of the protein machinery is achieved via post-translational modification or allosteric control. Thirdly, however, is strict regulation of metabolic flux. As has been found to be the case with insulin and glucose transport, the propagation of substrates throughout a biological system often serves as a control point for additional metabolic reactions.

In the rapidly expanding field of metabolomic research, such understanding is truly being achieved. Using high throughput analysis such as High Performance Gas and Liquid chromatography (HPLC), and Mass spectrometry (MS), a quantitative and qualitative profile of the metabolites and their propagation reveals an accurate, though complex fingerprint of cellular function. Metabolomics, the study of all the metabolites present in a cell at a specific moment provides a snapshot of the molecular machinery operative under a given condition. It also allows for a better understanding of the macromolecules mediating the production of the metabolites. The mapping of the genome has opened many avenues to explore in terms of what a cell does to live, adapt and die. It has not, however, elucidated the leap to the functional proteome and organization of the metabolome itself.
‘Omic’ research aims at the non-targeted identification of all gene products (transcripts, proteins, metabolites) present in a biological sample. By their nature, these profiles reveal both previously unknown and quantitative dynamics to biological systems. Metabolomics, having the potential to rapidly screen and characterize the small molecular weight organic compounds, is crucial to understanding the dynamics of systems biology. The integration of methods based on GS/MS and LC/MS for the comprehensive identification and quantification of metabolites has attained a robustness that is comparable or even better than conventional mRNA or protein profiling technologies. Analysis of metabolic networks and the regulation of metabolite flux with respect to specified environmental or genetic perturbations will permit the investigation of dynamic interactions in metabolic networks and the discovery of novel correlations of biochemically characterized pathways hitherto unknown.

Aerobic Metabolism
Aerobic metabolism involves the strategic oxidation of macromolecules into monomers for both energy reward and supply of building blocks for subsequent anabolism. Such metabolism is comprised of the specific substrates and proteins required to efficiently pass electrons to oxygen to form ATP of for direct formation of high phosphate compounds. In such a system, the ratio of ATP to ADP and the redox status of the cell become biomarkers for optimal or dysfunctional metabolism. Depending on the needs of the cell and the supply of oxygen, substrates, ATP and cofactors, the entire integrated scheme facilitates the funnelling of substrates, the activation or suppression of specific metabolic reactions and the alteration of gene expression.

Carbohydrate Metabolism
In the context of oxidative stress, aerobic metabolism, and the response to environmental stress, carbohydrate metabolism is of profound importance. Carbohydrates themselves comprise most of the organic matter on earth and within the cell, and serve as energy stores, fuels, and metabolic intermediates. Secondly, ribose and deoxyribose sugars form the backbone of nucleic acid structure, inducing conformational flexibility of both DNA and RNA. Thus, they facilitate both storage and expression of genetic information. Beyond this, carbohydrates play pivotal roles in the structural elements of cell walls themselves, are linked to many proteins and lipids to infer polar anionic potential, and for cell-cell recognition via surface interactions.

The main role of carbohydrates, however, must always remain as substrates for the main metabolic networks known to drive energy production and metabolism in all organisms: glycolysis, gluconeogensis, the TCA cycle, the Pentose-phosphate Pathway (PPP), and the glyoxylate cycle. These pathways are of supreme importance in mediating the energy status of the cell, optimal cellular functions during growth and differentiation, and the stress response. Specifically, the reactions of the TCA cycle have been revealed to play a central role in all of cellular metabolism. Energy levels are maintained via substrate level phosphorylation, fatty acid oxidation and oxidative phosphorylation, while myriad anaplerotic reactions replenish stores of required macromolecules and intermediates. By means of such an integrated network, the growth of prokaryotes in nearly any growth medium can be facilitate

The citric acid cycle, is the final common pathway for the oxidation of fuel molecules, be it amino acid, fatty acid or carbohydrate. Thus, the aim of an optimal metabolism is to convert most fuel molecules into acetyl CoA for energy harvesting, or to funnel excess energy into the production of metabolic intermediates for biosyntheses. For example, the majority of carbon atoms in porphyrins are derived from succinyl CoA, while many amino acids are derived from α-ketoglutarate and oxaloacetate. Thus, the regulation of the cycle becomes supremely important if TCA cycle intermediates must be replenished. In the case of oxaloacetate depletion, since mammals lack direct conversion of acetyl CoA to TCA cycle precursors, the enzyme pyruvate carboxylase (PC) is employed as an anaplerotic means of maintaining optimal metabolism.

Control of the TCA cycle is achieved primarily through regulation of three main enzymes: CS, ICDH, and αKGDH. In the case of CS, ATP is a known allosteric inhibitor, marking the cell as having ample energy reserves increasing the Km for Acetyl CoA and hindering citrate formation. This will block further energy yields from the TCA. ICDH is found to be allosterically stimulated by ADP, and the binding of ADP, NAD+, and Mg+2 is mutually cooperative. Logically then, ATP and NADH display inhibitory allosteric effects on the enzyme. The third control site is found to follow similar patterns of regulation. The αKGDH is inhibited by succinyl CoA and NADH, its own products, as well as by high ATP levels. PDH may very well serve as a fourth control site in the TCA cycle, showing significant inhibition via phosphorylation. Such a blockade would halt further energy production when ATP concentrations are more than sufficient. In brief, one finds that funnelling of two carbon compounds into the TCA and the rate of the cycle and its anaplerotic counterparts are all tightly linked to the energy status and health of the organism.

The in depth analysis of the metabolites and enzymes of the TCA cycle and peripheral carbohydrate metabolism in an organism under severe stress gives an accurate assessment of how a stress has affected metabolism, and how metabolism has in turn responded. It reveals much of the complex mechanisms of toxicity while shedding light on the causes of the characteristic phenotypic responses to certain diseased states. At the root of all stress, endogenous or exogenous, is a pressure exerted upon one of any of the following three: The energy status of the cell, the specific enzymes of the metabolic profile, or the integrity of direly needed substrates and intermediates. The response to such a stress will then encompass the ability of peripheral metabolic networks of the integrated scheme to counteract such deleterious effects. The benefits inferred by such integration are exemplified in the knowledge that a stress on one region of metabolism affects all regions in some way, eliciting the appropriate response.

Oxidative Stress, Metabolism, and the Reductive Environment
The relevant literature on oxidative stress and ROS production ideally aims to assess the primary and secondary antioxidant defences against a known oxidant or pro-oxidant, and quantifies toxic end products when possible. It is becoming increasingly evident that beyond the normal antioxidant defence, a larger metabolic concern of an organism during extreme stress is maintenance of reducing power to fuel the entire detoxification process. Studies have shown decreased activities of G6PDH and NADP+-ICDH and ME during periods of hyperglycaemia, characterized by elevated oxidative damage. It has been speculated that these enzymes, as the sole source of NADP+-NADPH conversion, play crucial roles beyond simple carbohydrate metabolism. During growth, the high respiratory activity and ROS formation during early growth and maturation would require an adequate supply of NADPH to limit oxidative damage. In fact, NADPH becomes the sole reductive buffer in the dangerous balance of high ROS production and high ATP demand in youth. Interestingly, Darwinian-directed evolution provides the odds of survival against oxidative stress in favour of younger organisms, still in their prime for faithful passing of genes. Thus antioxidant defence is at its greatest. As an organism reaches an age whereby it no can longer be depended upon to pass on such favourable genes, antioxidant defence is found to degrade.

In parasites exposed to oxidative stress, increased mRNA transcripts and protein levels of NADP+ dependent ICDH were shown to be up regulated. Surprisingly, there is little research aimed at understanding the metabolic response to oxidative stress through directed reductive adaptation. The redox status of the cell allows for all major cellular biochemistry to occur. The oxidative component relates to the harvesting of energy from oxidizable substrates by the machinery of catabolism. The reductive environment, by comparison, encompasses anabolism in times of energy abundance or cellular biosynthetic needs, and fuelling the antioxidant defence system. The redox status of the cell is maintained by the thiol redox status, described by the ratios of GSH, GSSG, and protein sulfhydryl group integrity, but more importantly by the pyridine nucleotides, NADPH/NADP+ and NADH/NAD+. In HeLa cells recent studies show that when subjected to both peroxide and thiol oxidants, though the GSH/GSSG ratio can be a marker for oxidative stress, it is the NADPH supply that determines whether the turnover of the tri-peptide can even occur. The PPP is by far the main metabolic avenue for NADPH production, and the thus redox balance for the cell. The pathway has been observed to modify easily to meet the demands of the cell. NADPH can be supplied, but as well, ribose can be produced for nucleotide synthesis. As well, both ATP and NADPH can be produced. NADP+-dependent ICDH and ME, and G6PDH in particular have been shown in many species to play a key role in the generation of cytosolic NADPH, and their activities and expression are found to be higher in more oxidative muscle fibres. As well, the same trend follows aging patterns, and the enzymes are found in abundance proximal to the GPx system in skeletal muscle. In fact, studies involving UV induced oxidative damage show E.coli mutant lacking both NADP+-ICDH and ME to be highly susceptible to oxidative damage.

Novel mechanisms for NADPH production within a biological system are thus rapidly being revealed as potent sources of antioxidant fuel and the criteria by which an organism survives the dangers of aerobic respiration. More direct approaches are possible as well, in the form of NAD+ and NADH kinases, capable of producing NADP+ and NADPH respectively. Would such an avenue be favourable at the expense of the cofactors for ETC energy metabolism? In the case of severe oxidative stress, whereby ETC has been shown to be highly susceptible to inactivation, the uses of such cofactors become limited, and the dangers become large. A cell must again make the choice between energy reward and adaptation.

Studies involving E.coli show traces of such metabolic networks. Upon knockouts of phosphoglucose isomerase, the glyoxylate shunt was found to be activated, as is the case with plant seed-stem maturation. Interestingly, in glucose limited mutants, only a minor fraction of isocitrate was converted to α ketoglutarate, while the majority was funnelled through glyoxylate.

Glycolysis is involved in the catabolism of glucose, and thus is found to be operative in all differentiated cell types in higher organisms. In the case of retina, it is the only ATP –producing pathway, in that it is non-oxidative. The fate of pyruvate itself, as the precursor to acetyl CoA, is tightly linked to cellular oxygen tension. Under anaerobic conditions, skeletal muscle has been shown to convert pyruvate to lactate and NAD+ is regenerated to fuel further glycolytic activity. Organisms grown on acetate or lipids however, utilise the glyoxylate bypass of the TCA cycle and employ ICL, and MS. This pathway is not operative in all living organisms, and is notoriously unnecessary in glucose fed bacteria. It provides the cell with a bypass for isocitrate oxidation, preventing NADH production from downstream enzymes of the TCA, and confers the ability to utilize two carbon metabolites. Thus it is involved in the synthesis of anaplerotic precursors, the curtailing of excess NADH production during periods of ETC sluggishness, and the means by which organisms can continue optimal biology on varying carbon media. The activation of the glyoxylate shunt can be explained on the basis of intracellular redox metabolism. The flux of metabolites is of supreme importance in this newly delineated metabolism. In wild type E.coli, the ICDH reaction was the major producer of NADPH, accounting for more than 60%. In the mutant E. coli lacking ICDH, the PPP generated a large amount of NADPH to compensate. Overproduction of NADPH, however, remains deleterious as there is a limited cellular capacity for the re-oxidation of NADPH. Regulation of all metabolic changes occurring under stress must be kept stringent, indeed. Flexibility in adaptive prowess is truly the key to survival.

The PPP diverts metabolites from glycolysis and serves, as mentioned above, two main purposes. Oxidatively, it generates NADPH, while non-oxidatively, it generates biosynthetic precursors for nucleotide formation. It can also be entered from other glycolytic intermediates, like fructose-6 phosphate. Edwards and Palsoon were the first to successfully block the first reaction of the PPP in E. coli, the conversion of glucose 6 phosphate to D-6 phosphate glucone lactone. This is the entire step for the oxidative branch. Deletion of this reaction completely blocks the oxidative branch, but seemed to affect metabolic output only minimally, a reduction of only 1% under aerobic glucose fed conditions. Prior to the deletion, 2/3 of the NADPH needed was supplied by the PPP and most of the NADH required for ATP synthesis was supplied by the TCA cycle and its high energy phosphate bond formation. Upon deletion, the major systemic reorganizations of metabolic flow were found to be aimed at simply maintaining NADPH stores. To compensate for the blocked PPP, most NADPH production was shifted through an increased flux of a TCA/Glyolytic/Gluconeogenic type integrated cycle, capable of consuming any excess NADH produced from TCA, and producing NADPH from malic enzyme. This was the first microbial example of such a transhydrogenase network, once observed in some plant life. Interestingly, the non-oxidative branch of the PPP can still be entered by other metabolites of glycolysis, thus still serving the biosynthetic needs of the cell. Another key change involved the increased flux through glycolytic reactions and the PK enzyme to absorb the reduced flux through PPP.

It is clear now that despite the elaborate and redundant array of antioxidant defence inherent in all aerobes, the driving force of the biological response to oxidative stress is maintenance of reducing power. In that this is the case, an understanding of how the integrated metabolic scheme can compensate for antioxidant deficits will bear much fruit.

