Microbes vary strategically in their metalation of mononuclear enzymes.

Studies have determined that nonredox enzymes that are cofactored with Fe(II) are the most oxidant-sensitive targets inside Escherichia coli. These enzymes use Fe(II) cofactors to bind and activate substrates. Because of their solvent exposure, the metal can be accessed and oxidized by reactive oxygen species, thereby inactivating the enzyme. Because these enzymes participate in key physiological processes, the consequences of stress can be severe. Accordingly, when E. coli senses elevated levels of H2O2, it induces both a miniferritin and a manganese importer, enabling the replacement of the iron atom in these enzymes with manganese. Manganese does not react with H2O2 and thereby preserves enzyme activity. In this study, we examined several diverse microbes to identify the metal that they customarily integrate into ribulose-5-phosphate 3-epimerase, a representative of this enzyme family. The anaerobe Bacteroides thetaiotaomicron, like E. coli, uses iron. In contrast, Bacillus subtilis and Lactococcus lactis use manganese, and Saccharomyces cerevisiae uses zinc. The latter organisms are therefore well suited to the oxidizing environments in which they dwell. Similar results were obtained with peptide deformylase, another essential enzyme of the mononuclear class. Strikingly, heterologous expression experiments show that it is the metal pool within the organism, rather than features of the protein itself, that determine which metal is incorporated. Further, regardless of the source organism, each enzyme exhibits highest turnover with iron and lowest turnover with zinc. We infer that the intrinsic catalytic properties of the metal cannot easily be retuned by evolution of the polypeptide.

Evidence that protein thiols are not primary targets of intracellular reactive oxygen species in growing Escherichia coli.

The oxidizability of cysteine residues is exploited in redox chemistry and as a source of stabilizing disulfide bonds, but it also raises the possibility that these side chains will be oxidized when they should not be. It has often been suggested that intracellular oxidative stress from hydrogen peroxide or superoxide may result in the oxidation of the cysteine residues of cytoplasmic proteins. That view seemed to be supported by the discovery that one cellular response to hydrogen peroxide is the induction of glutaredoxin 1 and thioredoxin 2. In this study we used model compounds as well as alkaline phosphatase to test this idea. Our results indicate that molecular oxygen, superoxide, and hydrogen peroxide are very poor oxidants of N-acetylcysteine and of the protein thiols of alkaline phosphatase in vitro. Copper could accelerate thiol oxidation, but iron did not. When alkaline phosphatase was engineered to remain in the cytoplasm of live cells, unnaturally high concentrations of hydrogen peroxide were required to oxidize it to its active, disulfide-dependent form, and toxic levels of superoxide had no effect. At the same time, far lower concentrations of these oxidants were sufficient to poison key metalloenzymes. The elimination of glutaredoxin 1 and thioredoxin 2 did not change these results, raising the question of why E. coli induces them during peroxide stress. In fact, when catalase/peroxidase mutants were chronically stressed with hydrogen peroxide, the absence of glutaredoxin 1 and thioredoxin 2 did not impair growth at all, even in a minimal medium over many generations. We conclude that physiological levels of reduced oxygen species are not potent oxidants of typical protein thiols. Glutaredoxin and thioredoxin must either have an alternative purpose or else play a role under culture conditions that differ from the ones we tested.

How a natural antibiotic uses oxidative stress to kill oxidant-resistant bacteria.

Natural products that possess antibiotic and antitumor qualities are often suspected of working through oxidative mechanisms. In this study, two quinone-based small molecules were compared. Menadione, a classic redox-cycling compound, was confirmed to generate high levels of reactive oxygen species inside Escherichia coli. It inactivated iron-cofactored enzymes and blocked growth. However, despite the substantial levels of oxidants that it produced, it was unable to generate significant DNA damage and was not lethal. Streptonigrin, in contrast, was poorer at redox cycling and did not inactivate enzymes or block growth; however, even in low doses, it damaged DNA and killed cells. Its activity required iron and oxygen, and in vitro experiments indicated that its quinone moiety transferred electrons through the adjacent iron atom to oxygen. Additionally, in vitro experiments revealed that streptonigrin was able to damage DNA without inhibition by catalase, indicating that hydrogen peroxide was not involved. We infer that streptonigrin can reduce bound oxygen directly to a ferryl species, which then oxidizes the adjacent DNA, without release of superoxide or hydrogen peroxide intermediates. This scheme allows streptonigrin to kill a bacterial cell without interference by scavenging enzymes. Moreover, its minimal redox-cycling behavior avoids alerting either the OxyR or the SoxRS systems, which otherwise would block killing. This example highlights qualities that may be important in the design of oxidative drugs. These results also cast doubt on proposals that bacteria can be killed by stressors that merely stimulate intracellular O2- and H2O2 formation.

