The Diversity of Microbial Responses to Nitric Oxide and Agents of Nitrosative Stress: Close Cousins but Not Identical Twins

Lesley A.H. Bowman1, Samantha McLean1, Robert K. Poole1 and Jon M. Fukuto2

1Department of Molecular Biology and Biotechnology, The University of Sheffield, Sheffield, United Kingdom 2Department of Chemistry, Sonoma State University, Rohnert Park, California, USA

ABSTRACT

Nitric oxide and related nitrogen species (reactive nitrogen species) now occupy a central position in contemporary medicine, physiology, biochemistry, and microbiology. In particular, NO plays important antimicrobial defenses in innate immunity but microbes have evolved intricate NO-sensing and defense mechanisms that are the subjects of a vast literature. Unfortunately, the burgeoning NO literature has not always been accompanied by an understanding of the intricacies and complexities of this radical and other reactive nitrogen species so that there exists confusion and vagueness about which one or more species exert the reported biological effects. The biological chemistry of NO and derived/ related molecules is complex, due to multiple species that can be generated from NO in biological milieu and numerous possible reaction targets. Moreover, the fate and disposition of NO is always a function of its biological environment, which can vary significantly even within a single cell. In this review, we consider newer aspects of the literature but, most importantly, consider the underlying chemistry and draw attention to the þ distinctiveness of NO and its chemical cousins, nitrosonium (NO ),

nitroxyl (NO , HNO), peroxynitrite (ONOO ), nitrite (NO2 ), and nitrogen dioxide (NO2). All these species are reported to be generated in biological systems from initial formation of NO (from nitrite, NO synthases, or other sources) or its provision in biological experiments (typically from NO gas, S-nitrosothiols, or NO donor compounds). The major targets of NO and nitrosative damage (metal centers, thiols, and others) are reviewed and emphasis is given to newer “-omic” methods of unraveling the complex repercussions of NO and nitrogen oxide assaults. Microbial defense mechanisms, many of which are critical for pathogenicity, include the activities of hemoglobins that enzymically detoxify NO (to nitrate) and NO reductases and repair mechanisms (e.g., those that reverse S-nitrosothiol formation). Microbial resistance to these stresses is generally inducible and many diverse transcriptional regulators are involved—some that are secondary sensors (such as Fnr) and those that are “dedicated” (such as NorR, NsrR, NssR) in that their physiological function appears to be detecting primarily NO and then regulating expression of genes that encode enzymes with NO as a substrate. Although generally harmful, evidence is accumulating that NO may have beneficial effects, as in the case of the squid-Vibrio light-organ symbiosis, where NO serves as a signal, antioxidant, and specificity determinant. Progress in this area will require a thorough understanding not only of the biology but also of the underlying chemical principles.

Abbreviations ...... 137 1. Overview ...... 138 2. Historical Perspective ...... 139 3. Origins of Reactive Nitrosative Species in Biology ...... 140 3.1. Nitrite Reduction and Denitrification ...... 140 3.2. Nitrate-Derived Stress ...... 141 3.3. NO Synthases and the Nitrosative Burst ...... 142 3.4. Non-NOS Sources of NO in Microbes ...... 145 3.5. The Combined Reactive Species Response ...... 147 4. The Biological Chemistry of NO and Related Species ...... 148 4.1. NO, Its Redox Chemistry, and NO2 ...... 148 4.2. The Reaction of NO with Superoxide Anion ...... 151 4.3. Reaction with Metal Centers ...... 152 4.4. Products of NO Reduction ...... 153 4.5. The Reactions of HNO with Biological Targets ...... 154 5. Laboratory Methods ...... 156 5.1. The Use of Nitrogen Oxide Donors ...... 156 5.2. NO ...... 157 5.3. S-Nitrosothiols ...... 159 5.4. Other Donors ...... 162 5.5. HNO Donors ...... 162 NITRIC OXIDE AND AGENTS OF NITROSATIVE STRESS 137

5.6. Use of ONOO ...... 163 5.7. NO2 ...... 164 5.8. NO2 ...... 166 5.9. Other Nitrogen Oxides ...... 166 6. Bacterial Responses to RNS: Effectors and Regulators ...... 166 6.1. Targets of RNS in Microorganisms ...... 166 6.2. Microbial Defenses: The Microbe Strikes Back ...... 167 6.3. Microbial Globins ...... 168 6.4. NO and RNS Reductases ...... 174 6.5. Other Proteins Implicated in NO Tolerance ...... 176 6.6. Beneficial Effects of NO in Microbial Symbioses ...... 177 6.7. Microbial Responses to ONOO Stress ...... 178 7. Microbial Sensing of NO and Gene Regulation ...... 181 7.1. Introduction ...... 181 7.2. Fnr ...... 181 7.3. NorR ...... 182 7.4. NsrR ...... 182 7.5. Others (DOS, FixL, GCS) ...... 183 8. Global and Systems Approaches to Understanding Responses to NO andRNS...... 183 8.1. Methodology ...... 184 8.2. Outcomes from Global Transcriptomic Approaches ...... 188 8.3. Responses of Other Microbes to RNS ...... 193 8.4. Proteomics ...... 195 9. Conclusions ...... 197 References ...... 198

ABBREVIATIONS

CPTIO 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-l- oxyl-3-oxide EPR electron paramagnetic resonance þ FNR ferredoxin-NADP reductase Fnr fumarate and nitrate reduction regulator, encoded by fnr gene GSH glutathione GSNO S-nitrosoglutathione GSSG glutathione disulfide (oxidized glutathione) GTN glyceryltrinitrate Hcy homocysteine iCAT isotope-coded affinity tag iTRAQ isobaric tags for relative and absolute quantitation NHE normal hydrogen electrode 138 LESLEY A.H. BOWMAN ET AL.