Limiting the Oxidizing Power
The metabolism of an aerobe is primed for oxidative phosphorylation as it is the primary means of energy harvesting. This being the case, under the constant bombardment of oxidative stress, UV radiation, toxic pollutants, and the abundance of heavy metals in the environment, oxidative phosphorylation ceases to become the sole avenue for energy metabolism. The cell is given a choice between ATP formation and adaptation to the stress. It is noteworthy that in almost all analyses to date, NADH producing enzymes of TCA cycle and the NADH consuming components of the ETC are significantly reduced in activity. Regardless of whether ETC has been inactivated by ROS deliberately or not, the cell has chosen to reroute metabolism towards the reductive environment. Oxidative metabolism is perhaps best represented by a single cofactor: NADH. This metabolite carries electrons from oxidized substrates of metabolism to Complex I, and thus during the oxidative stress response, NADH levels must be kept at a minimum. In many cases, alternate oxidases are found to be higher in activity under oxidative stress, allowing for NADH to be consumed with the concomitant formation of H2O.37 It is feasible that NAD+-dependent enzymes have also been strategically inactivated to prevent further NADH production. Given the predicament of energy needs and NADH limitation, one would hope to find secondary energy pathways in operation, comprised of glycolytic activity and substrate level phosphorylation. As well, one might find a bypassed TCA cycle with glyoxylate enzymes working diligently to restore reductive balance. In the context whereby NADH has become a potential pro-oxidant, mechanisms for limiting its production, funnelling it towards H2O production, or conversion to NADPH become the main goals of metabolism. Many recent studies from aerobes of various phenotypes show such common trends during oxidative stress. NAD+-dependent enzymes are reduced in activity and expression, NADPH producing enzymes increase in activity and expression, ETC components show marked reduction while alternate oxidases are highly expressed. Furthermore, an operative glyoxylate shunt is often found, allowing for bypassing of TCA’s oxidative components while facilitating its reductive component.

α-Ketoglutarate as an ROS scavenger
In nearly all forms of neurodegeneration, bearing hallmarks of oxidative stress, α-KGDH has been found to have significantly reduced activity. Furthermore, clinical trials involving the administration of α-keto acids, specifically α ketoglutarate and pyruvate, show unheralded efficiency in counteracting the progression of toxicity. Studies both in vivo and in vitro have illustrated the capacity for these endogenous compounds to non-enzymatically decarboxylate in the presence of O2.- and H2O2. The products of such reactions are carbon dioxide and a readily oxidizable and useable substrate, such as succinate or acetate. The inactivation of α KGDHC during oxidative stress is suspect, in that given its sulfhydryl component on the lipoic arm of its E1 subunit, the enzyme could act as an efficient ROS sensor. The hypothesis follows that in sensing ROS and losing functionality in its lipoic side chain, the enzyme becomes a blockade along the oxidative branch of the TCA cycle, while a pool of a potential ROS scavengers forms ahead of it. Though much research is required to fully elucidate the connection of the enzyme, its substrate, and the mechanisms of ROS detoxification, the connection itself may someday yield a cure to unchecked cellular oxidative damage.

The TCA cycle enzymes are more sensitive to oxidative stress in that they are localized in the mitochondria and in most bacterial membranes where ROS are notoriously formed. α-KGDH is truly a housekeeping enzyme of the TCA. It is essential in maintaining both NADH levels and thus ATP production from oxidative phosphorylation. Of equal importance, it is involved in glutamate and thus glutamine syntheses, the major nitrogen storage centers and source of major neurotransmission in higher organisms. Many pro-oxidants and toxic end products of lipid peroxidation have been shown both to inhibit the enzyme and mimic the overall toxicity of both PD and Alzheimer’s disease. In further studies, the association of oxidative stress, reduced enzyme activity and decreased ferritin levels illustrates a rather novel interesting antioxidant system. Thus, ketoacids can play an important role in the abating of oxidative stress.

THESIS OBJECTIVES
A living system can evoke three possible responses when confronted with an extreme environment. It may succumb, lay dormant, or adapt. Our laboratory has been delineating the molecular mechanisms associated with the latter scenario. We have recently demonstrated the role of oxalate in the adaptation to Al stress and the involvement of α-ketoglutarate in the tolerance to gallium toxicity. In these two cases, the detoxification strategies entailed the participation of various metabolic pathways aimed at maintaining the levels of these two metabolites. Hence, it became evident that to adjust to an abnormal situation, an organism has to invoke a holistic strategy. In this study the main goal is to evaluate the metabolic shift that enables P.fluorescens to survive in an environment rich in oxidative stress. A body of literature exists on various enzymes such as catalase and SOD that are critical for survival under an oxidative situation. However, the homeostasis of the main ingredient, namely NADPH, that helps maintain a reductive environment, is not well established. The metabolic networks that mediate the production of this reducing factor have been studied. Furthermore, the role of NADH and α-ketoglutarate in diminishing oxidative tension has also been explored.

MATERIALS AND METHODS
List of Reagents and Equipment
2-Thiobarbituric acid; Sigma Chemical Company (St.Louis, Missouri)
2,4-Dinitophenol; ICN Biochemicals (Cleveland, Ohio)
2,6-Dichloroindophenol; Sigma Chemical Company (St.Louis, Missouri)
5,5’-Dithio-bis-(2-nitrobenzoic acid); Sigma Chemical Company (St.Louis, Missouri)
Accumet pH meter 910; Fisher Scientific (Unionville, Ontario)
Acrylamide; Bio-Rad Laboratories (Mississauga, Ontario)
Acetyl coenzyme A; Sigma Chemical Company (St.Louis, Missouri)
Adenosine 5’ triphosphate (ATP); Sigma Chemical Company (St.Louis, Missouri)
Adenosine 5’ diphosphate (ADP); Sigma Chemical Company (St.Louis, Missouri)
α-Ketoglutaric acid; ICN Biochemicals (Cleveland, Ohio)
Alpha-innotech Gel Documentation system and software; Fisher Scientific (Unionville, Ontario)
Ammonium chloride (NH4Cl); Sigma Chemical Company (St.Louis, Missouri)
Ammonium molybdate; Fisher Scientific (Unionville, Ontario)
Ammonium persulphate (APS); Bio-Rad Laboratories (Mississauga, Ontario)
Ammonium sulphate (NH4)2SO4; Sigma Chemical Company (St.Louis, Missouri)
Avidin-peroxidase conjugate for western antibody detection; Fisher Scientific (Unionville, Ontario)
Bacto-Agar; Difco Laboratories (Detroit, Michigan)
Bio-Rad Protein Assay; Bio-Rad Laboratories (Mississauga, Ontario)
Bio-Rad Mini-Protein II Dual Slab Cell; Bio-Rad Laboratories (Mississauga, Ontario)
Bio-Rad Silver Stain kit; Bio-Rad Laboratories (Mississauga, Ontario)
Bis (N,N’-bis-methylacrylamide); Bio-Rad Laboratories (Mississauga, Ontario)
Bis(2-hydroxyethyl)imino-tris(hydroxymethyl)methane; Sigma Chemical Company (St.Louis, Missouri)
Bovine serum albumin (BSA); Sigma Chemical Company (St.Louis, Missouri)
Calcium chloride; BDH Laboratory Chemicals Division (Toronto, Ontario)
Centrifuge Model J2-MI; Beckman Instruments (Mississauga, Ontario)
Chemiglow Reagent; Fisher Scientific/Alpha Innotech (Unionville, Ontario)
Chloramphenicol; Sigma Chemical Company (St.Louis, Missouri)
Citric-2,4-13C2 acid; Isotech Inc. (Miamisberg, Ohio)
Citric acid monohydrate; Sigma Chemical Company (St.Louis, Missouri)
Coenzyme A (sodium salt); Sigma Chemical Company (St.Louis, Missouri)
Coomassie G 250; Sigma Chemical Company (St.Louis, Missouri)
Coomassie R 250; Sigma Chemical Company (St.Louis, Missouri)
Deuterium, 99.9 atom %D; Sigma Chemical Company (St.Louis, Missouri)
D-glucose; Sigma Chemical Company (St.Louis, Missouri)
D,L-isocitric acid trisodium salt; ICN Biochemicals (Cleveland, Ohio)
Ethylenediaminetetraacetic acid disodium salt (EDTA); BDH Laboratory Chemicals Division (Toronto, Ontario)
€-amino-n-caproic acid; Sigma Chemical Company (St.Louis, Missouri)
Ferric chloride (FeCl3.6H2O); Fisher Scientific (Unionville, Ontario)
Fumaric acid; Fisher Scientific (Unionville, Ontario)
Glacial acetic acid; CanLab (Toronto, Ontario)
Glucose-6-phosphate (disodium salt); Sigma Chemical Company (St.Louis, Missouri)
Glucose-6-phosphate dehydrogenase EC. 1.1.1.49 (from porcine heart); Sigma Chemical Company (St. Louis Missouri)
Glutamic acid (monosodium salt); Sigma Chemical Company (St.Louis, Missouri)
Glutathione recudtase EC.1.6.4.2; Fisher Scientific (Unionville, Ontario)
Glycerol; Sigma Chemical Company (St.Louis, Missouri)
Glycine; Sigma Chemical Company (St.Louis, Missouri)
Glyoxylic acid (monohydrate); Sigma Chemical Company (St.Louis, Missouri)
Gyratory waterbath shaker model G-76; New Brunswick Scientific (Edison, New Jersey)
Hybond™-P: PVDF membrane; Amersham Pharmacia Biotech (Piscataway, NJ, USA)
Hydrochloric acid (HCl); CanLab (Toronto, Ontario)
Hydrogen peroxide (30%(w/w) solution); Sigma Chemical Company (St.Louis, Missouri)
Iodonitrotetrazolium chloride; Sigma Chemical Company (St.Louis, Missouri)
Isocitrate dehydrogenase EC 1.1.1.42 (from porcine heart); Sigma Chemical Company (St.Louis, Missouri)
Isocitrate lyase EC
Magnesium chloride hexahydrate (MgCl2.6H2O); BDH Laboratory Chemicals Division (Toronto, Ontario)
Magnesium chloride tetrahydrate; BDH Laboratory Chemicals Division (Toronto, Ontario)
Malachite green (oxalate salt); Sigma Chemical Company (St.Louis, Missouri)
Malic acid; BDH Laboratory Chemicals Division (Toronto, Ontario)
Malic dehydrogenase EC. 1.1.1.40 (from porcine heart); Sigma Chemical Company (St. Louis, Missouri)
Malonic acid (disodium salt); Sigma Chemical Company (St.Louis, Missouri)
Menadione (sodium bisulfite); Sigma Chemical Company (St.Louis, Missouri)
2-mercaptoethanol; Sigma Chemical Company (St.Louis, Missouri)
n-Dodecyl β-D-maltoside; Sigma Chemical Company (St.Louis, Missouri)
Nicotinamide adenine dinucleotide (oxidized form); Sigma Chemical Company (St.Louis, Missouri)
Nicotinamide adenine dinucleotide (reduced form); Sigma Chemical Company (St.Louis, Missouri)
Nicotinamide adenine dinucleotide phosphate (oxidized form); Sigma Chemical Company (St.Louis, Missouri)
Nicotinamide adenine dinucleotide phosphate (reduced form); Sigma Chemical Company (St.Louis, Missouri)
N,N,N’,N’-Tetramethylenediamine (TEMED); Bio-Rad Laboratories (Mississauga, Ontario)
Nitroblue Tetrazolium salt; Sigma Chemical Company (St.Louis, Missouri)
Oxaloacetic acid; Sigma Chemical Company (St.Louis, Missouri)
Oxalic acid dehydrate; ICN Biochemicals (Cleveland, Ohio)
P-anisidine; Sigma Chemical Company (St.Louis, Missouri)
Peroxidase EC 1.11.1.7; Sigma Chemical Company (St.Louis, Missouri)
Phenazine methosulphate; Sigma Chemical Company (St.Louis, Missouri)
Phenylmethylsulphonylfluoride (PMSF); Sigma Chemical Company (St.Louis, Missouri)
Phosphoenolpyruvate (PEP); Sigma Chemical Company (St.Louis, Missouri)
Ponceau S; Sigma Chemical Company (St.Louis, Missouri)
Potassium phosphate monobasic (KH2PO4); Sigma Chemical Company (St.Louis, Missouri)
Pseudomonas fluorescens ATCC 13525; American Type Culture Collection (Rockville, Maryland)
Pyruvic acid (sodium salt crystalline); Sigma Chemical Company (St.Louis, Missouri)
Rifampicin; Sigma Chemical Company (St.Louis, Missouri)
Sodium carbonate anhydrous; Mallinckrodt Inc. (Kentucky)
Sodium phosphate dibasic (Na2HPO4); Sigma Chemical Company (St.Louis, Missouri)
Sodium hydroxide (NaOH); Fisher Scientific (Unionville, Ontario)
Sodium dodecyl sulphate (SDS); Sigma Chemical Company (St.Louis, Missouri)
Sodium molybdate dihydrate; BDH laboratory Chemicals Division (Toronto, Ontario)
Spectrophotometer model DU-65; Beckman Coulter, Inc. (Fullerton, Ontario)
Spectrophotometer model Ultrospec 3000; Amersham Pharmacia Biotech (Baie d’Urfe, Quebec)
Succinic acid; BDH Laboratory Chemicals Division (Toronto, Ontario)
Sucrose; Sigma Chemical Company (St.Louis, Missouri)
Sulphuric acid (H2SO4); CanLab (Toronto, Ontario)
Tricarbyllylic acid; Sigma Chemical Company (St.Louis, Missouri)
Tris(hydroxymethyl)aminomethane (Tris) HCl and Tris base (Trizma Base); Sigma Chemical Company (St.Louis, Missouri)
Tween-20; Bio-Rad Laboratories (Mississauga, Ontario)
Ultracentrifuge Model L8-M; Beckman Coulter, Inc. (Fullerton, Ontario)

Organism and growth conditions
The bacterial strain Pseudomonas fluorescens 13525 was obtained from the American Type Culture Collection (ATCC). The microbe was kept on a mineral medium containing citric acid and solidified by 2% agar. The sterile agar test tubes were maintained at 4oC. The bacteria were sub-cultured bi-weekly. DDH2O was utilized in all experiments.