Shear rate sensitizes bacterial pathogens to H2O2 stress.

Cells regularly experience fluid flow in natural systems. However, most experimental systems rely on batch cell culture and fail to consider the effect of flow-driven dynamics on cell physiology. Using microfluidics and single-cell imaging, we discover that the interplay of physical shear rate (a measure of fluid flow) and chemical stress trigger a transcriptional response in the human pathogen Pseudomonas aeruginosa. In batch cell culture, cells protect themselves by quickly scavenging the ubiquitous chemical stressor hydrogen peroxide (H2O2) from the media. In microfluidic conditions, we observe that cell scavenging generates spatial gradients of H2O2. High shear rates replenish H2O2, abolish gradients, and generate a stress response. Combining mathematical simulations and biophysical experiments, we find that flow triggers an effect like "wind-chill" that sensitizes cells to H2O2 concentrations 100 to 1,000 times lower than traditionally studied in batch cell culture. Surprisingly, the shear rate and H2O2 concentration required to generate a transcriptional response closely match their respective values in the human bloodstream. Thus, our results explain a long-standing discrepancy between H2O2 levels in experimental and host environments. Finally, we demonstrate that the shear rate and H2O2 concentration found in the human bloodstream trigger gene expression in the blood-relevant human pathogen Staphylococcus aureus, suggesting that flow sensitizes bacteria to chemical stress in natural environments.

Excess copper catalyzes protein disulfide bond formation in the bacterial periplasm but not in the cytoplasm.

Copper avidly binds thiols and is redox active, and it follows that one element of copper toxicity may be the generation of undesirable disulfide bonds in proteins. In the present study, copper oxidized the model thiol N-acetylcysteine in vitro. Alkaline phosphatase (AP) requires disulfide bonds for activity, and copper activated reduced AP both in vitro and when it was expressed in the periplasm of mutants lacking their native disulfide-generating system. However, AP was not activated when it was expressed in the cytoplasm of copper-overloaded cells. Similarly, this copper stress failed to activate OxyR, a transcription factor that responds to the creation of a disulfide bond. The elimination of cellular disulfide-reducing systems did not change these results. Nevertheless, in these cells, the cytoplasmic copper concentration was high enough to impair growth and completely inactivate enzymes with solvent-exposed [4Fe-4S] clusters. Experiments with N-acetylcysteine determined that the efficiency of thiol oxidation is limited by the sluggish pace at which oxygen regenerates copper(II) through oxidation of the thiyl radical-Cu(I) complex. We conclude that this slow step makes copper too inefficient a catalyst to create disulfide stress in the thiol-rich cytoplasm, but it can still impact the few thiol-containing proteins in the periplasm. It also ensures that copper accumulates intracellularly in the Cu(I) valence.

The vulnerability of radical SAM enzymes to oxidants and soft metals.

Radical S-adenosylmethionine enzymes (RSEs) drive diverse biological processes by catalyzing chemically difficult reactions. Each of these enzymes uses a solvent-exposed [4Fe-4S] cluster to coordinate and cleave its SAM co-reactant. This cluster is destroyed during oxic handling, forcing investigators to work with these enzymes under anoxic conditions. Analogous substrate-binding [4Fe-4S] clusters in dehydratases are similarly sensitive to oxygen in vitro; they are also extremely vulnerable to reactive oxygen species (ROS) in vitro and in vivo. These observations suggested that ROS might similarly poison RSEs. This conjecture received apparent support by the observation that when E. coli experiences hydrogen peroxide stress, it induces a cluster-free isozyme of the RSE HemN. In the present study, surprisingly, the purified RSEs viperin and HemN proved quite resistant to peroxide and superoxide in vitro. Furthermore, pathways that require RSEs remained active inside E. coli cells that were acutely stressed by hydrogen peroxide and superoxide. Viperin, but not HemN, was gradually poisoned by molecular oxygen in vitro, forming an apparent [3Fe-4S]+ form that was readily reactivated. The modest rate of damage, and the known ability of cells to repair [3Fe-4S]+ clusters, suggest why these RSEs remain functional inside fully aerated organisms. In contrast, copper(I) damaged HemN and viperin in vitro as readily as it did fumarase, a known target of copper toxicity inside E. coli. Excess intracellular copper also impaired RSE-dependent biosynthetic processes. These data indicate that RSEs may be targets of copper stress but not of reactive oxygen species.