NOC-5 1-hydroxy-2-oxo-3-(3-aminopropyl)-3-isopropyl-1- triazene NOC-7 1-hydroxy-2-oxo-3-(N-methyl-3-aminopropyl)-3- methyl-1-triazene NOR-3 ()-(E)-ethyl-2-[(E)-hydroxyimino]-5-nitro-3- hexeamide DEA/NO diethylamine NONOate DETA diethylenetriamine NONOate NONOate norV, norW genes involved in nitric oxide reduction and its regulation (norR) NOS NO synthase PTIO 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3 oxide RNS reactive nitrogen species SNAP S-nitroso-N-acetyl-D,L-penicillamine SNO S-nitrosothiol SNOCAP S-nitrosothiol capture SNP sodium nitroprusside SOD superoxide dismutase

1. OVERVIEW

Nitric oxide (NO) is a small and freely diffusible species once known primarily as a toxic component of air pollution. In physiology and biochemistry, it was well known as a poison and ligand for heme proteins. The discovery of the enzymic generation of NO in mammalian systems and its cell signaling functions represents a watershed moment in the evolution of our understanding of biological signal transduction. The importance of NO as a molecule of real biological significance cannot, however, have escaped the attention of any microbiologist, although the realization is relatively recent. To illustrate this point, consider that a multi-authored, edited book Microbial Gas Metabolism published in 1985 (Poole and Dow, 1985) contained only three index entries for ‘nitric oxide’, namely, ‘denitrification’, ‘mass spectrum cracking pattern’ and ‘reaction with cytochrome d.’ The general topics addressed by these specific terms illustrate well the dominant interests of microbiologists in NO a quarter of a century ago—NO as a possible (but far from proven) intermediate in the microbe- catalyzed conversion of nitrate to dinitrogen, the biochemical analysis and NITRIC OXIDE AND AGENTS OF NITROSATIVE STRESS 139 detection of the gas, and the use of NO as an experimental tool in the study of heme proteins. In the intervening years, a large literature has grown up around NO and related species, fueled by the recognition that NO plays important antimicrobial defenses in innate immunity and that, in turn, and not unexpect- edly, microbes have intricate NO-sensing and defense mechanisms. Unfortunately, the burgeoning publication of information on NO has not always been accompanied by an understanding of the intricacies and complexities of NO and other “agents of nitrosative stress” so that there exist some confusion and vagueness about which one or more species exert the reported, interesting biological effects. There is, for example, a tendency for authors to write “NO” when it is actually meant “in a generic sense.” It should not be necessary to write “NO radical” to eliminate the possibil- þ ity that one is actually meaning NO or some other congener. There is only one NO. Butler and Nicholson (2003) suggest that “there are not many small molecules about which a whole book could be written,” although in biology, we doubt the veracity of this. Consider the gases oxygen, carbon dioxide, methane, and nitrogen. Nevertheless, we agree that NO is certainly one such species: several books could be, and have been, written on this one gas. As a result, we are forced to be brief and focused when revisiting some familiar aspects of NO in microbiology and apologize for any over- sights or omissions. Our objective in this review is not only to review newer aspects of this vast literature but, most importantly, to consider the underlying chemistry and draw attention to the distinctive chemistry of NO and þ its chemical cousins, NO ,NO , HNO, ONOO ,NO2 , and NO2.

2. HISTORICAL PERSPECTIVE

The modern era of NO research may be considered to begin in the 1980s when NO was identified as the endothelium-derived relaxing factor (EDRF). This remarkable story and its culmination in a Nobel Prize and the designation of NO as “molecule of the year” by Science in 1992 are covered well elsewhere, especially in accounts by the laureates (Furchgott, 1999; Ignarro, 1999, 2005; Murad, 1999). NO was also shown to participate in the regulation of the nervous and immune systems, and it was soon discovered that NO also plays a vital role in the resistance of mammalian hosts to microbial infections. Activated macrophages were shown to form nitrite and nitrate from arginine (Iyengar et al., 1987; Marletta et al., 1988) 140 LESLEY A.H. BOWMAN ET AL. via the formation of NO (Stuehr et al., 1989) and to have a powerful cyto- static effect in vitro on the fungal pathogen Cryptococcus neoformans (Granger et al., 1986). Activated macrophages also destroyed the intracellular parasite Leishmania major in vitro by an L-arginine-dependent mechanism (Green et al., 1990) and mice infected with L. major developed exacerbated disease when the lesions were injected with the NOS inhibitor L-NMMA, providing the first compelling evidence for the attenuation by NO of an infectious microorganism in vivo (Liew et al.,1990). A direct role for NO against intracellular bacteria was soon established, initially with Mycobacterium bovis (Flesch and Kaufmann, 1991). Shortly after, we showed that NO dramatically upregulated expression of the Escherichia coli flavohemoglobin (Poole et al., 1996), and an enzymic function in NO detoxification was demonstrated by Gardner et al. (1998). A globin mutant was NO sensitive unambiguously demonstrating a physiological role (Membrillo-Hernández et al., 1999). In murine macrophages, NO was shown to have a role in bacterial clearance (Shiloh et al., 1999; Vazquez- Torres et al., 2000). Also, flavohemoglobin-catalyzed NO detoxification by Salmonella enterica serovar Typhimurium protected the bacterium from NO-mediated killing in human macrophages (Stevanin et al., 2002). Flavohemoglobin (Hmp) was shown to catalyze the reaction of NO with oxygen to give innocuous nitrate via a dioxygenase (Gardner et al., 1998, 2000, 2006) or denitrosylase (Hausladen et al., 1998a, 2001) mechanism, and further gene reporter experiments showed that hmp gene transcription is activated on exposure of bacteria to NO or nitrosating agents (Poole et al., 1996; Poole and Hughes, 2000; Gilberthorpe et al., 2007). Mutants were used to demonstrate unequivocally the key role of flavohemoglobin in defense against NO not only in vitro (Membrillo-Hernández et al., 1999) but also in vivo (Stevanin et al., 2002). Other globins intensively studied (Wu et al., 2003) now include the two globins in each of Mycobacterium tuberculosis (Couture et al., 1999; Pathania et al., 2002) and Campylobacter jejuni (Lu et al., 2007a,b). These are covered further in Section 8.3.