Agar Media
In 250mL of DDH2O was added Na2HPO4 (2.4g); KH2PO4 (1.2g); NH4Cl (0.4g); MgSO4.7H2O (0.08g); citric acid monohydrate (1.6g) and 400µL of trace elements. The trace element solution consisted of FeC3.6H2O (2µM); MgCl2.4H2O (1µM); Zn(NO3)2.6H2O (0.05µM); CaCl2 (1µM); CoSO4.7H2O (0.25µM); CuCl2.2H2O (0.1µM); NaMoO4.2H2O (0.1µM). The pH of the trace element solution was adjusted to 2.75 with dilute HCl to prevent precipitation of the metals and the solution was stored at 4oC. The pH of the agar media was raised to 6.8 with dilute NaOH and the final volume was brought to 400mL with DDH2O. The solution was gently heated and Bactoagar® (6.6g) was added and stirred until completely dissolved. Approximately 7 to 10mL were placed in test tubes and capped for slant. Following autoclave sterilization for 20 min at 17lbs/in2, 121oC, the test tubes were laid on an angle and allowed to solidify at room temperature. Slants were stored in the refrigerator at 4oC.

Pre-culture Media
The media used for the pre-culture growth solution contained: Na2HPO4 (6.0g); KH2PO4 (3.0g); NH4Cl (0.8g); MgSO4.7H2O (0.2g); citric acid monohydrate (4.0g); trace element solution (1.0mL), per litre of DDH2O. The pH was raised to 6.8 with dilute NaOH and the media was separated into 100mL aliquots in 250mL Erlenmeyer flasks. The flasks were capped with foam plugs and autoclaved for 20 min at 17lbs/in2, 121oC. The pre-culture media was inoculated with a loop of Pseudomonas fluorescens stored on agar slants. Late-logarithmic phase growth was attained following 24-48 hrs of incubation.

Culture and Cell Growth
The media used for liquid culture contained: Na2HPO4 (6.0g); KH2PO4 (3.0g); NH4Cl (0.8g); MgSO4.7H2O (0.2g); citric acid monohydrate (4.0g); trace element solution (1mL) in 600mL of double distilled water. The pH of the medium was raised to 6.8 with dilute NaOH and the volume was adjusted to 1L with DDH2O. The media was separated in 200mL amounts in 500mL Erlenmeyer flasks and autoclaved for 20 min at 17lbs/in2, 121oC. For the oxidative stress experiments 100µM of menadione bisulfite from a 100mM stock was added to autoclaved control media. The media was then inoculated with 1mL of the pre-cultured bacteria. The cultures were incubated at 26oC in a gyratory water bath shaker model G76 (New Brunswick Scientific) at 140 rpm. The media without added menadione constituted the control media. The media was separated into 200mL amounts in 500mL Erlenmeyer flasks, covered with foam plugs and autoclaved for 20min at 121oC.

Harvesting of P.fluorescens
Pseudomonas fluorescens were collected from the growth medium by centrifugation at 10,000 x g for 15 min at 4oC. The supernatant was removed and 0.85% NaCl was used to wash and suspend the bacterial pellet. The bacteria were then centrifuged again at 10,000 x g for 15 min and the procedure was repeated. A Bio-Rad Bradford Protein assay was then used to assess cellular growth.

Preparation of Cell Free Extract (CFE) from Whole Cells
Following bacterial harvesting by the methods outlined above, they cells were resuspended in a cell storage buffer containing 50mM Tris-HCl, 5mM MgCl2, 1mM PMSF, 1mM DTT at pH 7.3. The cells were disrupted by sonication using a Brunswick sonicator, power level 4 for 15 sec for 4 intervals. Cells were stored on ice between intervals for approximately 3 min.

The supernatant fraction of CFE was collected and centrifuged at 180,000 g for 60 min. at 4oC to yield the membrane and soluble components. The membrane fraction was resuspended in cell storage buffer and stored at 4oC for immediate use or frozen in maltoside suspension for later use. The soluble fraction was removed and centrifuged again at 180,000 g for 2 hrs to insure a membrane free system. The final pellet was discarded as membranous cellular debris, and the soluble fraction was either stored at 4oC for immediate use or frozen for later use. Samples of both fractions were kept at 4oC for a maximum of three days.

Isolation of spheroplasts of P.fluorescens
The inner membrane of P.fluorescens was isolated by a modified version of the methods described by Mizuno and Kageyama. The cells were harvested by the methods mentioned above and then washed with 20% (w/v) sucrose and ice-cold reagents were slowly added to the suspension in an ice bath in the following order; 9mL of 2M sucrose, 10mL of 0.1M Tris-HCl (pH 7.8 at 25oC), 0.8mL of 1% Na-EDTA (pH 7.0), and 1.8mL of 0.5% lysozyme. The mixture was then warmed to 30oC within a period of 5 min. and kept in the gyratory water bath at this temperature for 1 hr. The suspension was then centrifuged to remove the spheroplasts at 10,000 g for 30min at 30oC. The spheroplasts were incubated with the lysis buffer containing 40mL of 50mM Tris, 5 mM MgCl2, 1mM PMSF, and 1mM DTT. The spheroplast membranes were then recovered by centrifugation at 100,000 g for 30 min. and washed in the same buffer.

13C NMR analyses of citrate metabolism in CFE
13C NMR analyses were performed on the CFE using a Varian Gemini 2000 spectrometer operating at 50.38 MHz for 13C. Experiments were conducted with a 5mm dual probe (35o pulse, 1-s relaxation delay, 8 kilobytes of data). Chemical shifts were referenced to shifts of standard compounds observed under the same conditions. Membrane and soluble fraction equivalents of 2mg/mL of protein were obtained from control CFE grown 22 hrs or menadione-stressed CFE grown 30-32 hrs, placed in a 10mM phosphate buffer, 10% D2O. The reactions were initiated in 1.5mL conical tubes by the addition of 5mM labelled citrate [2,4-13C2], and if required for enzyme activity, 0.5mM of the respective cofactor was also included. Following 60 min. incubation at 26oC, the reactions were subjected to 13C NMR proton decoupled analyses.

1H NMR analyses of citrate metabolism in CFE of P.fluorescens
1H NMR analyses were obtained using the Varian Gemini 2000 spectrometer operating at 200MHz for 1 hr. Enzymatic reactions were assayed in 1H NMR buffer (10mM phosphate, 5mM MgCl2 and pH 7.4) with 500ug membrane or soluble fractions, 2-5mM substrate, and if required, 0.5-1.0mM of the respective cofactor. The experiments were performed in 1.5mL conical tubes and the reactions were stopped by placing the tubes in a boiling water bath for 3 min. The formation of any precipitate was removed by centrifugation at 20,000 g for 15 min. The supernatant was lyophilized and dissolved in 500uL deuterium oxide (D2O), 99.9 atom %D. H2O resonance was effectively suppressed with the aid of the homodecoupler set to the signal corresponding to H2O. Predominantly, the following NMR parameters were found to be effective: Decoupler modulation mode (dmm=ccc), where c=continuous; decoupler modulation (dm=nyn), where n=no and y=yes; decoupler low power (dlp=2000); the first delay (d1=0); the second delay (d2=5); the first pulse (p1=2); and the acquisition time (at=1). The number of transients varied among samples. It was found that all parameters could be varied to achieve maximum water signal suppression. Experiments were executed with a 5mm dual probe placed at a 90o pulse angle, and 8 kilobytes of data.

Monitoring end-products of oxidative stress
Measurement of oxidized lipids
Thiobarbituric acid (TBA) is known to react with aldehyde equivalents resulting from the oxidation of lipids. Thus, the amount of thriobarbituric acid reactive species (TBARS) was measured in the membrane and inner membrane fractions of control and menadione-stressed cells at various growth intervals. To milligrams of membrane protein equivalent was heated with 15% trichloroacetic acid (TCA), 0.375% TBA/0.25N HCl in a final volume of 1.0mL for 15 min. Following the development of a pinkish color, samples were centrifuged for 10min at 10,000 x g. The supernatant was isolated and the absorbance was measured at 532nm. Blanks did not contain any membrane component. The extinction coefficient was €= 1.56 E 105 M-1cm-1.

Measurement of oxidized proteins
The protein carbonyl content was assessed in soluble fractions of control and menadione-stressed CFE as an indirect assessment of oxidized proteins.44 One milligram of soluble protein equivalent was allowed to react with 2%DNPH in a final volume of 1.0 mL for 60min. 200ul of 50%TCA was added to each sample to precipitate the proteins. The proteins were then spun at 14,000 rpm for 10min. The supernatant was discarded and washed with a solution of 10%TCA and re-centrifuged three times. The pelleted proteins were then washed in a solution of ethylacetate:ethanol in a 1:1 ratio and centrifuged three times. The final precipitate was dissolved in 1.0mL of 6M guanidine and the absorbance was measured at 370nm. The extinction coefficient for hydrazones was 21.5nmol*L-1cm-1.44

Monitoring ROS production in CFE
H2O2 measurement in cellular fractions exposed to menadione
The amount of H2O2 was measured in membrane and soluble fractions in a buffer containing 25mM Tris-HCl/5mM MgCl2, pH 7.3, subjected to 5mM citrate. Briefly, 2mg CFE equivalents were added to 4 units of peroxidase and 10mM p-anisidine in a final volume of 1.0mL. The reaction was allowed for 30min. and the resulting absorbance was measured at 458nm. The amount of H2O2 produced was measured and quantified. (p-anisidine €=1.173 M-1 cm-1).45

O2.- measurement in cellular fractions exposed to menadione
The amount of O2.- anion was measured in the membrane and soluble fractions of control and stressed cells in a buffer containing 25mM Tris-HCl/5mM MgCl2 buffer, pH 7.3. To the reaction mixture was added 0.12mM INT. The reaction was allowed to react for 1 hr and the absorbance of reduced formazan was measured at 485nm. The extinction coefficient of 11mM-1cm-1 for INT was used.

Monitoring Enzyme Activity in CFE
The CFE from Pseudomonas fluorescens were isolated as previously described. The protein content of each fraction was measured by the method of Bradford using the kit supplied by Bio-Rad. The methods utilized to monitor the various enzymatic activities are described below. For spectrophotometric analyses the specific activity and standard deviation were calculated for each enzyme. All experiments were performed at least three times and in duplicate. Critical controls involved reaction solutions with inhibitors or deprived of substrate or cofactor.

Catalase Activity
The activity of CAT, EC 1.11.1.6, was measured with the aid of the reagent p-anisidine, and the absorbance at 458nm was monitored. 200µg of protein equivalent from control or menadione-stressed proteins were incubated with 15mM H2O2. 10mM p-anisidine was added immediately in a final volume of 1.0mL and the absorbance was measured after 60min. Blanks were prepared similarly lacking the H2O2 component.

Superoxide Dismutase Activity
The activity of SOD, EC 1.15.1.1, was measured with the aid of the reagent INT, having an oxidized absorbance of 485nm €=11mM-1cm-1. Two hundred micrograms of control or menadione-stressed protein were incubated with 5mM menadione. 15µL of Iodonitrotetrazolium violet (INT), (4mg/mL stock) was added for a final volume of 1.0mL and the absorbance was measured after 60 min. Blanks were prepared similarly lacking the presence of menadione. Menadione was used to obtain a standard curve.

Assay for Aldehydes and Ketoacids
The reagent 2,4-dinitrophenylhydrazine (DNPH) was used to assess the levels of aldehydes and ketoacids.49,50 DNPH reacts readily with the vast majority of biologically relevant aldehydes and ketones to yield 2,4-dinitrophenylhydrazones. The reaction is performed initially under acidic condition, 5mM DNPH in 2N HCl, and then addition of 1N NaOH to deprotonate and colourize the 2,4-dinitrophenylhydrazones. The absorbance was then monitored at 450nm, €=16,000M-1cm-1.

The enzymatic reactions were performed in a final volume of 1ml containing 50mM Tris buffer, pH 7.3 with 5mM MgCl2. Prior to stopping the reaction, the samples were separated into 2 x 0.5mL fractions whereby 0.1mL 2,4 DNPH, 5mM in 2N HCl, was added to stop the reaction. The samples were allowed to stand at room temperature for 15 min. The sample was then diluted with 1mL of double distilled water and 1mL of NaOH (1N). The absorbance was measured at 450nm resulting from the presence of dinitrophenylhydrazone after 10min. Appropriate controls and blanks were prepared and the respective keto acids and aldehydes of interest were used as standards.