Escherichia coli Uses a Dedicated Importer and Desulfidase To Ferment Cysteine.

CyuA of Escherichia coli is an inducible desulfidase that degrades cysteine to pyruvate, ammonium, and hydrogen sulfide. Workers have conjectured that its role may be to defend bacteria against the toxic effects of cysteine. However, cyuA sits in an operon alongside cyuP, which encodes a cysteine importer that seems ill suited to protecting the cell from environmental cysteine. In this study, transport measurements established that CyuP is a cysteine-specific, high-flux importer. The concerted action of CyuP and CyuA allowed anaerobic E. coli to employ cysteine as either the sole nitrogen or the sole carbon/energy source. CyuA was essential for this function, and although other transporters can slowly bring cysteine into the cell, CyuP-proficient cells outcompeted cyuP mutants. Cells immediately consumed the ammonia and pyruvate that CyuA generated, with little or none escaping from the cell. The expression of the cyuPA operon depended upon both CyuR, a cysteine-activated transcriptional activator, and Crp. This control is consistent with its catabolic function. In fact, the cyuPA operon sits immediately downstream of the thrABCDEFG operon, which allows the analogous fermentation of serine and threonine; this arrangement suggests that this gene cluster may have moved jointly through the anaerobic biota, providing E. coli with the ability to ferment a limited set of amino acids. Interestingly, both the cyu- and thr-encoded pathways depend upon oxygen-sensitive enzymes and cannot contribute to amino acid catabolism in oxic environments. IMPORTANCE Cysteine is a singularly reactive amino acid; in high concentrations, it can disrupt cytoplasmic metabolism. This phenomenon prompted the view that the cyuPA operon of Escherichia coli serves to detoxify cysteine by degrading it. The present study indicates, however, that the natural purpose of that operon is to provide a concise route of cysteine fermentation. CyuP is the first dedicated cysteine importer to be functionally validated among the bacteria, and CyuA constitutes a cysteine desulfidase. Intriguingly, the CyuA iron-sulfur cofactor is inactivated by oxygen so that cysteine is, uniquely, a carbon source that is usable only in anoxic environments. Presumably, this constraint is tolerable because cysteine is scarce in oxic habitats. It also avoids sulfide release, which could interfere with aerobic respiration. Cysteine joins just serine and threonine as amino acids that E. coli is known to ferment, underscoring that this facultative bacterium is oriented toward the fermentation of carbohydrates.

Escherichia coli induces DNA repair enzymes to protect itself from low-grade hydrogen peroxide stress.

Escherichia coli responds to hydrogen peroxide (H2 O2 ) by inducing defenses that protect H2 O2 -sensitive enzymes. DNA is believed to be another important target of oxidation, and E. coli contains enzymes that can repair oxidative lesions in vitro. However, those enzymes are not known to be induced by H2 O2 , and experiments have indicated that they are not necessary for the cell to withstand natural (low-micromolar) concentrations. In this study, we used H2 O2 -scavenging mutants to impose controlled doses of H2 O2 for extended time. Transcriptomic analysis revealed that in the presence of 1 µM cytoplasmic H2 O2 , the OxyR transcription factor-induced xthA, encoding exonuclease III. The xthA mutants survived a conventional 15-min exposure to even 100 times this level of H2 O2 . However, when these mutants were exposed to 1 µM H2 O2 for hours, they accumulated DNA lesions, failed to propagate, and eventually died. Although endonuclease III (nth) was not induced, nth mutants struggled to grow. Low-grade H2 O2 stress also activated the SOS regulon, and when this induction was blocked, cell replication stopped. Collectively, these data indicate that physiological levels of H2 O2 are a real threat to DNA, and the engagement of the base-excision-repair and SOS systems is necessary to enable propagation during protracted stress.