3. ORIGINS OF REACTIVE NITROSATIVE SPECIES IN BIOLOGY

3.1. Nitrite Reduction and Denitrification

The major source of NO in man is via the action of NOS (see Section 3.3), but other sources should be briefly considered. Nitrite is protonated under NITRIC OXIDE AND AGENTS OF NITROSATIVE STRESS 141 acidic conditions (as in the stomach) and the resulting nitrous acid will yield NO and other nitrogen oxides; the beneficial effects of acidified nitrite in killing ingested pathogens, gastric mucosal integrity and other effects are discussed elsewhere (Lundberg et al., 2004). Acidified nitrite is sometimes, but perhaps not ideally, used as a source of NO in bacterial experiments in vitro (as covered elsewhere in this review). The bacterial reduction of nitrite is a key reaction in anaerobic bacterial metabolism and the subject of an immense literature (Potter et al., 2001; Lundberg et al., 2004). There are three classes of nitrite reduction. In the first (denitrification), reduction of nitrite to NO is catalyzed by either copper-containing NirK or cytochrome cd1 nitrite reductase, NirS. The periplasmic enzymes involved have been extensively characterized and are outside the scope of this contribution (for an authoritative review, see Potter et al., 2001). Second, when bacteria utilize nitrite as a terminal electron acceptor, NADH- and siroheme-dependent reduction is the primary pathway, encoded in E. coli by the nirBDCcydG operon. Third, nitrite can be reduced to ammonia by a widely distributed cytochrome c nitrite reductase Nrf (Potter et al., 2001), which can also, however, produce low levels of NO when nitrite is in excess under anoxic conditions (Corker and Poole, 2003).

3.2. Nitrate-Derived Stress

Nitrate is often regarded mainly as a water pollutant and its presence in the diet of man is seen as an unfortunate consequence of the use of nitrogen fertilizers in agriculture. Increasingly stringent regulations to limit nitrate intake suggest that nitrate has wholly undesirable effects including formation of N-nitrosamines, infantile methemoglobinemia, carcinogenesis, and possibly teratogenesis (McKnight et al., 1999). However, nitrosamine formation is via nitrite (nitrosation chemistry) not nitrate; nitrite is the cul- prit. An alternative view is that the products of nitrate metabolism have beneficial effects, especially in host defense. The argument is made elsewhere (Lundberg et al., 2004) that nitrate-reducing commensal bacteria play a symbiotic role in mammalian nitrate reduction to nitrite, NO, and other products. Nitrate reduction to nitrite and thence to NO are the topics of a vast literature, beyond our scope (for a review, see Lundberg, 2008). It is clear, though, that large amounts of NO and other reactive nitrogen species (RNS) are generated in vivo from salivary nitrite in the acidic stomach (Benjamin et al., 1994) and may contribute to killing of ingested pathogens (Lundberg et al., 2004). Depending on nitrite concentration in 142 LESLEY A.H. BOWMAN ET AL. the saliva (McKnight et al., 1999), the NO concentration in the stomach headspace gas may be around 20 ppm (McKnight et al., 1997).