Isocitrate Lyase (ICL) Activity
ICL activity, EC 4.1.3.1, was assayed in 25mM Tris-HCl buffer, pH 7.3, containing 5mM MgCl2, 2mM isocitrate, and 0.1mg/mL soluble protein. Blanks and controls were prepared in a similar manner lacking the substrate, isocitrate. Enzyme activity was determined spectrophotometrically by monitoring the production of glyoxylate with 2,4-DNPH at 450nm. The increase in intensity of color has been shown to be proportional to the glyoxylate produced. Glyoxylate was used as a standard.

NAD+-dependent Isocitrate Dehydrogenase (ICDH-NAD+) Activity
ICDH-NAD+, EC 1.1.1.41, catalyzes the oxidative decarboxylation of isocitrate to form α-ketoglutarate. ICDH-NAD+ activity was assayed in 25mM Tris-HCl/5mM MgCl2 buffer, pH 7.3, containing 4mM isocitrate and 0.5mM NAD+, 8mM malonate (for ICL inhibition), and 0.4mg/mL membrane protein. Blanks were prepared in a similar manner lacking the substrate, isocitrate. ICDH-NAD+ activities were determined by measuring the formation of α-ketoglutarate. The amount of ketoacid produced was determined spectrophotometrically using 2,4-DNPH and α-ketoglutarate served as the standard.

α-Ketoglutarate Dehydrogenase (α-KGDH) Activity
α-KGDH activity, EC 1.2.4.2, was assayed in 25mM Tris-HCl buffer, pH 7.3, containing 5mM MgCl2. To the buffer was added 0.3mM α-ketoglutarate, 0.1mM Coenzyme A and 0.5mM NAD+ and finally 0.2mg/mL membrane protein. The disappearance of α-ketoglutarate was followed colorimetrically with the aid of DNPH. Blanks were prepared in a similar manner lacking the substrate, α-ketoglutarate, and a 0.3mM solution of the specific substrate was used as a standard.

Glutamate Dehydrogenase (GDH) Activity
The enzyme GDH, EC 1.4.1.2, catalysed the oxidative deamination of glutamate, with the aid of the cofactor NAD+. The enzyme was monitored in the membrane fractions of control and stressed bacteria. The activity buffer consisted of 25mM Tris containing 5mM MgCl2, pH 7.3, added to 2.0mM glutamate, 0.5mM NAD+, and 0.2mg of protein in a final volume of 1.0mL. The formation of α-ketoglutarate was followed colorimetrically with the aid of DNPH. Blanks were prepared in a similar manner lacking the specific substrate, glutamate. A solution of 1.0mL containing 0.3mM α-ketoglutarate served as standard.

The enzyme was monitored in the soluble fractions (EC 1.4.1.3) of control and stressed bacteria as well. The reactions, blanks and standards were prepared in the same manner, with 0.2mg of soluble protein and the cofactor NADP+ at 0.5mM. Again, a solution of the keto-acid produced served as standard.

Malate Synthase (MS) Activity
MS activity, EC 2.3.3.9, was determined spectrophotometrically by monitoring the disappearance of coenzyme A in the presence of dithiobenzoic acid.51 In this method 0.2mg/mL of soluble protein equivalent was incubated with 1mM glyoxylate, 0.1mM acetyl-CoA, 0.1mM DTNB in 25mM Tris-HCl/5mM MgCl2, pH 7.3. The increase in absorbance from the formation of free thionitrobenzoate ion was monitored at 10 sec intervals for 10 min at 412 nm. An extinction coefficient of €=13.6 mM-1cm-1 was used.

Pyruvate Dehydrogenase (PDH) Activity
PDH, EC 1.2.4.1, catalysed the oxidative decarboxylation of pyruvate to acetyl-CoA, with the concomitant release of NADH from NAD+. The activity of this enzyme was monitored in the membrane fraction of CFE from control and stressed cells by measuring the consumption of pyruvate with the aid of DNPH. Membrane protein equivalents of 0.4mg/mL were incubated with 0.2mM pyruvate, 0.1mM CoA, and 0.5mM NAD+ in a final volume of 1.0mL. The absorbance was measured at 450nm and pyruvate was used the standard. Blanks and experimental controls were prepared in a similar manner lacking the substrate, pyruvate, and membranes respectively.

Succinate Dehydrogenase (SDH) Activity
SDH, EC 1.3.5.1, catalyses the oxidation of succinate to fumarate. Flavine adenine dinucleotide (FAD) is covalently bound to the enzyme, thus for the catalysis to occur, electrons from reduced flavin are passed onto through the electron transport chain (ETC). To monitor the activity of the enzyme, 2,6-Dichlorophenol indophenol (DCPIP) was used as an artificial electron acceptor. DCPIP has been shown to absorb strongly at 600nm, with €=22,000 M-1 cm-1 when oxidized, and colorless when reduced. The decrease in intensity of the color measured at 600nm is proportional to the measure of SDH activity.52 Briefly, in a final volume of 1.0 mL, 25mM Tris-HCl, 5mM MgCl2, 10mM succinate, 12.5mg/mL DCPIP, and 5mM KCN (to inhibit the ETC), were added. The reaction was initiated by addition of 0.2mg/ml of membrane protein equivalent and the absorbance was then measured at 600nm at 10 sec intervals over 100 sec.

Malic Enzyme (ME) Activity
The oxidative decarboxylation of malate to pyruvate is catalyzed by ME, EC 1.1.1.38, requiring the cofactor NADP+ as co-substrate. The reduction of NADP+ was determined by following the formation of NADPH at 340nm.53 The reaction was carried out at room temperature, pH 7.3 and consisted of 25mM Tris-HCl, 5mM MgCl2, 2mM malate, 0.5mM NADP+, and 0.2mg/mL soluble proteins in a final volume of 1.0mL. The absorbance at 340nm was recorded over 5 min at 10 second intervals. The specific activity was calculated using the molar extinction coefficient for NADPH (6.22mmol/L for a path length of 1.0cm). Alternatively, the formation of pyruvate could be monitored with the aid of DNPH.

Pyruvate Kinase (PK) Activity
PK, EC 2.7.1.40, catalyzes the transfer of a phosphoryl group from phosphoenolpyruvate (PEP) to ADP to produce ATP. The activity of PK was assessed by monitoring the appearance of pyruvate using DNPH. The assay was carried out at room temperature, pH 7.6, and consisted of 50mM Tris-HCl, 5mM MgCl2, 0.2mg/mL soluble protein, 0.25mM PEP and 1mM ADP. The keto-acid was measured spectrophotometrically at 450nm with the aid of DNPH. Pyruvate served as the standard. Blanks were prepared in similar fashion lacking the substrate, PEP. Experimental controls contained 50mM Tris-HCl, 5mM MgCl2, 0.25mM PEP and 1mM ADP.

Glucose-6-phosphate Dehydrogenase (G6PDH) Activity
The oxidation of glucose-6-phosphate to form phosphoglucono-δ-lactone is catalyzed by G6PDH, EC 1.1.1.49, requiring the cofactor NADP+ as a co-substrate. The reduction of NADP+ was determined by monitoring the abosorbance at 340nm.53 The following modifications were performed: In 25mM Tris-HCl, 5mM MgCl2, pH 7.3, with 1mM glucose-6-phosphate, 0.5mM NADP+, 0.2mg/mL of soluble protein was added for a final volume of 1.0mL. The absorbance at 340nm was recorded over 5 min at 10 second intervals. The specific activity was calculated using the molar extinction coefficient for NADPH (6.22 mmol/L for a path length of 1.0cm).
Isocitrate Dehydrogenase (ICDH-NADP+) Activity

The oxidative decarboxylation of isocitrate to form α-ketoglutarate using NADP+ as a co-substrate is catalyzed by ICDH-NADP+, EC 1.1.1.44. The reduction of NADP+ was determined by monitoring the formation of NADPH at 340nm.53 The following modifications were performed: In 25mM Tris-HCl, pH 7.3, 5mM MgCl2, 2mM isocitrate, 0.5mM NADP+, 0.1mg/mL of soluble protein was added. The reduction was also performed in the presence of 4mM malonate (for ICL inhibition). The absorbance at 340nm was plotted over 100 sec for 10 sec intervals. The specific activity was calculated using the molar extinction coefficient for NADPH (6.22 mmol/L for a path length of 1.0cm). Alternatively, α-ketoglutarate formation was monitored via the DNPH assay.

Malate Dehydrogenase (MDH) Activity
The oxidation of malate to oxaloacetate using NAD+ as a cofactor is catalyzed by MDH, EC 1.1.1.37. 2,4-DNPH was utilized to monitor the formation of oxaloacetate at 450nm. The assay consisted of 25mM Tris-HCl, pH 7.3, 5mM MgCl2, 1mM malate, 0.5mM NAD+, and 0.2mg/mL of membrane protein equivalent over 7 min. Oxaloacetate served as the standard. Blanks were prepared in the same manner lacking the substrate, malate.

Fumarase (FUM) Activity
The conversion of fumarate to malate is catalyzed by FUM, EC 4.2.1.2. 2,4-DNPH was utilized to monitor the formation of oxaloacetate. The assay consisted of 25mM Tris-HCl/5mM MgCl2, pH 7.3, 1mM fumarate, 0.5mM NAD+, and 0.2mg/mL membrane protein equivalent over 7min. Oxaloacetate served as the standard. The omission of fumarate from the above mixture served as blanks. The activity of FUM was calculated taking into consideration the activity of MDH, the downstream enzyme directly responsible for keto acid formation.

Aconitase (ACN) Activity
The activity of ACN, EC 4.2.1.3, was determined in the soluble fraction of CFE. Precautions were taken to increase the stability of ACN in the soluble fraction. Ten percent tricarballylic acid was added to the whole cells prior to sonication to maintain protein integrity. The assay consisted of 25mM Tris-HCl, 5mM MgCl2, 10mM substrate (citrate), and 0.2mg/mL soluble protein. The reaction was monitored at 240nm for the formation of cis-aconitate as previously described.54 Aconitate served as the standard. Blanks were prepared in a similar fashion however, omitting the substrate from the mixture.

Phosphoenolpyruvate Carboxykinase (PEPCK)
PEPCK, EC 4.1.1.49, catalyses the ATP dependent, reversible decarboxylation of oxaloacetate yielding PEP and CO2. The disappearance of oxaloacetate was followed colorimetrically with the aid of 2,4-DNPH at 450nm, and oxaloacetate served as the standard. The assay was carried out at room temperature, and consisted of 50mM Tris-HCl, pH 7.6, 5mM MgCl2, 0.2mM oxaloacetate, and 0.2mg/mL membrane protein equivalent. However, DNPH was added prior to the addition of protein to ensure that no enzymatic reaction took place.

List of commonly used Buffers
Cell Storage Buffer
50mM Tris-HCl
1mM EDTA
1mM PMSF
1mM DTT
15 mM MgCl2
pH 7.3

NMR buffer Activity Buffer

10mM sodium phosphate 25mM Tris-HCl
5mM MgCl2 5mM MgCl2
pH 7.3 pH 7.3

Statistical Analyses
The student t test was calculated to determine the significance of the difference in specific activity of various enzymes in control compared to menadione-stressed bacteria.

T-TEST
If calculated t value exceeds the tabulated value of 2.78 for n=3 then the means are significantly different and p is said to be ≤ 0.05.55
Enzymatic Activities at Various Growth Intervals
P.fluorescens were grown on either a control medium, or in a menadione-supplemented media. At specified time intervals the bacterial cells were harvested and the CFE were isolated as described before. A Bradford assay was performed to determine the protein content from the CFE, and the specified activities of different enzymes were monitored as indicated above or through gel electrophoresis methods that will be outlined in the following sections.

O2.- and The Modulation of Enzymatic Activities
P.fluorescens was grown in menadione-stressed media to late-logarithmic stage of growth and 10mg protein equivalent of whole cells were transferred to 100ml of either control medium, a control medium containing rifampicin (50µg/mL), or a control medium containing chloramphenicol (50µg/mL), and allowed to grow for a further 15 hours. Alternatively, P.fluorescens was grown in control media to late-logarithmic stage growth and 10mg protein equivalent of whole cells were transferred to 100ml of menadione-supplemented media and allowed to grow a further 15 hrs. The CFE were assessed for activities of various enzymes either by the methods mentioned above, or by electrophoretic techniques.

Electrophoresis
Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE)
1mm spacers were used to make small gels for the Bio-Rad MiniProtean™ 2 system. The final volume of one separating gel was 5.8ml, thus 2.9ml 4% acrylamide and 2.9 ml 16% acrylamide solutions per gel were used to create a linear gradient (4-16%) using the BioRad gradient former for a broad range separation. Appropriate combs were placed in the un-polymerized stacking component and solidified sample wells were overlaid with 1 X Gel Buffer.