When anaerobes encounter oxygen: mechanisms of oxygen toxicity, tolerance and defence.

The defining trait of obligate anaerobes is that oxygen blocks their growth, yet the underlying mechanisms are unclear. A popular hypothesis was that these microorganisms failed to evolve defences to protect themselves from reactive oxygen species (ROS) such as superoxide and hydrogen peroxide, and that this failure is what prevents their expansion to oxic habitats. However, studies reveal that anaerobes actually wield most of the same defences that aerobes possess, and many of them have the capacity to tolerate substantial levels of oxygen. Therefore, to understand the structures and real-world dynamics of microbial communities, investigators have examined how anaerobes such as Bacteroides, Desulfovibrio, Pyrococcus and Clostridium spp. struggle and cope with oxygen. The hypoxic environments in which these organisms dwell - including the mammalian gut, sulfur vents and deep sediments - experience episodic oxygenation. In this Review, we explore the molecular mechanisms by which oxygen impairs anaerobes and the degree to which bacteria protect their metabolic pathways from it. The emergent view of anaerobiosis is that optimal strategies of anaerobic metabolism depend upon radical chemistry and low-potential metal centres. Such catalytic sites are intrinsically vulnerable to direct poisoning by molecular oxygen and ROS. Observations suggest that anaerobes have evolved tactics that either minimize the extent to which oxygen disrupts their metabolism or restore function shortly after the stress has dissipated.

How Microbes Defend Themselves From Incoming Hydrogen Peroxide.

Microbes rely upon iron as a cofactor for many enzymes in their central metabolic processes. The reactive oxygen species (ROS) superoxide and hydrogen peroxide react rapidly with iron, and inside cells they can generate both enzyme and DNA damage. ROS are formed in some bacterial habitats by abiotic processes. The vulnerability of bacteria to ROS is also apparently exploited by ROS-generating host defense systems and bacterial competitors. Phagocyte-derived O2- can toxify captured bacteria by damaging unidentified biomolecules on the cell surface; it is unclear whether phagocytic H2O2, which can penetrate into the cell interior, also plays a role in suppressing bacterial invasion. Both pathogenic and free-living microbes activate defensive strategies to defend themselves against incoming H2O2. Most bacteria sense the H2O2via OxyR or PerR transcription factors, whereas yeast uses the Grx3/Yap1 system. In general these regulators induce enzymes that reduce cytoplasmic H2O2 concentrations, decrease the intracellular iron pools, and repair the H2O2-mediated damage. However, individual organisms have tailored these transcription factors and their regulons to suit their particular environmental niches. Some bacteria even contain both OxyR and PerR, raising the question as to why they need both systems. In lab experiments these regulators can also respond to nitric oxide and disulfide stress, although it is unclear whether the responses are physiologically relevant. The next step is to extend these studies to natural environments, so that we can better understand the circumstances in which these systems act. In particular, it is important to probe the role they may play in enabling host infection by microbial pathogens.

How Microbes Evolved to Tolerate Oxygen.

Ancient microbes invented biochemical mechanisms and assembled core metabolic pathways on an anoxic Earth. Molecular oxygen appeared far later, forcing microbes to devise layers of defensive tactics that fend off the destructive actions of both reactive oxygen species (ROS) and oxygen itself. Recent work has pinpointed the enzymes that ROS attack, plus an array of clever protective strategies that abet the well known scavenging systems. Oxygen also directly damages the low-potential metal centers and radical-based mechanisms that optimize anaerobic metabolism; therefore, committed anaerobes have evolved customized tactics that defend these various enzymes from occasional oxygen exposure. Thus a more comprehensive, detailed, and surprising view of oxygen toxicity is coming into view.

During Oxidative Stress the Clp Proteins of Escherichia coli Ensure that Iron Pools Remain Sufficient To Reactivate Oxidized Metalloenzymes.