3.3. NO Synthases and the Nitrosative Burst

3.3.1. The NOS Family

NO synthases (NOSs) are highly regulated multidomain metalloenzymes that catalyze the conversion of L-arginine (L-Arg) to L-citrulline and NO with the consumption of NADPH and O2. They were first identified in mammals, and three forms are identified: endothelial NOS (eNOS or NOSIII), neuronal NOS (nNOS or NOSI), and inducible NOS (iNOS or NOSII) (reviewed in Alderton et al., 2001). The nitrosative burst, triggered by pathogenic agonists and inflammatory mediators (Lowenstein et al., 1993), succeeds the oxidative burst and is mediated by NO production following activation of iNOS. The first two isoforms are constitutively expressed and are calcium dependent, whereas iNOS is stimulated to produce NO at markedly higher levels than the constitutive isoforms following infection (Lowenstein and Padalko, 2004). iNOS is most important in a microbial context and is known to occur in a wide variety of stimulatable cells such as macrophages, neutrophils, vascular smooth cells, and glial cells in the CNS (Bogdan, 2001). On microbial infection, the NO produced has diverse, apparently conflicting functions; on the one hand, NO exerts antimicrobial and anti-inflammatory host defense effects and, on the other, proinflammatory and cytotoxic activities (Fang, 2004). The host defense function is exemplified by the effects of NO in microbial infections, whereas NO-mediated inflammation and pathogenesis are known in certain diseases including arthritis, encephalitis, ulcerative colitis, and viral infections. These enzymes are homodimeric with each monomer containing an N- terminal oxygenase domain with binding sites for heme, L-arginine, and tetrahydrobiopterin (H4B) and a C-terminal reductase domain, which has binding sites for FMN, FAD, and NADPH (Stuehr et al., 2009). The reductase domain transfers electrons sourced from NADPH via flavin carriers to heme in the oxygenase domain. This drives the oxidation of L-arginine in þ the presence of O2 to NO, citrulline, and NADP (Daff, 2010). Electron flow is strictly between the reductase domain of one monomer and the oxygenase domain of the other (Siddhanta et al., 1996). As iNOS is confined to the cytoplasm, NO must diffuse to the phagosome to react with internalized microorganisms. Unlike O2 NITRIC OXIDE AND AGENTS OF NITROSATIVE STRESS 143 generation, the onset of NO production is late in the defensive process, starting around 8 h postinfection as demonstrated in murine macrophages challenged with S. enterica (Eriksson et al., 2003). NO makes a significant contribution to the host’s innate immune response, exemplified by a recent study revealing that macrophages derived from murine bone marrow generate an approximately 2-log reduction in C. jejuni viability relative to iNOS mutants over a 24-h period (Iovine et al., 2008). In a recent example of the intricacies of iNOS function, a newborn mouse model (Mittal et al., 2010) of meningitis revealed that infection with E. coli K1 resulted in iNOS expression in the brain. iNOS / mice, however, were resistant to infection and showed normal brain morphology and a reduced inflammatory response. An iNOS inhibitor (ami- noguanidine) also prevented meningitis and brain damage, and further, peritoneal macrophages and polymorphonuclear leukocytes from such mutant mice showed enhanced killing of bacteria. These data suggest that NO from iNOS is actually beneficial for E. coli K1 survival in the macrophage and suggests a therapy for neonatal meningitis.

3.3.2. Bacterial NOS

NOS enzymes are now also recognized in several bacteria (Table 1) and, more controversially, plants (for a review, see Wilson et al., 2008). The prokaryotic NOS enzymes (Crane et al., 2010) are in many ways similar to their mammalian counterparts, catalyzing the conversion of L-Arg to NO o via the intermediate N -hydroxy-L-arginine (NOHA), but the role(s) of the NO so formed is far less clear. In 1994, the first report of a bacterial NOS-like activity was published (reviewed in Crane et al., 2010). However, the first definitive evidence for NOS-like proteins came from genome mining just over 10 years ago, revealing bacterial ORFs with high sequence similarity to mammalian NOS. Most recent data suggest that bacterial and mammalian NOS enzymes have similar reactivities with almost identical catalytic active sites. This has greatly facilitated research into the core features of all NOS proteins because many bacterial NOS are readily expressed in E. coli and provide protein for crystallographic, various spectroscopic, and kinetic studies. The main differences between NOS of mammals and bacteria reside in cofactor specificity and the nature of the reductase partners for these enzymes (Crane et al., 2010). Not all bacterial NOS contain the pterin cofactor (tetrahydrobiopterin, H4B) associated with mammalian NOS. Table 1 Bacterial NO synthasesa.

Biological function Reductase Comments Referenceb

Nocardia species First report of bacterial Chen and Rosazza NOS, but no obvious NOS (1994, 1995) gene homologue Lactobacillus No obvious NOS gene Morita et al. (1997) fermentum homologue Salmonella No obvious NOS gene Choi et al. (2000) enterica homologue Typhimurium Staphylococcus NO or NOS protect cells from NOS homologue Hong et al. (2003); aureus H2O2 Gusarov and Nudler (2005) Deinococcus 1. Reaction with tryptophan Active with surrogate Adak et al. (2002b); radiodurans tRNA synthetase II to form mammalian reductase Buddha et al. (2004); 4-nitro-Trp-tRNATrp , involved Patel et al. (2009) in protein or secondary metabolite synthesis? 2. Response to UV radiation exposure, with NO upregulating transcription of growth factor? Bacillus subtilis, 1. NO or NOS protects cells Dedicated reductase not First crystal structure of a Gusarov and Nudler Bacillus anthracis from H2O2 required (?) bacterial NOS (2005); Shatalin et al. 2. Protection against (2008); Gusarov et al. antibiotics (see text) (2009) Streptomyces NOS encoded on No obvious reductase Kers et al. (2004) species, S. pathogenicity island associated partner proteins turgidiscabies with potato scab disease. NOS encoded by NOS involved in thaxtomin (toxin) pathogenicity island production (see text) Sorangium Covalently bound Agapie et al. (2009) cellulosum unique reductase aNOSs have also been reported in eukaryotic microbes, notably the protozoa Entamoeba histolytica and Toxoplasma gondii, but neither genome contains an NOS homologue (Crane et al., 2010). bFor further details, see Crane et al. (2010). NITRIC OXIDE AND AGENTS OF NITROSATIVE STRESS 145