Following the polymerization of the gel, the sample wells were dried with filter paper and samples were applied and gently overlaid with Blue Cathode Buffer. The inner chamber of the electrophoretic unit was also filled with blue cathode buffer, while the Anode Buffer filled up the rest of the tank. A voltage of 80 V was applied for the migration of the proteins through the stacking gel, and was then incrementally increased over the span of the gel towards 200 V. At the half-way point of the moving front across the resolving gel, the Blue Cathode was removed from the inner chamber and replaced with colorless cathode buffer to aid in visualization of the proteins. Electrophoresis was stopped once the moving front had moved out of the gel.56 In circumstances whereby localizations of the specific protein is known across the 4-16% gradient, or when the enzyme will simply not catalyze in the presence of its substrates and cofactors, variations of the acrylamide concentration in the gel and methods based on single-concentration gels can be employed to focus on the protein of interest. Low-end concentrations of gels tend to be fragile, but are able to resolve heavier proteins much more effectively. High-end concentrations, alternatively, are designed to separate proteins with low molecular masses. Ponceau S (0.001%) in the Cathode Buffer was utilized to monitor enzymes when Coomassie was ineffective at maintaining native conditions.

BN PAGE Buffers
Blue Cathode Buffer (1L) Colorless Cathode Buffer (1L)
8.96g Tricine (50mM) 8.96g Tricine (50mM)
3.138g BisTris (15mM) 3.138g BisTris (15mM)
0.2g Coomassie blue G 250 pH 7.0 at 4oC
pH 7.0 at 4oC

3 X Gel Buffer (50mL) Anode Buffer (1L)
9.84g aminocaproic acid (1.5M) 10.45g BisTris (50mM)
1.567g BisTris (150mM) pH 7.0 at 4oC
pH 7.0 at 4oC

Coomassie Blue Staining Solution Destaining Solution
50% methanol 50% methanol
10% acetic acid 10% acetic acid
0.2% Brilliant Blue R 250

4-16% Blue Native PAGE Gel Setup

4% 16% Stacking Gel
Acryl-Bis mix 234µL 937µL 273µL
(49.5%T, 1.5%C)

3x Gel Buffer 967µL 967µL 1136µL

Water * 1699µL 223µL 2000µL

75% Glycerol 0 773µL 0

10%APS 9.7µL 7.6µL 30µL

TEMED 1.0µL 0.8µL 2.5µL

*Water is deionized distilled water (DDH2O)

In-Gel Activity Staining for Soluble Enzymes
Soluble fractions of CFE were isolated from P.fluorescens grown in control and menadione-stressed media at various growth intervals and conditions. Samples were prepared for gel electrophoresis by dilution of fractions with 3X Blue Native (BN) buffer and water to a final concentration of 4mg/mL protein equivalent and 1X BN buffer (50mM BisTris, 500mM €-amino-n-caproic acid, pH 7.0) respectively.

To each sample lane 20-80µg of protein were loaded, 60µg being of predominant use, and electrophoresed under Blue native conditions. Following BN-PAGE the gels were incubated in equilibration buffer (25mM Tris-HCl, pH 7.3, 5mM MgCl2) for at least 15 min. The gels were then placed in the appropriate activity buffer (equilibration buffer with the desired pH, substrate, cofactor, and/or enzymes for coupled reactions, inhibitors or activators), and incubated for various times. The activity in the gel was visualized using phenazine methosulfate (PMS) and iodonitrotetrazolium (INT), or DCPIP for catalysis involving the oxidation of reduced cofactors (NADH, FADH2), and in the case of CAT, p-anisidine was utilized. Enzymatic reactions that require NAD+ and/or NADP+, which are then converted to both NADH and NADPH respectively are easily stained within the gel. The tetrazolium salt (INT) is then converted to an insoluble pink formazan precipitate in the presence of the electron donor (ex: NADH) and PMS. Under such conditions, care is taken to avoid light exposure.

NADPH Producing Enzymes: ICDH, ME, G6PDH
The activity of these enzymes was visualized using INT. The tetrazolium is readily reduced by either NADH or NADPH in the presence of PMS to form the insoluble formazan precipitate that will localize directly at the site of enzymatic activity on the gel slab. The gels were placed in equilibration buffer (25mM Tris-HCl, pH 7.3, 5mM MgCl2) as well as 0.4mg/mL PMS, 0.4mg/mL INT, 0.1mM NADP+ and the following substrate depending on the respective enzyme to be detected: 1mM isocitrate for ICDH-NADP+, 5mM malate for ME, and 5mM glucose-6-phosphate for G6PDH activity. Upon visualization of a pink precipitate at the site of enzyme catalysis the gel(s) was placed in destaining solution (50% methanol, 10% acetic acid). This stops catalysis and serves in removing excess Coomassie G 250 from the gel, leaving a clear gel and pink band(s) at the site of enzyme activity. The total volume of reaction mixture per lane was 1.5mL and gel slabs are to be cut for identification of control and stressed protein lanes. Multiple bands per reaction must be identified by any means necessary. Cutting of the un-reacted gel and showing precipitation still occurs with the required reactants proves specificity of the enzyme in the elimination of enzyme products or contaminants from the rest of the gel.

Detection of CAT Activity in BN-PAGE
The in-gel staining of CAT activity was visualized with the aid of p-anisidine. Following electrophoresis, gels were placed in equilibration buffer (25mM Tris-HCl, 5mM MgCl2, pH 7.3) along with 10mM p-anisidine and 35mM H2O2. The total volume of the reaction mixture per lane was set at 1.5mL. When peroxide is cleaved by CAT in the gel, p-anisidine is oxidized to develop a pinkish precipitate directly on the site of catalysis. However, unlike tetrazoliums and indophenol that precipitate within the gel, allowing for crude washing and destaining methods prior to gel-documentation, p-anisidine stains on the surface of the gel and care must be taken in handling the gel piece following the reaction. Upon visualization of a pink precipitate at the site of enzyme catalysis (60min) the gel was either scanned or documented.

Detection of ACN Activity in BN-PAGE
The in-gel activity of ACN was visualized using ICDH from porcine heart in an enzyme coupled reaction. The gels were placed in equilibration buffer (25mM Tris-HCl, 5mM MgCl2, pH 7.3) containing 60 units of ICDH, 0.5mM NADP+, 10mM citrate along with 0.4mg/mL PMS and 0.4mg/mL INT. Total volume per lane was set at 1.5mL and the band corresponding to ACN was observed within 15 min of incubation. It was observed that the use of tricarballylic in cell storage buffer during sonication increased the stability and catalytic function of aconitase considerably in subsequent experiments, owing to the fragility of the Fe-S clusters under our experimental conditions.

Detection of ICL Activity in Native PAGE and BN-PAGE
The in gel-activity of ICL was visualized using a 12% native PAGE with Ponceau S staining or a 4-16% BN-PAGE. The band was visualized with the aid of LDH Type II and NAD+ to draw electron flow through ICL rather than ICDH. It is unclear how Coomassie Blue in fact affects the activity of the enzyme but regardless bands were visualized within 60min of incubation. The gel was placed in an equilibration buffer (25mM Tris-HCl, 5mM MgCl2, pH 7.3) containing 5mM isocitrate, 0.5mM NAD+, 10 units LDH, 0.4mg/mL INT and 0.4mg/ml PMS. Total volume per lane was 1.5mL and band was observed within 30min of incubation.

Detection of MS Activity in BN-PAGE
The in-gel activity of MS was visualized on a 4-16% BN-PAGE. Gels were incubated in an equilibration buffer (25mM Tris-HCl, 5mM MgCl2, pH 7.3) containing 5mM glyoxylate, 5mM acetyl CoA, 0.5mM NAD+, 5 units MDH, 0.4mg/mL PMS and INT. Total volume per lane was 1.5mL and band was observed within 60 min of incubation.

Detection of GR activity in BN-PAGE
The in-gel activity of GR was assumed to be of great importance in understanding the underlying mechanisms of adaptation of P.fluorescens to oxidative stress. An 8% BN-PAGE was utilized for optimal protein migration and band intensity, and following electrophoresis, gels were incubated in equilibration buffer (25mM Tris-HCl, 5mM MgCl2, pH 7.3) containing 5mM oxidized glutathione (GSSG), 0.5mM NADPH, 0.0167mg/mL DCPIP, and 0.4mg/mL INT. Total volume perlane was 1.5mL and a band was observed within 2 hrs of incubation.

Detection of PEPCK activity in BN-PAGE
The in-gel activity of PEPCK was visualized on a 4-16% BN PAGE. Gels were incubated in equilibration buffer (25mM Tris-HCl, 5mM MgCl2, pH 7.3) containing 10mM PEP, 1mM ADP, 10mM HCO3 -, 5 units MDH, 0.5mM NADH, 0.4mg/mL INT and 0.0167mg/mL DCPIP. Total volume per lane was 1.5mL and a band was observed within 1 hr.57

Detection of PK activity
The in-gel activity of PK was visualized on a 4-16% BN-PAGE. Gels were incubated in equilibration buffer (25mM Tris-HCl, 5mM MgCl2, pH 7.3) containing 1mM PEP, 1mM ADP, 0.5mM NADH, 5 units MDH, 0.4mg/mL INT and 0.0167mg/mL DCPIP. Total volume per lane was 1.5mL and band was observed within 6 hrs.

Activity Stain in Blue Native Gels for Membrane Enzymes
Following the isolation of membrane fractions as described in the above methods, the proteins were solubilized for electrophoresis using the detergent dodecylmaltoside: samples were prepared by diluting the membrane fraction with 3 X BN buffer, 10% dodecylmaltoside, and water to give a final concentration of 4mg/mL protein equivalent in 1 X BN buffer (50mM BisTris, 500mM aminocaproic acid, pH 7.0) and 1% dodecylmaltoside. The samples were then incubated on ice for 60 min with intermittent mixing. To each sample well of a mini slab gel (BioRad), a range of 20-80µg of protein was effectively loaded and run, whereby 60µg of protein equivalent was used predominantly for enzyme reactions. The gel unit was set up inside of a refrigerator at 4oC to allow for high amperage during the process without denaturing the proteins themselves. Following BN-PAGE the gels were incubated in equilibration buffer (25mM Tris-HCl, pH 7.3, 5mM MgCl2) for 15 min. Whereas the majority of soluble fraction enzymes seem to perform catalysis best with the aid of the cofactor NADP+, membrane fraction enzymes predominantly required NAD+ as a cofactor for kinetically efficient reactions. Thus, unless otherwise specified, membrane gels were placed in equilibration buffer with 5mM specific substrate for the protein of interest, 0.4mg/mL PMS, 0.4mg/mL INT, and 0.5mM NAD+. Upon visualization of a pink formazan precipitate at the site of enzyme catalysis, the gel(s) was placed in destaining solution (50% methanol, 10% acetic acid). This effectively stopped further catalysis and served to remove the Coomassie G 250 from the gel, leaving transparent gel background and distinguished pink band(s) localized at the enzyme of interest.

Detection of SOD Activity in BN-PAGE
For the detection of SOD, both soluble and membrane fractions were electrophoresed and reacted in hopes of characterizing both the known soluble and insoluble isoforms reported in the literature. Gels were incubated in a volume of 1.5mL per lane of reaction buffer containing 0.5mg/mL INT, and 15mM menadione. The detection of SOD was evident within 6 hrs of incubation and appeared as achromatic bands against a deeply colored gel. Gels were subsequently scanned.

Detection of α-KGDH Activity in BN-PAGE
For the detection of α-KGDH, the gel was incubated in a volume of 1.5mL per lane of reaction buffer containing 0.1mM CoA, 0.5mM NAD+, 5mM α-ketoglutarate, 0.4mg/mL PMS and 0.4mg/mL INT. The detection of α-KGDH was evident within 40 min. To prevent improper staining of the entire gel, the level of CoA for the reaction was kept to a minimum for catalysis to avoid CoA serving as reductant for INT.

Detection of MDH Activity in BN-PAGE
For the detection of MDH, the gel was incubated in a volume of 1.5mL per lane of reaction buffer containing 0.5mM NAD+, 5mM malate, 0.4mg/mL PMS and 0.4mg/mL INT. The detection of MDH was evident within 20 min.

Detection of GDH Activity in BN-PAGE
For the detection of GDH, both soluble and membrane fractions were electrophoresed and probed in hopes of characterizing both known isoforms of the enzyme: soluble NADP+ dependent GDH, and membrane bound NAD+ dependent GDH. The gels were incubated in a volume of 1.5mL per lane of reaction buffer containing 0.5mM NAD+ or NADP+, 0.4mg/mL PMS and INT, and 5mM glutamate. The detection of both isoforms were evident within 30 min.

Detection of Complex I and NADHOX Activity in BN-PAGE
For the detection of Complex I, the gel was incubated in a volume of 1.5mL per lane of equilibration buffer (25mM Tris-HCl, 5mM MgCl2, pH 7.3) containing 0.5mM NADH and 0.4mg/mL INT. Unlike any other gel reactions, detection of ETC components requires the added use of KCN in the activity buffer at 5mM to hinder the entire ETC system. The enzyme will serve as the electron bridge from NADH to the tetrazolium, negating the use of PMS. The NADH oxidase, and alternative oxidase system to the ETC, will also precipitate the formazan in this reaction. Thus two strong bands were evident from the reaction, Complex I appearing much higher on the gel slab and appearing within 4 hours, while the NADH oxidase appeared mid-gel within 5 min.