Hydrogen peroxide (H2O2) is formed in natural environments by both biotic and abiotic processes. It easily enters the cytoplasms of microorganisms, where it can disrupt growth by inactivating iron-dependent enzymes. It also reacts with the intracellular iron pool, generating hydroxyl radicals that can lethally damage DNA. Therefore, virtually all bacteria possess H2O2-responsive transcription factors that control defensive regulons. These typically include catalases and peroxidases that scavenge H2O2 Another common component is the miniferritin Dps, which sequesters loose iron and thereby suppresses hydroxyl-radical formation. In this study, we determined that Escherichia coli also induces the ClpS and ClpA proteins of the ClpSAP protease complex. Mutants that lack this protease, plus its partner, ClpXP protease, cannot grow when H2O2 levels rise. The growth defect was traced to the inactivity of dehydratases in the pathway of branched-chain amino acid synthesis. These enzymes rely on a solvent-exposed [4Fe-4S] cluster that H2O2 degrades. In a typical cell the cluster is continuously repaired, but in the clpSA clpX mutant the repair process is defective. We determined that this disability is due to an excessively small iron pool, apparently due to the oversequestration of iron by Dps. Dps was previously identified as a substrate of both the ClpSAP and ClpXP proteases, and in their absence its levels are unusually high. The implication is that the stress response to H2O2 has evolved to strike a careful balance, diminishing iron pools enough to protect the DNA but keeping them substantial enough that critical iron-dependent enzymes can be repaired.IMPORTANCE Hydrogen peroxide mediates the toxicity of phagocytes, lactic acid bacteria, redox-cycling antibiotics, and photochemistry. The underlying mechanisms all involve its reaction with iron atoms, whether in enzymes or on the surface of DNA. Accordingly, when bacteria perceive toxic H2O2, they activate defensive tactics that are focused on iron metabolism. In this study, we identify a conundrum: DNA is best protected by the removal of iron from the cytoplasm, but this action impairs the ability of the cell to reactivate its iron-dependent enzymes. The actions of the Clp proteins appear to hedge against the oversequestration of iron by the miniferritin Dps. This buffering effect is important, because E. coli seeks not just to survive H2O2 but to grow in its presence.

Escherichia coli K-12 Lacks a High-Affinity Assimilatory Cysteine Importer.

The most direct route by which microbes might assimilate sulfur would be by importing cysteine. However, alone among the amino acids, cysteine does not have well-characterized importers. We determined that Escherichia coli can rapidly import cysteine, but in our experiments, it did so primarily through the LIV ATP-driven system that is dedicated to branched-chain amino acids. The affinity of this system for cysteine is far lower than for Leu, Ile, and Val, and so in their presence, cysteine is excluded. Thus, this transport is unlikely to be relevant in natural environments. Growth studies, transcriptomics, and transport assays failed to detect any high-affinity importer that is dedicated to cysteine assimilation. Enteric bacteria do not contain the putative cysteine importer that was identified in Campylobacter jejuni This situation is surprising, because E. coli deploys ion- and/or ATP-driven transporters that import cystine, the oxidized form of cysteine, with high affinity and specificity. We conjecture that in oxic environments, molecular oxygen oxidizes environmental cysteine to cystine, which E. coli imports. In anoxic environments where cysteine is stable, the cell chooses to assimilate hydrogen sulfide instead. Calculations suggest that this alternative is almost as economical, and it avoids the toxic effects that can result when excess cysteine enters the cell.IMPORTANCE This investigation discovered that Escherichia coli lacks a transporter dedicated to the assimilation of cysteine, an outcome that is in striking contrast to the many transporters devoted to the other 19 amino acids. We ascribe the lack of a high-affinity cysteine importer to two considerations. First, the chemical reactivity of this amino acid is unique, and its poorly controlled import can have adverse consequences for the cell. Second, our analysis suggests that the economics of biosynthesis depend sharply upon whether the cell is respiring or fermenting. In the anoxic habitats in which cysteine might be found, the value of import versus biosynthesis is strongly reduced compared to that in oxic habitats. These studies may explain why bacteria choose to synthesize rather than to import other useful biomolecules as well.

Do reactive oxygen species or does oxygen itself confer obligate anaerobiosis? The case of Bacteroides thetaiotaomicron.