In the latter enzymes, H4B delivers electrons to the active site for oxygen activation in two steps. In the first, oxygen bound to heme is activated by a single electron transfer, and in the second (final) stage of the reaction, NO is liberated. The mechanism of action of the mammalian enzymes is beyond our scope but well covered in reviews (Daff, 2010). It is worth not- ing, however, that a common feature of bacterial NOS is that the NO dissociation rate is 15- to 25-fold slower than in the mammalian enzyme (Adak et al., 2002a; Wang et al., 2004). This favors oxidation of NO to more reactive species raising the intriguing possibility that synthesis of these alternative species (HNO, NO ) is the true biological function, as discussed below. Nonetheless, NO does appear to be the major product in at least three cases (Johnson et al., 2008; Shatalin et al., 2008; Patel et al., 2009). A case of special interest in the context of the present review is that of Streptomyces turgidiscabies, where the NOS is encoded on a pathogenicity island associated with potato scab disease (Crane et al., 2010). NOS has been shown by mutation studies to be involved in production of thaxtomin (a plant toxin), and it is thought that NOS might be involved in a biosynthetic nitration reaction (Johnson et al., 2008). Indeed, a feeding study with 15N-Arg showed that the thaxtomin nitro group nitrogen does originate from the terminal guanidinium nitrogen of Arg, strongly implicating an NOS activity. Importantly, however, NO will not itself react directly to nitrate substrates such as the tryptophanyl moiety of thaxtomin, whereas þ þ oxidation products of NO (NO ,NO2 , ONOO ,NO2; see Section 4) are known to nitrate aromatic groups (Hughes, 2008). What are the potential targets of endogenously generated NO and related reactive species in bacteria? The examples in Table 2 include transcription factors, biosynthetic enzymes, metalloproteins, and kinases. How- ever, the true biological roles of NOS-derived NO in microbes remain elusive. The fact that NOS enzymes are restricted to certain species and genera suggests a complexity that we do not yet understand.

3.4. Non-NOS Sources of NO in Microbes

Classically, NO has been regarded as a mammalian signaling molecule or a component of the host’s innate immune response to infection. However, a number of papers have demonstrated that bacteria also have the capacity to synthesize NO. This ability was first demonstrated by Hollocher and others: bacteria grown anaerobically with nitrate produced NO from nitrite and the NO was detected by nitrosation of 2,3-diaminophthalene. Table 2 Bacterial targets of NO and nitrosative stress.

Target Basis Examples Selected examples

Heme cofactors Iron nitrosylation Hemes of cytochromes, globins Fu et al. (2009); Pixton et al. (2009) Diverse S-nitrosation Binding of NO moiety 5S-nitrosylated proteins in Qu et al. (2011) events to Cys residue to give Helicobacter pylori S-nitrosothiol 10S-nitrosylated proteins in E. coli Brandes et al. (2007) 29S-nitrosylated proteins in Rhee et al. (2005) M. tuberculosis Unidentified S-nitrosation events in Wang et al. (2011) Moraxella catarrhalis Free and Zn-bound Cys thiols in Bourret et al. (2011) Borrelia burgdorferi Transcription factors Heme binding DosS/DosT Kumar et al. (2007) and sensor kinases Shewanella oneidensis H-NOX- Price et al. (2007) histidine kinase pair SNO formation E. coli OxyR Hausladen et al. (1996) Fe–S cluster reaction E. coli SoxR, Fnr Ding and Demple (2000); with NO Cruz-Ramos et al. (2002); Landry et al. (2010); Smith et al. (2010), for an overview, see Tonzetich et al. (2010) Actin Actin nitrosation GSNO nitrosates key proteins Flamant et al. (2011) involved in S. flexneri invasion, perhaps actin and GTPase Outer membrane SNO formation Bacillus subtilis Morris et al. (1984) proteins NITRIC OXIDE AND AGENTS OF NITROSATIVE STRESS 147

The responsible enzymes have been identified as nitrite reductase (Corker and Poole, 2003) or nitrate reductase (Ji and Hollocher, 1989; Gilberthorpe and Poole, 2008). There are parallels with the recently described ability of mammalian neuroglobin to generate NO from nitrite, which is suggested to be a primordial hypoxia- and redox-regulated source of NO for physiological functions (Tiso et al., 2011). The generation of NO by Salmonella has been demonstrated not only in vitro but also inside infected cancer cells and in implanted tumors in live mice using mic- rosensors. In strains mutated in hmp and norV, the bacteria generated more NO and killed cancer cells more effectively in a nitrate-dependent manner (Barak et al., 2010).

3.5. The Combined Reactive Species Response

ROS and RNS act in concert, exemplified by murine macrophages doubly immunodeficient in iNOS and NAPDH oxidase (Phox) that were completely unable to quash S. enterica growth (Vazquez-Torres et al., 2000). In other words, the roles of iNOS and Phox are nonredundant (Nathan and Shiloh, 2000). An earlier study also clearly demonstrated syn- ergistic killing of E. coli by the combination of NO and H2O2 (Pacelli et al., 1995). Additionally, NO and O2 can rapidly react to form the highly toxic species peroxynitrite (ONOO-) (Reaction 5), which will be discussed in Section 4.2. On account of diffusion constraints, particularly the negative charge on O2 ,ONOO generation tends to colocalize with NADPH oxidase. Peroxynitrite is often stated to be the principal nitrating agent for tyrosine residues in proteins, yet the yield in vitro of nitrated tyrosine at pH 7.4 is less when O2 and NO are cogenerated than when a bolus of ONOO is given. Goldstein et al. (2000) demonstrated that maximal nitrosation of tyrosine occurred when NO and O2 fluxes were equal. Although ONOO is formed in vivo from these two radicals, and the temporal concurrence of these precursors, as is found at inflammatory sites, favors ONOO formation in vivo,ONOO itself is not likely to be the nitrating species. It is generally thought that decomposition of the conjugate acid, peroxynitrous acid (ONOOH), gives the two one-electron oxidants nitrogen dioxide (NO2) and hydroxyl radical (HO ), either of which is capable of oxidizing tyrosine to the tyrosyl radical. Subsequent addition of NO2 to the tyrosyl radical then gives nitrotyrosine (e.g., Gunaydin and Houk, 2009). Thus, this mechanism of tyrosine nitration is highly dependent on the presence of NO2, and since NO2 can be formed from peroxynitrite-independent pathways, tyrosine nitration is not necessarily a marker for ONOO formation. 148 LESLEY A.H. BOWMAN ET AL.