Detection of SDH (Complex II) in BN-PAGE
For the detection of SDH, the gel was incubated in a volume of 1.5mL per lane of reaction buffer containing 5mM KCN, 20mM succinate, 0.4mg/mL INT, and 0.2mg/mL PMS. KCN and PMS were found to significantly increase the rate of the reaction and the appearance of the bands, which was evident after 30 min of incubation.

Detection of PC in BN-PAGE
For the detection of PC either a 4-16% BN PAGE or a 7.5% BN-PAGE could be used to characterize its in-gel activity. Gels were incubated in equilibration buffer (25mM Tris-HCl, 5mM MgCl2, pH 7.3) containing 5mM pyruvate, 1mM ATP, 5mM HCO3-, 0.5mM NADH, 8 units MDH, 0.4mg/mL INT, and 0.0167mg/mL DCPIP. Total volume per lane was 1.5mL and bands were visualized six hours following incubation. Interestingly, the NADHOX activity band will appear on any gel incubated with NADH and INT, whether in the presence of KCN or not, thus precaution must be taken to fully identify the band of interest from such experiments, either by MW analysis or critical controls.

Detection of NAD+ Kinase (NADK) and NADH Kinase (NADHK) in BN-PAGE
The NADK catalyzes the conversion of NAD+ to NADP+, thus supplying the NADPH producing enzymes with their required cofactor. The NADHK will catalyze an even more favourable conversion of NADH to NADPH, a key goal of an organism in its attempt to reach a reducing environment. For the detection of the NADHK, both soluble and membrane fractions were probed. The gels were incubated in an equilibration buffer (25mM Tris-HCl, 5mM MgCl2, pH 7.3) containing 1mM NADH, 5mM GSSG, 5 units GR from Fisher, 0.0167 mg/mL DCPIP, and 0.4mg/mL INT. Total volume per lane was 1.5 mL, and bands appeared within 4 hrs.

For the detection of NADK, both soluble and membrane fractions were probed. The gels were incubated in an equilibration buffer (25mM Tris-HCl, 5mM MgCl2, pH 7.3) containing 1mM NAD+, 5mM Glucose 6 phosphate, 10 units G6PDH, 3mM ATP, 0.4 mg/mL PMS, and 0.4 mg/mL INT. Total volume per lane was 1.5 mL and bands appeared within 4 hrs.

2D BN-PAGE and Protein Quantification
The protein of interest was first detected catalytically in two lanes of the first dimension of BN-PAGE: Lane 1 corresponding to control cells, Lane 2 corresponding to menadione-stressed cells. The corresponding bands were carefully sliced from the orginal BN-PAGE gel slab and inserted into sample wells of a 2D BN-PAGE, either 4-16%, or at a specific concentration known to accommodate the size of the enzyme of interest. Alternatively, sliced gel fragments can be loaded directly onto the polymerized resolving gel of the second dimension gel and the unpolymerized stacking gel may be poured around it and allowed to solidify. After polymerization, the gels were run as previously described. Any air pockets surrounding gel fragments will hinder protein mobility and overall efficiency of the experiment. Enzyme activity was detected once more by methods previously described, or gel can be stained with Coomassie Brilliant Blue to analyze relative protein concentrations in control and menadione-stressed cells. Slab gels were fixed and stained with 10% acetic acid, 50% methanol, and 0.2% Coomassie Brilliant Blue R-250. The gels were left in staining solution overnight. The gels were then destained in a similar solution lacking only the R-250 component. Destaining was allowed to proceed until band intensity of interest was visibly desirable.

2D and 3D SDS PAGE
Bands corresponding to the specific activity of an enzyme of interest in either 1D or 2D BN-PAGE were carefully spliced from the original dimension and incubated for 60 min in 1 X electrophoresis buffer containing 0.1% mercaptoethanol. Gel fragments were then loaded between two plates containing and SDS gel, again, either in sample wells set into a polymerized stacking gel, or directly against the resolving/stacking interface, with stacking polymerized around them. Gels were run through a discontinuous buffer system and gels were then stained for protein levels using Coomassie or silver stain. In one lane, a broad range SDS marker was run to align proteins of interest with approximate MW. The nature of isoenzymes and the subunits associated with a given protein was readily identified.

SDS PAGE
SDS-PAGE was performed predominantly with a concentration of 10% for heavier proteins while concentrations of 6% and 8% were found to be useful for proteins with lower molecular masses. The resolving gel concentrations were: 10% T and 0.8% C, 0.375M Tris-HCl pH 8,8, 0.1% SDS, 0.06% TEMED, and 0.03% APS. The concentrations of the stacking gel were: 4% T and 0.8% C, 0.1% SDS, 0.625M Tris-HCl (pH 6.8), 0.06% TEMED, and 0.03% APS. The electrode buffer (pH 8.3) contained 0.025 M Tris, 0.192 M glycine, and 0.1% SDS. Electrophoresis was carried out with a constant voltage of 200 V until the Coomassie blue marker from either one sample well or residual staining from gel fragments of a previous dimension reached the bottom of the gel.

LIST OF SDS BUFFERS
30% Acrylamide Stock Solution 4 X Tris/SDS pH 8.8

29.2g Acrylamide 1.5M Tris Base
0.8g Bisacrylamide 0.4% SDS
pH adjusted to 8.8 with 11N HCl

4 X Tris/SDS pH 6.8 5 X Electrophoresis Buffer

1.5M Tris Base 15.1 g Tris Base
0.4% SDS 72.0g Glycine
pH adjusted to 6.8 with 11N HCl 5.0g SDS

Immunoblotting of Proteins Separated by SDS-PAGE
Following SDS-PAGE, a Western blot experiment was performed on specific enzymes to characterize their protein levels using enzyme specific primary antibodies and species-specific peroxidase-conjugate secondary antibodies. In the case of PC and ACC, two enzymes containing covalently-bound biotin moieties, an avidin-peroxidase conjugate was employed as the secondary antibody. The stacking gel of the SDS PAGE was removed and the orientation of the resolving gel was marked by the cut on a corner. The gel was then soaked in the protein transfer buffer for at least 10-20min. Hybond™-P (PVDF membrane) was pre-wet by placing it in 100% methanol for 10 sec followed by 5 min of washing in distilled water prior the equilibration in protein transfer buffer for at least 10 min. The electroblotting cassette was assembled according to the instructions provided by BioRad laboratories. The proteins were transferred overnight at 4oC with a constant voltage of 20 V. The PVDF membranes were then removed and non-specific binding sites for antibodies were blocked by soaking the membranes in 5% Blotto (5% skim milk in TTBS: 20mM Tris-HCl, 0.8% NaCl, 1% Tween 20, pH 7.6). After 60 min incubation, the membranes were washed, 1 x 5 min, with an excess volume of TTBS. The blot was then incubated for 60 min with the primary antibody at the optimized dilution from literature provided with the antibody (Ex. 1/5000 for α-KGDH in 5% Blotto). The membranes were then washed briefly with excess TTBS followed by 2 x 5 min washes in the same buffer. Following 60 min incubation with the appropriate dilution of the secondary antibody (Ex. 1/10 000 in 5% Blotto), the blots were washed 1 x 15 min and 4 x 5 min with excess volumes of TTBS. In the case of PC and ACC, the membranes were placed in Blotto to block all non-biotin containing binding sites for the avidin reagent. After washing, the avidin-peroxidase conjugate was added at the step that formerly involved incubation with the secondary, non-specific antibody of a normal Western blot procedure.

LIST OF WESTERN BLOT BUFFERS
Protein Transfer Buffer (1L) Tris Buffered Saline (TBS) (1L)
3.03g Tris-base 2.42g Tris-base
14.4g glycine 8g NaCl
200ml methanol adjust pH to 7.6 with 2N HCl
store at 2-8oC store at 2-8oC

Tween Tris Buffered Saline (TTBS) 5% Blotto
Dilute required volume of TweenTM 20 5% (w/v) skim milk
in TBS to give a 0.1% (v/v) solution in TTBS
store at 2-8oC

Chemiluminescence Detection
The detection of the desired proteins was achieved with the use of ChemiglowTM (Alpha Innotech). The detection reagents, Solution A (luminol plus enhancer) and Solution B (stable peroxide solution), were mixed in a 1:1 ratio and were allowed to equilibrate at room temperature on the surface of the membrane containing the transferred proteins. Excess volumes were used to ensure that all of the membrane was covered and no sections were allowed to air-dry. Though the working solution is stable up to 24 hrs, an incubation of the membrane in the working solution for 5 min is usually sufficient. Following incubation, membranes were carefully placed within a plastic protector (Plastic Cling Wrap) and all air bubbles were forced out to prevent drying of the membrane. The blots were then visualized with the aid of a Gel Documentation system from Alpha Innotech.

Quantification of Bands
Often either protein levels or activity bands were quantified by either Scion Image V. 4.0.2 (Scion Corp), or through a gel documentation system provided by Alpha Innotech and its densitometry function.

RESULTS
I: Growth and Antioxidant Status during menadione stress
A: Growth profile of P.fluorescens exposed to Menadione
When P.fluorescens was stressed with menadione (100µM), the rate of cell growth was slower as the stressed cells reached the stationary phase at least 5 hrs later than the control cells. In fact, initial growth in the menadione medium was observed at 15 hrs of incubation while in the control culture, significant growth was recorded at 10 hrs. However, at stationary phase of growth, the biomass in both cases were relatively similar.

These results suggest that the bacteria were able to adapt to the O2.- . Data points shown are an average of three independent protein assays (n=3) p ≤ 0.05.

NMR studies: Disparate metabolic profile
To probe for the metabolic remodelling associated with this adaptation process, 13C NMR spectroscopy was performed. Disparate 13C NMR spectra were observed when CFE from the control and menadione-stressed cells at the same growth phase were incubated with labelled citrate

The 13C NMR of menadione-stressed cultures was found to have a signal characteristic of citrate, 43ppm, but much weaker than the standard NMR spectra of the same concentration of labelled citrate (data not shown). As well, peaks indicative of succinate at 32ppm and 181ppm, were observed. The control CFE displays the expected signals for citrate, cis-aconitate, α-ketoglutarate and succinate, depicting an optimally functioning metabolism. These peaks were compared to their respective standards (data not shown).

B: Oxidative Stress
Markers of Oxidative Stress
Though it is widely known that menadione is a potent O2.-generator in biological systems, it was still useful to assess the relative levels of oxidized lipids and oxidized proteins in menadione-stressed CFE. Both the TBARS and protein carbonyl assessment revealed that there were approximately three fold the level of oxidized lipids and two fold the level of oxidized proteins in the CFE of menadione-stressed cells (Table 1).

Table 1: Oxidized Lipids and Proteins in control and stressed cells
Control Menadione-stress
Oxidized Lipids (nmol malondialdehyde/mg protein) 0.24 ± 0.04 0.67 ± 0.04
Oxidized Proteins (nmol of carbonyl/mg protein 0.026 ± 0.003 0.075 ± 0.003
n=3 ± SD p ≤ 0.05
O2.- and H2O2 measurements in CFE exposed to menadione

When CFE fractions were incubated with peroxidase and p-anisidine the absorbance at 458nm was reflective of the relative amounts of H2O2 present, as molecular oxygen liberated from the peroxidase reaction oxidizes p-anisidine. CFE fractions were also incubated with INT and comparisons were made at 485nm as reflections of the comparative O2.- concentrations inherent in cellular fractions. Not surprisingly, H2O2 and O2.- levels were found to be two fold higher under menadione-stress. Though ROS are notoriously short lived, experiments were performed immediately after sonication and significant changes were recorded as signs of oxidative stress.

Table 2: ROS concentrations in control and stressed cells
Control Menadione-stress
Peroxide Concentration (µmol/mg protein) 9.5 ± 0.3 40 ± 0.3
Superoxide Concentration (nmol/mg protein) 11.4 ± 0.5 36.9 ± 0.5
n=3 ± SD p ≤ 0.05

C: Proteomic Studies
As the menadione-stressed cells had a disparate metabolic profile and had signs of oxidative stress, it was important to evaluate the proteins that were enabling this organism to survive. PAGE, BN-PAGE and SDS-PAGE techniques were utilized to perform proteomic studies, (Figure 28).
1 2 3 4 5 6 7
Control Stress BSA 60ug 20ug 40ug 60ug

1 2 3
Control Menadione SDS marker

Panel A: BN-PAGE analysis of soluble CFE from Control and menadione cultures. Lanes 1,2 and 3 correspond to 60µg equivalents of membrane CFE (control), menadione-stress, and the BSA marker respectively. Cells were harvested at late-logarithmic phase of growth. Lanes 4, 5, 6, and 7 correspond to 60µg protein equivalent of soluble CFE control, 20µg, 40µg and 60µg protein equivalent soluble menadione-stress, respectively. Panel B: SDS-PAGE analysis of membrane CFE protein from control and menadione-stress cultures. Lanes 1, 2 and 3 correspond to 60µg protein equivalents membrane CFE control culture, meandione culture, and a broad range MW marker.

D: Enzymes involved in the detoxification of ROS
CAT, SOD and GR
As there was significant change in protein properties of control and menadione-stressed cells, key enzymes involved in combating ROS were evaluated. CAT, a known scavenger for H2O2 is widely distributed in many organisms. The activity of the enzyme was almost five fold higher in menadione-stressed cells by spectroscopic methods. Menadione is a O2.- generator, thus the activity of SOD, an enzyme responsible for the dismutation of O2.- was examined. A 6-fold increase in activity was observed in the soluble fractions of the menadione-stressed cells (Table 3). These increases in enzymatic activities were further confirmed by BN-PAGE. GR was required to fully illustrate the antioxidant response. In-gel activity staining of GR was performed, and it was found to be higher under stress.