Bacteroides thetaiotaomicron was examined to determine whether its obligate anaerobiosis is imposed by endogenous reactive oxygen species or by molecular oxygen itself. Previous analyses established that aerated B. thetaiotaomicron loses some enzyme activities due to a high rate of endogenous superoxide formation. However, the present study establishes that another key step in central metabolism is poisoned by molecular oxygen itself. Pyruvate dissimilation was shown to depend upon two enzymes, pyruvate:formate lyase (PFL) and pyruvate:ferredoxin oxidoreductase (PFOR), that lose activity upon aeration. PFL is a glycyl-radical enzyme whose vulnerability to oxygen is already understood. The rate of PFOR damage was unaffected by the level of superoxide or peroxide, showing that molecular oxygen itself is the culprit. The cell cannot repair PFOR, which amplifies the impact of damage. The rates of PFOR and fumarase inactivation are similar, suggesting that superoxide dismutase is calibrated so the oxygen- and superoxide-sensitive enzymes are equally sensitive to aeration. The physiological purpose of PFL and PFOR is to degrade pyruvate without disrupting the redox balance, and they do so using catalytic mechanisms that are intrinsically vulnerable to oxygen. In this way, the anaerobic excellence and oxygen sensitivity of B. thetaiotaomicron are two sides of the same coin.

Cystine import is a valuable but risky process whose hazards Escherichia coli minimizes by inducing a cysteine exporter.

The structure of free cysteine makes it vulnerable to oxidation by molecular oxygen; consequently, organisms that live in oxic habitats have acquired the ability to import cystine as a sulfur source. We show that cystine imported into Escherichia coli can transfer disulfide bonds to cytoplasmic proteins. To minimize this problem, the imported cystine is rapidly reduced. However, this conversion of cystine to cysteine precludes product inhibition of the importer, so cystine import continues into cells that are already sated with cysteine. The burgeoning cysteine pool is itself hazardous, as cysteine promotes the formation of reactive oxygen species, triggers sulfide production and competitively inhibits a key enzyme in the isoleucine biosynthetic pathway. The Lrp transcription factor senses the excess cysteine and induces AlaE, an export protein that pumps cysteine back out of the cell until transcriptional controls succeed in lowering the amount of the importer. While it lasts, the overall phenomenon roughly doubles the NADPH demand of the cell. It comprises another example of the incompatibility of the reduced cytoplasms of microbes with the oxic world in which they dwell. It also reveals one natural source of cytoplasmic disulfide stress and sheds light on a role for broad-spectrum amino acid exporters.

A conserved motif liganding the [4Fe-4S] cluster in [4Fe-4S] fumarases prevents irreversible inactivation of the enzyme during hydrogen peroxide stress.

Organisms have evolved two different classes of the ubiquitous enzyme fumarase: the [4Fe-4S] cluster-containing class I enzymes are oxidant-sensitive, whereas the class II enzymes are iron-free and therefore oxidant-resistant. When hydrogen peroxide (H2O2) attacks the most-studied [4Fe-4S] fumarases, only the cluster is damaged, and thus the cell can rapidly repair the enzyme. However, this study shows that when elevated levels of H2O2 oxidized the class I fumarase of the obligate anaerobe Bacteroides thetaiotaomicron (Bt-Fum), a hydroxyl-like radical species was produced that caused irreversible covalent damage to the polypeptide. Unlike the fumarase of oxygen-tolerant bacteria, Bt-Fum lacks a key cysteine residue in the typical "CXnCX2C″ motif that ligands [4Fe-4S] clusters. Consequently H2O2 can access and oxidize an iron atom other than the catalytic one in its cluster. Phylogenetic analysis showed that certain clades of bacteria may have evolved the full "CXnCX2C″ motif to shield the [4Fe-4S] cluster of fumarase. This effect was reproduced by the construction of a chimeric enzyme. These data demonstrate the irreversible oxidation of Fe-S cluster enzymes and may recapitulate evolutionary steps that occurred when microorganisms originally confronted oxidizing environments. It is also suggested that, if H2O2 is generated within the colon as a consequence of inflammation or the action of lactic acid bacteria, the inactivation of fumarase could potentially impair the central fermentation pathway of Bacteroides species and contribute to gut dysbiosis.

Evolutionary adaptations that enable enzymes to tolerate oxidative stress.