4. THE BIOLOGICAL CHEMISTRY OF NO AND RELATED SPECIES

4.1. NO, Its Redox Chemistry, and NO2

The biological chemistry of NO and derived/related molecules is potentially complex due to a multitude of species that can be generated from NO in a biological milieu and the multiple possible reaction targets associated with these derived species (for an overview, see Lehnert and Scheidt, 2010). Moreover, the fate and disposition of NO is always a function of its biochemical environment, which can vary significantly even within a single cell. The redox relationship between NO and related/ derived nitrogen oxides is given in Fig. 1 and all of the species shown have been reported to be generated in biological systems from initial formation of NO. Herein will be described the chemical properties of NO and derived agents important to their biological functions and effects. The biological utility of NO is based on its unique chemistry. First and foremost, NO has an unpaired electron and is, therefore, considered to be a free radical (for the sake of simplicity, we have adopted the definition of Halliwell and Gutteridge (2007)) for the term “free radical” which is any species that can exist independently (i.e., free) and contains one or more unpaired electron. The presence of an unpaired electron is immediately evident from the Lewis dot depiction for NO (Fig. 2). However, the unpaired electron is not associated solely with the nitrogen atom of NO (as indicated by the Lewis structure), but rather is delocalized throughout

− − − − − +e +e +e +e +2e − + + NO3 +2H NO NO HNO H NO NH OH NH (+ H O) − − − − − 2 − − 2 − − 3 2 − − −e e e e 2e −e +e H O −H O 2 H2O 2 − +e − + NO2 − NO2 +2H −e

Figure 1 Redox relationship between biologically relevant nitrogen oxides. Note: protons omitted from several processes for the sake of simplification.

N + O N O

Figure 2 Lewis dot depiction of NO. NITRIC OXIDE AND AGENTS OF NITROSATIVE STRESS 149 the molecule in a p* antibonding orbital as indicated by the molecular orbital diagram (Fig. 3). As discussed below, the free radical nature of NO as well as the orbital location on the unpaired electron are important factors in the biological chemistry of NO. Although the term free radical is often associated with extreme reactivity (e.g., strong oxidant), this is not the case with NO. The lack of oxidizing capability of NO is evidenced by its relatively low þ reduction potential (E ¼0.55 V vs. NHE for the NO,H /HNO couple; Bartberger et al., 2002; Shafirovich and Lymar, 2002). For comparison, the hydroxyl radical (HO , a prototypical strong one-electron oxidant) þ has a reduction potential of 2.3 V versus NHE for the HO,H /H2O couple at pH 7 (Sawyer, 1991). Consistent with the idea that NO is a poor one-electron oxidant, NO is also poor at H-atom abstraction since the bond formed from this reaction (the HNO bond) is weak, approximately 47 kcal/mol (Dixon, 1996). Again, for comparison, when HO abstracts a hydrogen atom, the strength of the bond made (HOH) is very strong, 119 kcal/mol. Thus, NO is generally difficult to reduce and, therefore, poor at initiating radical chemistry via oxidation pathways. However, NO will

σ∗

π∗ 2p

σ∗

σ N O

Figure 3 Molecular orbital diagram for NO. Schematic depiction of molecular orbitals shown. 150 LESLEY A.H. BOWMAN ET AL. rapidly react with existing radicals. As such, NO has been found to be a potent antioxidant capable of quenching otherwise deleterious/oxidizing radical chemistry (Rubbo et al., 1995). NO also reacts with dioxygen (which has two unpaired electrons and so, according to the definition above, is considered a free radical or, in this case, a diradical). The reaction of NO with O2 results in the generation of nitrogen dioxide (NO2) (Reaction 1).

2NO þ O2 ! 2NO2 ð1Þ

Like NO, NO2 is a free radical species. However, unlike NO, NO2 is a fairly strong oxidant, as indicated by a reduction potential of 1.04 (vs. NHE) for the NO2/NO2 couple (Stanbury, 1989). Therefore, reaction of NO with O2 takes two relatively weak oxidants (the reduction potential for the O2/O2 couple is 0.33 V (Sawyer, 1991)) and forms a reasonable one-electron oxidant. NO2 is known to oxidize a variety of biologically relevant functional groups such as cysteine thiols, tyrosines, and polyunsatu- rated fatty acids, just to name a few. In the absence of NO2-reactive species (i.e., reductants), the fate of NO2 generated via the reaction of NO with O2 is to react with another NO (since they are both radicals) to give dinitrogen trioxide (N2O3) (Reaction 2). ( NO2 þ NO + N2O3 ð2Þ

N2O3 (which is not a radical) is electrophilic and in aqueous systems will react with H2O to give two equivalents of nitrite (NO2 ) (Reaction 3). If other nucleophiles are present (e.g., thiols, amines), in a reaction analogous to the reaction of H2O, these nucleophiles can be nitrosated by N2O3 (Reaction 4).