CAT and SOD activities in control and stressed cells.
Cytosolic Enzyme Control Stress
CAT (µmol/µg/min) 38.6 ± 1.3 192 ± 4.5
SOD (ηmol/µg/min) 2.7 ± 0.2 16 ± 0.8
n=3 ± SD p ≤ 0.05

A 1 2

B
1 2

C
1 2

1 2 3 4

(Soluble CFE was analyzed)
Lane 1: cells grown in menadione-media (stress). Lane 2: menadione-stressed cells transferred to control media. Lane 3: cells grown in control medium. Lane 4: control cells transferred to menadione medium.

II: THE SOURCE OF NADPH
A: NADPH producing enzymes
In the effort to attain a reductive environment to combat and adapt to an oxidative stress, more is required of an organism than simply having the classical components of the antioxidant defence. NADPH is the main reducing equivalent used by the majority of aerobic organisms to feed antioxidant processes and regenerate reduced macromolecules and ROS scavengers from prior oxidation. If this is the case, an analysis of the major NADPH producing enzymes of the TCA cycle and PPP was deemed necessary to understand the full scope of the organism’s response to oxidative stress. All kinetic studies performed implied a large increase in the activity of NADPH producing enzymes under menadione stress.

In the case ICDH-NADP+, BN-PAGE analysis revealed the presence of two isoforms of ICDH-NADP+ under menadione stress, both having markedly higher activity than control cells. Subsequent regulation and protein level studies revealed that the activity and protein expression of the ICDH-NADP+ isoforms were tightly tethered to the presence of ROS.

15h 24h 30h 25h 30h 35h 40h

Control Menadione-stress

B Control Menadione-stress

C 1 2 3 4

In the case of G6PDH, BN-PAGE analysis revealed the presence of three isoforms of the protein under menadione-stress. All isoforms were found to have markedly higher activity than control cells (Figure 33) across the time profile. Slicing of the unreacted gel slab prior to incubation of each gel fragment showed that each isoform was kinetically substrate-specific for Glucose-6-phosphate. Following this, a second dimension BN-PAGE was performed and Coomassie Blue staining showed increased protein expression for the isoforms as well under menadione stress.

Figure 33: BN PAGE analysis of G6PDH activity in control and menadione-stressed cells at various growth intervals. Lanes 1,2 and 3 correspond to soluble CFE from cells grown in a citrate medium (control) for 15, 24 and 30 hrs respectively. Lanes 4, 5 and 6 correspond to soluble CFE from cells grown in a menadione-stressed medium for 24, 30, 35 and 45 hrs respectively. A, B, and C the isoforms of G6PDH.

A
1 2

B
1 2

Figure 34: 2D BN PAGE analysis of G6PDH protein expression in control and menadione-stressed cells. Panel A: Lanes 1 and 2 correspond to the soluble CFE from control and soluble CFE from menadione cultures. Panel B: Lanes 1 and 2, (Band B from Figure 33) was analyzed. Coomassie Blue staining was utilized.

The analysis of ME was performed through BN-PAGE activity and Coomassie blue protein staining to complete the assessment of the NADPH production in menadione-stressed cultures at various growth intervals. ME is of great interest in that it is also a producer of pyruvate, a keto acid with potential peroxide scavenging properties, and a metabolite that links the TCA cycle, gluconeogenesis and glycolysis, (Figure 35, 36). Though no isoforms were detected, the enzyme showed markedly higher activity in stressed cultures than the control ones.

1 2 3

A final NADPH producing enzyme, GDH-NADP+ was was studied because its activity would serve two major purposes, creation of a keto acid pool for potential H2O2 scavenging and formation of NADPH. Much like ME, GDH-NADP+ facilitated the conversion of glutamate to α-ketoglutarate with the concommitant production of NADPH. BN PAGE activity assays revealed a markedly higher activity for the enzyme in the presence of menadione

The NAD+ kinase (NADK)
Following the observations that NADPH generation was significantly increased by enzymatic activity and protein expression, it was then important to analyze the status of the enzyme involved in the production of NADP+, the required cofactor for all enzymes of interest.
The in-gel analysis of NADK revealed that its activity and expression was significantly increased under menadione stress. As G6PDH was also utilized to couple this enzyme, a G6PDH band was also observed.

III: NADH Homeostasis
REGULATION OF OXIDATIVE METABOLISM and IRON PROTEINS IN P.fluorescens
A: Assessment of ETC components
As NADH is considered as a prooxidant due to its ability to generate ROS via oxidative phosphorylation, it was important to evaluate its production and utilization in the membrane fraction.

ROS and the ETC
The first enzyme of the ETC is the Complex I, a key metabolic and Fe containing protein. When exposed to menadione, this enzyme showed marked decrease in activity (Figure 40 Panel A). In analysis of protein expression levels by Coommassie staining of a 2D BN-PAGE revealed that protein expression was greatly reduced in the presence of menadione.

In analyzing the activity of Complex I, the activity of an alternative oxidase (AOX), the NADH oxidase was performed as well. Kinetically, the enzyme appeared within 5 min mid-way through the gel, while the much heavier Complex I came within 4 hours at the top of the 4-16% gel. The activity and expression of the NADH oxidase was found to be significantly higher under menadione stress and directly influenced by the prescence of menadione. The oxidase will appear in all reactions involving NADH and INT.

Given the effects of ROS on Complex I, an activity and protein expression assessment of Complex 2, succinate dehydrogenase (SDH) in P.fluorescens exposed to menadione was performed. Not surprisingly, both activity and protein expression in response to menadione was found to be significantly decreased.
B: Enzymatic regulation of NADH production
In a situation whereby ETC components have been inactivated, oxidative stress is prevalent throughout the biological system and a reductive environment is being created, the presence of excess NADH becomes a danger to the cell. Thus, with no ETC to take its electrons, NADH becomes a potent pro-oxidant in and of itself and thus its production and processing must be tightly regulated to prevent accumulation. Following this line of thought, the activity, and in some cases protein expression, were assessed for the major NADH producing enzymes ICDH-NAD+, α-KGDH, GDH-NAD+, and PDH.
BN PAGE analysis of ICDH-NAD+ revealed a pronounced decrease in activity for the enzyme, found to be dependent on bacterial growth time in the medium.

BN PAGE analysis of α-KGDH followed a similar trend showing significantly decreased activity as well as protein expression. α-KGDH is of increased importance in that it contains a sulfhydryl moiety that is highly susceptible to oxidative inactivation, while it is a link between the TCA and the nitrogen metabolism involving glutamate and glutamine production.

The analysis of GDH-NAD+ activity was performed using BN-PAGE analysis and the results revealed yet another NADH producing enzyme that has been inactivated. Contrasting this result was the prior observation that the soluble isoform of GDH, a producer of NADPH, showed markedly higher activity under menadione stress. For the membrane counterpart, enzymatic activity was lower.

The analysis of PDH activity was performed using BN-PAGE analysis and the results followed the preceding trend. Activity was found to be significantly reduced as well. It is interesting to note that these enzymes mediate the flux of metabolites to and from major metabolic schemes such as the TCA cycle, glutamate/glutamine metabolism, gluconeogenesis and glycolysis. Reduction of their activities and protein levels must satisfy a need that is larger than the optimal flow of metabolism.

IV: Conversion of NADH to NADPH
METABOLIC NETWORKS EVOKED BY ROS
A: The Glyxoylate Shunt
As citrate was the sole source of carbon, it was critical to determine how this tricarboxylic was metabolized. The glyoxylate shunt that utilizes isocitrate was evaluated. The activity and expression of ICL and MS were investigated.

BN PAGE analysis of ICL activity in control and menadione-stressed cells. Panel A: BN-PAGE activity over various growth intervals: Lanes 1, 2, and 3 correspond to soluble CFE from control cells grown 15, 24, and 30 hrs respectively. Lanes 4, 5, 6, and 7 correspond to soluble CFE from menadione-stressed cells grown 25, 30, 35, and 45 hrs respectively. Lane 8 corresponds to the BSA MW marker. Band A, ICL, Band B and C, ICDH-NADP+ isoenzymes. Panel B: Influence of menadione on ICL activity: Lanes 1, 2, 3, and 4 correspond to soluble CFE from cells grown in a control medium, soluble CFE from control whole cells transferred to a menadione medium, soluble CFE from cells grown in a menadione medium, soluble CFE from menadione-stressed cells transferred to a control medium. Lanes 5 and 6 correspond to a duplicate of ICL activity in the presence of a known inhibitor, malonate.

Influence of menadione on ICL protein levels. Coomassie staining of ICL on 2D BN-PAGE is shown: Lanes 1, 2, 3, 4, and 5 correspond to a BSA MW markers, commercial ICL purchased from Sigma, soluble control CFE, soluble menadione-stressed CFE, and soluble aluminum-stressed CFE respectively.

The activity of MS was assessed by BN-PAGE as well over various growth intervals and in the presence or absence of menadione.

Panel A: BN-PAGE analysis of MS activity over various growth intervals. Lanes 1, 2, and 3 correspond to soluble CFE from cells grown in control medium for 15, 24, and 30 hrs. Lanes 4, 5, 6, and 7 correspond to soluble CFE from cells grown in a menadione medium for 25, 30, 35 and 45 hrs. Panel B: BN-PAGE analysis of the influence of menadione on MS activity. Lanes 1, 2, 3, 4 correspond to soluble CFE from cells grown in control media, soluble CFE of control cells transferred to a menadione medium, soluble CFE from cells grown in menadione media, and soluble CFE from menadione-stressed cells transferred to a control.

B: Pyruvate Carboxylase and ROS
Since oxaloacetate is a ketoacid that may contribute to the decrease in oxidative tension, the metabolism of this metabolite was followed.
The analysis of PC was performed using BN-PAGE for activity assays, SDS PAGE for protein expression analysis, and Western blotting with the Avidin-peroxidase conjugate for biotinylated proteins. A marked increase in both activity and expression was observed. (Figures 50, 51)

BN-PAGE analysis of PC activity in control and menadione-stressed cells. Panel A: Lanes 1, 2, and 3 correspond to membrane CFE from cells grown in a control medium for 15, 24, and 30 hrs. Lanes 4, 5, 6, 7 correspond to membrane CFE from cells grown in menadione media for 25, 30, 35, and 45 hrs. Band B: NADHOX activity. Lane 8 corresponds to the BSA MW markers.

PC activity and protein expression. Panel A: in-gel activity staining of PC in the membrane CFE. Lane 1: control cells; 2: menadione-stressed cells. Panel B: 2D SDS-PAGE of PC in a 7.5% acrylamide gel where activity bands from 1D BN-PAGE were utilized. Lane 1: SDS PAGE MW markers (116kDa top band, 21.5kDa bottom band); 2: membrane CFE from control cells; 3:membrane CFE from menadione-stressed cells. Panel C: Western blot of PC as detected by avidin-conjugated horseradish peroxidase. Lane 1: membrane CFE from citrate cells; 2: membrane CFE from menadione-stressed cells. Band A: PC; B: ACC.

The activity of MDH was then assessed by BN PAGE activity assays and the results showed an increase in MDH activity under menadione stress.

N-PAGE analysis of MDH activity. Lanes 1, 2, and 3 correspond to membrane CFE from cells grown in control media for 15, 24, and 30 hrs. Lanes 4, 5, 6, and 7 correspond to membrane CFE from cells grown in menadione media for 25, 30, 35, and 45 hrs.

BN PAGE activity assays revealed that the activity of PEPCK was in fact uncoupled from PC activity and markedly decreased in menadione-stressed cultures (Figure 53). This observation triggered the evaluation of PK, and enzyme that utilizes phosphoenolpyruvate. Its activity was found to be significantly enhanced in the menadione-stressed cultures.

Panel A: BN-PAGE analysis of PEPCK activity. Lanes 1 and 2 correspond to soluble control CFE and soluble menadione CFE. Panel B: 2D BN-PAGE expression of PEPCK. Lanes 1, 2, and 3 correspond to soluble control CFE, soluble menadione CFE, and soluble CFE from menadione-stressed cells transferred to a control medium. Activity bands obtained on a 1D BN-PAGE were excised and loaded.

BN-PAGE analysis of PK activity in control and menadione-stressed cells. Lanes 1, 2, and 3 correspond to soluble control protein, soluble menadione protein and the BSA MW markers.

C: NADHK ACTIVITY
NADH kinase (NADHK) and ROS stress
Given all of these massive alterations in the metabolic circuitry in an effort to create a reductive, antioxidant environment, it was deemed essential to analyze the activity of the NADHK, an enzyme that phosphorylates NADH directly to NADPH. This would satisfy two major goals of the cellular metabolism under ROS stress, namely NADPH production and NADH limitation.

The analysis of NADHK activity was performed in BN-PAGE and the results showed an increase in catalytic function and expression.

BN-PAGE analysis of NADHK activity in control and menadione-stressed cells. Lanes 1 and 2 correspond to membrane CFE control cells and membrane CFE menadione-stressed cells.