Biochemical mechanisms emerged and were integrated into the metabolic plan of cellular life long before molecular oxygen accumulated in the biosphere. When oxygen levels finaly rose, they threatened specific types of enzymes: those that use organic radicals as catalysts, and those that depend upon iron centers. Nature has found ways to ensure that such enzymes are still used by contemporary organisms. In some cases they are restricted to microbes that reside in anoxic habitats, but in others they manage to function inside aerobic cells. In the latter case, it is frequently true that the ancestral enzyme has been modified to fend off poisoning. In this review we survey a range of protein adaptations that permit radical-based and low-potential iron chemistry to succeed in oxic environments. In many cases, accessory domains shield the vulnerable radical or metal center from oxygen. In others, the structures of iron cofactors evolved to less oxidizable forms, or alternative metals replaced iron altogether. The overarching view is that some classes of biochemical mechanism are intrinsically incompatible with the presence of oxygen. The structural modification of target enzymes is an under-recognized response to this problem.

Where in the world do bacteria experience oxidative stress?

Reactive oxygen species - superoxide, hydrogen peroxide and hydroxyl radicals - have long been suspected of constraining bacterial growth in important microbial habitats and indeed of shaping microbial communities. Over recent decades, studies of paradigmatic organisms such as Escherichia coli, Salmonella typhimurium, Bacillus subtilis and Saccharomyces cerevisiae have pinpointed the biomolecules that oxidants can damage and the strategies by which microbes minimize their injuries. What is lacking is a good sense of the circumstances under which oxidative stress actually occurs. In this MiniReview several potential natural sources of oxidative stress are considered: endogenous ROS formation, chemical oxidation of reduced species at oxic-anoxic interfaces, H2 O2 production by lactic acid bacteria, the oxidative burst of phagocytes and the redox-cycling of secreted small molecules. While all of these phenomena can be reproduced and verified in the lab, the actual quantification of stress in natural habitats remains lacking - and, therefore, we have a fundamental hole in our understanding of the role that oxidative stress actually plays in the biosphere.

Quantification of Hydrogen Sulfide and Cysteine Excreted by Bacterial Cells.

Bacteria release cysteine to moderate the size of their intracellular pools. They can also evolve hydrogen sulfide, either through dissimilatory reduction of oxidized forms of sulfur or through the deliberate or inadvertent degradation of intracellular cysteine. These processes can have important consequences upon microbial communities, because excreted cysteine autoxidizes to generate hydrogen peroxide, and hydrogen sulfide is a potentially toxic species that can block aerobic respiration by inhibiting cytochrome oxidases. Lead acetate strips can be used to obtain semiquantitative data of sulfide evolution (Oguri et al., 2012). Here we describe methods that allow more-quantitative and discriminatory measures of cysteine and hydrogen sulfide release from bacterial cells. An illustrative example is provided in which Escherichia coli rapidly evolves both cysteine and sulfide upon exposure to exogenous cystine (Chonoles Imlay et al., 2015; Korshunov et al., 2016).

Endogenous superoxide is a key effector of the oxygen sensitivity of a model obligate anaerobe.

It has been unclear whether superoxide and/or hydrogen peroxide play important roles in the phenomenon of obligate anaerobiosis. This question was explored using Bacteroides thetaiotaomicron, a major fermentative bacterium in the human gastrointestinal tract. Aeration inactivated two enzyme families-[4Fe-4S] dehydratases and nonredox mononuclear iron enzymes-whose homologs, in contrast, remain active in aerobic Escherichia coli Inactivation-rate measurements of one such enzyme, B. thetaiotaomicron fumarase, showed that it is no more intrinsically sensitive to oxidants than is an E. coli fumarase. Indeed, when the E. coli enzymes were expressed in B. thetaiotaomicron, they no longer could tolerate aeration; conversely, the B. thetaiotaomicron enzymes maintained full activity when expressed in aerobic E. coli Thus, the aerobic inactivation of the B. thetaiotaomicron enzymes is a feature of their intracellular environment rather than of the enzymes themselves. B. thetaiotaomicron possesses superoxide dismutase and peroxidases, and it can repair damaged enzymes. However, measurements confirmed that the rate of reactive oxygen species production inside aerated B. thetaiotaomicron is far higher than in E. coli Analysis of the damaged enzymes recovered from aerated B. thetaiotaomicron suggested that they had been inactivated by superoxide rather than by hydrogen peroxide. Accordingly, overproduction of superoxide dismutase substantially protected the enzymes from aeration. We conclude that when this anaerobe encounters oxygen, its internal superoxide levels rise high enough to inactivate key catabolic and biosynthetic enzymes. Superoxide thus comprises a major element of the oxygen sensitivity of this anaerobe. The extent to which molecular oxygen exerts additional direct effects remains to be determined.