( þ N2O3 þ H2O + 2NO2 þ 2H ð3Þ

N2O3 þ Nuc ! Nuc NO þ NO2 ð4Þ

Thus, the combination of Reactions 1, 2, and 4 can result in the NO-mediated nitrosation of biological nucleophiles. For example, S-nitrosothiols (SNOs) can be generated from thiols via this chemistry. S-nitrosation and S-nitrosylation are terms often used interchangeably in the biological literature, yet it must be stressed that they have distinct þ meanings. S-nitrosation is a mechanism whereby NO -like species mediate an attack upon thiol side groups, whereas S-nitrosylation is a mechanistically ambiguous term for any process that results in the generation of an SNO. NITRIC OXIDE AND AGENTS OF NITROSATIVE STRESS 151

Thus, the term nitrosyl does not describe a mechanism but instead refers to NO bound to a metal, for example. It needs to be noted that the biological accessibility of this nitrosation chemistry is likely to be rare or, at the very least, very restricted due to the reaction kinetics. As shown in Reaction 1, two equivalents of NO are required for the O2-dependent generation of NO2. Thus, the kinetics of this reaction has a second-order dependence on NO (Ford et al., 1993). This second-order dependence indicates that this process is significant only at high concentrations of NO. Since both NO and O2 favorably partition into membranes where concentrations of both are likely to be significantly higher than in the aqueous compartments of cells, it has been proposed that nitrosation chemistry of the type described above may have special relevance in lipophilic/membrane environments (Liu et al., 1998). As an example of the effect that the second-order dependence on NO can have, for purely aqueous, aerobic (assuming 200 mMO2) solutions of NO, a 10 mM solution will degrade to one half its original concentration in approximately 1 minute, whereas a 10 nM solution of NO will degrade to half its concentration in over 70 h (note that the term “half-life” was not used since this term is only applicable to first-order processes).

4.2. The Reaction of NO with Superoxide Anion

One of the most highly studied reactions of NO is with the reduced dioxygen species superoxide (O2 ), generating, initially, peroxynitrite (ONOO ) (Reaction 5).

NO þ O2 ! ONOO ð5Þ There are numerous excellent reviews on the chemistry, biology, and (patho)physiology of this reaction and ONOO (see, e.g., Pryor and Squadrito, 1995; Beckman and Koppenol, 1996; Ferrer-Sueta and Radi, 2009). This reaction readily occurs via a near diffusion-controlled process. The pKa of peroxynitrous acid (ONOOH) is 6.8, indicating that, at pH 7, the anion is the predominant species. In pure aqueous solution at physiological pH, ONOO will eventually decompose to nitrate (NO3 ). This rearrangement occurs presumably via ONOOH and involves the generation of oxidizing intermediates (e.g., Gunaydin and Houk, 2008). Indeed, ONOO /ONOOH is capable of performing oxidation chemistry on, for example, thiols (i.e., cysteine), phenols (i.e., tyrosine), and other biologically relevant reducing species. A primary fate of ONOO in many biological systems is reaction with carbon dioxide (CO2) giving, initially, nitrosoperoxycarbonate (ONOOCOO ) (Reaction 6). Rearrangement of 152 LESLEY A.H. BOWMAN ET AL. this species generates nitrocarbonate (Reaction 7), which then hydrolyzes 2 to give NO3 and carbonate (CO3 ).

ONOO þ CO2 ! ONOO CðOÞO ð6Þ

ONOO CðOÞO ! O2NO CO2 ð7Þ As with ONOOH decomposition, the rearrangement of nitrosoperoxycarbonate to nitrocarbonate involves reactive intermediates that are also capable of oxidizing a number of biological molecules (e.g., Gunaydin and Houk, 2009).