2D BN-PAGE analysis of NADHK protein levels. Lanes 1 and 2 correspond to Coomassie staining of 2D BN-PAGE of membrane control and menadione-stressed CFE. The activity bands (Figure 55) were excised and loaded.

V: The Role of α-Ketoacids in ROS detoxification
α-KETOGLUTARATE AS AN ROS SCAVENGER
Results show that under oxidative stress, P.fluorescens will upregulate ICDH-NADP+ and down-regulate α-KGDH, allowing for a pool of α-ketoglutarate to form. The increased activity of GDH aids this accumulation as well, and thus based on present literature on the antioxidant properties of keto acids in general, the role of α-ketoglutarate as an ROS scavenger was assessed by NMR. and DNPH assays in the presence of H2O2 (data not shown). 13C NMR results reveal that α-ketoglutarate can act as a potent H2O2 scavenger in vitro, undergoing a decarboxylation to produce succinate and carbon dioxide. The NMR data from the control and menadione-stressed cells point to such a possibility.

C NMR spectra of α-ketoglutarate as an ROS scavenger. Panel A: α-ketoglutarate standard spectra. Panel B: succinate standard spectra. Panel C: reaction between H2O2 and α-ketoglutarate. Signals shown in Panel C represent methylene (32ppm) signals and carboxylic acid (181ppm) signals of succinate.

DISCUSSION

The preceding results point to specific and crucial metabolic networks that have been initiated with the aim of creating a reductive environment in P.fluorescens exposed to menadione, a generator of O2.-. The organism appeared to employ three major strategies to combat this oxidative situation: up-regulation of NADPH production, increased production of α-ketoglutarate, and a decrease in NADH formation. Hence metabolic circuits involving ICL, MS, ME, MDH, and uncoupled PC and PEPCK help transform NADH into NADPH, and increased direct enzymatic conversion of NAD+ and NADH to NADP+ and NADPH. This was further aided by the classical antioxidant arsenal such as CAT, SOD, and GR. In brief, a choice was made by the cell to forego the classical oxidative means of ATP synthesis and to favour the creation of a reducing environment necessary for survival.

I: Growth and Antioxidant Status in menadione stress
In the preliminary analysis of growth in the presence of menadione (100µM), the growth pattern displayed a distinct lag phase of approximately 5 hrs of growth, followed by a steep exponential phase, and at the stationary phase of growth the biomass was similar to the control culture. Recent literature shows that both bacterial and mammalian growth in the presence of ROS such as H2O2 or O2.- result in sluggish proliferation and delayed growth profiles. Hence it appears that P.fluorescens may have adapted to this oxidative environment.

Indeed NMR studies did reveal that citrate, the sole source of carbon, was metabolized in an entirely disparate manner in the menadione-stressed cells compared to the control. In the stressed cells, citrate was consumed however a significantly high accumulation of succinate and α-ketoglutarate was evident. Recent literature has shown the ability of keto acids to scavenge for ROS, resulting in non-enzymatic decarboxylation, an example being the favourable conversion of α-ketoglutarate to succinate. Such an alteration would allow for a truncated version of the TCA to proceed for NADPH production, while restricting oxidative mechanisms, and feeding pools of readily usable metabolites.

It is quite evident from the data on oxidized proteins and lipids that the microbe did incur oxidative injury due to menadione exposure. Hence, it is quite logical that the classical enzymes involved in ROS detoxification would be promoted.

The enzymes CAT, SOD, and GR were analysed. CAT is a known heme-containing detoxifier of H2O2, and its susceptibility to high levels of ROS via potential iron liberation is well documented. SOD, on the other hand, facilitates the dismutation of O2.- into H2O2. GR is an enzyme that is crucial to regeneration of reduced glutathione. It was not surprising to see the activities of these three enzymes elevated in menadione-stressed cultures. However, it is important to reiterate that NADPH is essential for the effective functioning of these enzymes. Indeed, the transfer of menadione-stressed cells to control cultures resulted in a decrease of these activities.

II: The Source of NADPH
In creating a reductive environment for adaptation to an oxidative stress, the strategy is, globally speaking, two fold. First, increased production of NADPH is required to fuel all antioxidant purposes. Secondly, a restriction of NADH production and metabolism through the ETC becomes equally paramount, in effect restricting oxidative mechanisms.
The major producers of NADPH are ICDH-NADP+, ME, and G6PDH. The activities and expressions of ME and GDH-NADP+ increased with time in the menadione cultures. Thus, there is a dedicated effort by menadione-stressed P.fluorescens to reconfigure its metabolism towards enhanced NADPH production. A similar observation has recently been reported. In this instance, it appears Al-stressed cells upregulate the expression of ICDH-NADP+ and G6PDH to survive the oxidative stress triggered by this trivalent metal.

Increased NADPH production would necessitate a higher or constant supply of NADP+. NADK is an enzyme that converts NAD+ into NADP+, and its increased activity serves dual purposes namely, Feeding the pool of required substrate for NADPH production, and restriction of the cofactor used in the production of NADH for the TCA cycle enzymes. Conceivably, this would both help promote a reductive environment while creating a limitation on NADH production, a potential source of ROS via oxidative phosphorylation. 2D BN PAGE analysis of the enzyme showed increased protein expression as well.

As previously mentioned, creating a reductive environment has to be coupled with strategies aimed at limiting oxidative pathways if an organism is to survive menadione-stress. In this study, it is clearly evident that the microbe has drastically reduced its oxidative reactions,. In such a situation, whereby the presence of menadione has inactivated the first two enzymes of the ETC, the potential for NADH to act as a pro-oxidant is sharply decreased, of course at the expense of ATP production. Hence, production and utilization of NADH were markedly diminished in an effort to restrict the creation of an oxidative environment.

An analysis was thus performed on the response of NADH producing enzymes of the TCA cycle to growth in menadione, and the results showed significant reduction in activity. In fact, for NAD+-ICDH, activity was found to be drastically reduced with respect to incubation time in the medium. Concurrently, the activity of α-KGDH was found to be markedly reduced with respect to incubation time and regulation of menadione concentrations. Furthermore, the expression was found to be reduced. Analysis of the activities of both NAD+-GDH and PDH displayed a similar trend of marked reduction in NADH production.

IV: Conversion of NADH to NADPH
Metabolic Networks Evoked by ROS
In the adaptation evoked by ROS in P.fluorescens, there is a clear shift of carbohydrate metabolism towards a reductive end. In truncating the oxidative mechanisms, a modified glyoxylate bypass was observed to allow the flux of metabolites towards the conversion of NADH to NADPH. The seed-to stem maturation of plant seeds, the survival of mycobacterium tuberculosis, as well as research from our own laboratory delineating the metabolic alterations evoked by the pro-oxidant aluminium, the use of the glyoxylate shunt has been shown to be modified to circumvent these challenges. In this instance, we do observe increased activity and expression of ICL. The increased activity of MS allowed the organism to create a pool of malate, not for gluconeogenesis, but to generate NADPH with the aid of enhanced ME expression.

Given this flux of metabolism towards malate, NADPH, and pyruvate, it became clear that pyruvate would accumulate or be utilized in other processes. As PDH has already been shown to be down-regulated, PC, the other effector of this metabolite, was significantly enhanced. SDS PAGE and Western Blot analysis revealed increased PC expression as well MDH was also found to be markedly more active in menadione-stressed cells, In the classical metabolism, the enzymes PC and PEPCK are consistently found to work in concert in an effort to fuel the gluconeogenic pathway for replenishing glucose stores. In the menadione-stressed cells, PEPCK activity and expression were lower in menadione cultures. Hence, it became quite evident that a novel metabolic pathway was at play in an effort to convert NADH to NADPH. Finally, the enzyme PK was found to have increased activity. What is then revealed here is a massive re-tailoring of classical carbohydrate metabolism, taking from enzymes involved in the glyoxylate shunt, TCA cycle, gluconeogenesis, and glycolysis with the aim of consuming excess NADH and furthering the production of NADPH. Both ICL and MS allow for a malate supply to fuel NADPH production through ME, while PC and MDH allow for the conversion of pyruvate to oxaloacetate and then back to malate while consuming NADH. A reduced PEPCK activity slows the flux of oxaloacetate towards phosphoenol pyruvate production, and yet should any be formed, the increased activity of PK allows for both ATP production via substrate-level phosphorylation and further pyruvate formation. Note as well that reduced PDH activity maintains this transhydrogenase network by preventing Acetyl CoA and NADH production, thus limiting the oxidizing power of the cell.
Despite the efficiency of such a network, there may still be two more potential means of using NADH. The NADHOX was found to have increased activity and expression. In numerous studies involving the interaction of ROS, NADH and an inoperable ETC, the use of an alternative oxidase system is often observed.10 The strategy is to absorb excess NADH through an oxidase to produce H2O, without any ATP production. The second avenue for dealing with NADH would be through the use of a direct transhydrogenase mechanism, employing the NADHK enzyme to convert NADH to NADPH. The activity and expression of this enzyme were higher in the menadione-stressed cells. Thus we find major re-routing of metabolites in the organism with the aim of limiting oxidative reactions, providing reducing equivalents for adaptation, and the elimination of excess NADH or its conversion to NADPH.

V: The Role of α-Ketoacids in ROS detoxification
The ability of an organism to shift its metabolism towards quenching an oxidative stress defines its adaptive prowess and ultimately decides its fate. Despite the previous strategies mentioned, there appears an important metabolic rearrangement in P.fluorescens subjected to menadione-stress geared towards the formation of an α-ketoglutarate pool. Here, we have found a novel isoform along with increased activity and expression of ICDH-NADP+. Concomitantly, the activity of α-KGDH was reduced in these cells, and was coupled to increased GDH-NADP+ activity and reduced GDH-NAD+ activity. Thus, one finds a metabolic network aimed at NADPH production and the creation of a ketoacid pool. 13C NMR studies confirmed the involvement of α-ketoglutarate as an ROS scavenger.

It is important to analyze the 13C NMR of the CFE now to fully understand the role of α –ketoglutarate in the detoxification of ROS. The nature of the labelled citrate and how it was metabolized imply that the accumulation of succinate evident under menadione-stress was due to the decarboxylation of α-ketoglutarate. As the labelled carbons were observed at 181 ppm and 32 ppm, it became clear that the succinate was produced via ROS-mediated decarboxylation. Furthermore, α-KGDH activity was low in menadione-stressed cells and the essential cofactor, CoASH was absent. α-Ketoacids have been shown in many cases to have successfully protected mammalian cells from oxidant-related diseases.68 Both pyruvate and α-ketoglutarate have been shown to reduce the oxidation of DNA by menadione and H2O2, and it is important to note that whereas ROS rarely accumulate in a system beyond the nanomolar range, these metabolites are maintained in the millimolar range.69 In this case, the increased flux towards the metabolite, coupled to decreased consumption by downstream enzymes has allowed for oxidative mechanisms to be bypassed during the scavenging of ROS.

CONCLUSION
It appears that under menadione-stress, global cellular metabolism in P.fluorescens has been reconfigured with the aim of creating a reductive environment, restricting NADH production and oxidative mechanisms, and the pooling of potential scavengers of ROS, namely α-ketoglutarate. An oxidative stress is a massive burden on a living system. Here, we have unravelled some important strategies aimed at neutralizing this challenge. Increased enzymatic production of NADP+ and NADPH, coupled to decreased production of NAD+ and NADH are critical. It is important to note that the analysis here is a snap-shot of the overall life of the organism, and it details only the metabolic reconfiguration deemed necessary to combat the stress.
There are definite draw-backs to such alterations in that the cell is deprived of its main sources of ATP, the NADH generating components of the TCA cycle, and the ETC. However, one must understand that under extreme stress conditions, the needs of the cell change drastically to adapt rather than die. In the case of NAD+ and NADH kinase activities, it is clear that ATP must be expended, but the energy use is justified in the end-products, NADP+ and NADPH. More profound, however, is the ease with which this organism is able to adjust its metabolic networks to survive. Of major significance is its ability to utilize various components of the TCA cycle, glyoxylate shunt, and gluconeogenesis to increase the NADPH pool and decrease NADH concentrations.

The metabolic shift evoked by menadione in P.fluorescens efficiently allows for a reducing environment to be attained, while maintaining stores of α-ketoglutarate and restricting the concentrations of NADH and the oxidizing power of the cell. It remains unclear the mechanism of interaction of ROS with the expression and activity parameters of enzymes and proteins, but the role of ROS as signalling molecules via sulfhydryl moiety oxidation has been reported. As well, its interaction with Fe-containing enzymes, especially those with an Fe-S cluster and their subsequent impact with the genetic machinery of the cell has also been well documented. In the case of ACN, the integrity of the Fe-S cluster is highly susceptible to denaturation, and in doing so, the protein then becomes a transcription factor for metal-scavenging and metal transport proteins. For both α-KGDH and PDH, the lipoic component of the enzyme complexes has a highly susceptible sulfhydryl moiety, and thus direct inactivation via sulfhydryl oxidation and downstream effects are quite likely.
This study illustrates the profound importance of metabolism and its plasticity in executing cellular functions. Metabolomic studies will reveal how the metabolites, proteome, and the genome are interlinked to ease inter- and intracellular communication.

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