4.3. Reaction with Metal Centers

In a reaction that is analogous to its reaction with O2 , NO also reacts with dioxygen-bound metal species such as oxyhemoglobin or oxymyoglobin to form NO3 . In both oxymyoglobin and oxyhemoglobin, the bound dioxygen has significant O2 character due to extensive donation of electrons from the metal to the bound O2. Thus, reaction of NO with the heme-bound O2 is analogous to the reaction of NO with “free” O2 (Reactions 8 and 9), although recent studies indicate no intermediacy of ONOO on the millisecond time scale in this chemistry (Yukl et al.,2009). ÂÃ II II III MbFe þ O2 ! MbFe O2 $ MbFe O2 ð8Þ ÂÃ II III III MbFe O2 $ MbFe O2 þ NO ! MbFe þ NO3 ð9Þ Along with its reaction with dioxygen and dioxygen-derived species, NO also binds metals. Most notable in biological systems is the reaction of NO with hemeproteins (e.g., Ford, 2010), although NO can bind other nonheme metalloproteins as well. Unlike O2 and CO, which will only bind to ferrous þ þ (Fe2 ) hemes, NO is capable of binding both ferric (Fe3 ) and ferrous hemeproteins (provided there is an open or exchangeable coordination site). The reaction with ferrous hemes results in a species that favors a 5-coordinate, square pyramidal geometry. Indeed, a major site of action of NO in mammalian systems is the hemeprotein soluble guanylate cyclase (sGC) which binds NO via its ferrous heme leading to the formation of a 5-coordinate ferrous nitrosyl that is presumably responsible for enzyme activation (although the process may be more complicated) (Poulos, 2006). Significantly, when O2 and CO bind most ferrous hemes, a 6-coordinate geometry is preferred making NO a unique ligand among these small molecule diatomic signaling NITRIC OXIDE AND AGENTS OF NITROSATIVE STRESS 153 species (Traylor and Sharma, 1992). Binding of NO to a ferric heme leads to the generation of a nitrosyl complex whereby the NO ligand becomes þ electrophilic (possesses nitrosonium (NO ) character). Attack of the bound NO by a nucleophile (such as water, thiols, etc.) then leads to reduction of the ferric iron to ferrous and generation of a nitrosated species (Reaction 10). ÂÃ þ FeIII NO $ FeII NO þ Nuc H ! FeII þ Nuc NO þ Hþ ð10Þ This process is referred to as reductive nitrosation whereby nitrosation of water (generating NO2 ) and thiols (generating nitrosothiols) can readily occur. Significantly, nitrosation via these metal catalyzed processes is not second order in NO and therefore is not as kinetically restricted as the autoxidation process described earlier. However, since a reduced metal is a product (i.e., FeII) and an oxidized metal (i.e., FeIII) is required for the chemistry, there will be a requirement for reoxidation of the metal if the process is to be catalytic. As mentioned above, NO2 can be generated via the autoxidation of NO (Reactions 1–3) or reductive nitrosation (Reaction 10, where the nucleophile is water). Significantly, Reactions 3 and 2 are reversible, indicating that NO can be formed from high concentrations of NO2 under acidic conditions. NO2 can also be reduced directly to make NO under appropriate conditions. The one-electron reduction of NO2 to NO is highly proton dependent and can be very favorable since the reduction þ potential for the NO2 ,H /NO couple is 0.98 V, versus NHE.

4.4. Products of NO Reduction

Thus far, the discussion of nitrogen oxide chemistry has concentrated on species that are oxidized relative to NO (i.e., NO2,N2O3,NO2 , ONOO , etc.). Indeed, in mammalian systems, oxidation appears to be the major fate of NO. However, reduction of NO is well established in prokaryotes. One- electron reduction of NO generates nitroxyl (NO /HNO). As mentioned þ previously, the reduction potential of the NO,H /HNO couple is only 0.55 V (vs. NHE at pH 7), indicating that direct, proton-assisted one- electron reduction of free NO is relatively difficult. The chemistry of NO / HNO has been the topic of numerous recent investigations and is reviewed elsewhere (e.g., (Fukuto et al., 2005; Miranda, 2005)). The pKa of HNO has been determined to be 11.4 (Shafirovich and Lymar, 2002), indicating that HNO is the predominant species at pH 7. A unique aspect of the acid–base chemistry of HNO is that the two equilibrium partners do not have the same 154 LESLEY A.H. BOWMAN ET AL.

electronic ground state. That is, HNO is a ground state singlet, while NO is 3 a ground state triplet ( NO ) (akin to O2) (Reaction 11).

HNO +( 3NO þ Hþ ð11Þ Thus, the protonation of NO and the deprotonation of HNO are slow compared to other acid–base processes due to the requirement for a spin- flip. It is generally thought that, in biological systems where HNO or NO are formed, no chemistry associated with the conjugate will be observed since other reactions are faster than protonation–deprotonation. Both the one- and two-electron standard reduction potentials for the þ þ HNO,H /H2NO and HNO,2H /NH2OH couples are reported to be approximately 0.7 and 0.9 V versus NHE, respectively (the two-electron þ process, the HNO,2H /NH2OH couple, at pH 7, has a reduction potential of approximately 0.3 V vs. NHE) (Shafirovich and Lymar, 2002; Dutton et al., 2005). These values predict that HNO could be easily reduced in biological systems. Nitroxyl itself, however, can be very reducing. The reported reduction potential for the NO/3NO couple is 0.81 V versus NHE, indicating that 3NO can be a very potent reducing agent (Bartberger et al., 2002). As mentioned earlier, the NH bond dissociation energy for HNO is also only approximately 47 kcal/mol. This low bond strength predicts that HNO would be a very good hydrogen atom donor.

4.5. The Reactions of HNO with Biological Targets

One of the most prevalent biological targets for HNO appears to be thiols and thiolproteins. Both experimental and theoretical work indicates that HNO is highly thiolphilic (Doyle et al., 1988; Bartberger et al., 2001). Attack of a nucleophilic thiol at the electrophilic nitrogen atom of HNO results in the formation of a fleeting N-hydroxysulfenamide (Fig. 4). The N-hydroxysulfenamide intermediate has two possible fates. In the presence of excess or vicinal thiols, the N-hydroxysulfenamide reacts to form a disulfide and hydroxylamine. A competing rearrangement can also occur resulting in the formation of a sulfinamide. Significantly, in biological systems, disulfides are generally considered to be readily reduced back to the corresponding thiols, whereas sulfinamides are likely to be resistant to reduction. Thus, reaction of HNO with thiols can result in either reversible or irreversible modifications. Another likely class of biological targets for HNO is metals. Akin to other small molecule metal ligands (e.g., O2, NO, CO, H2S, CN ), HNO NITRIC OXIDE AND AGENTS OF NITROSATIVE STRESS 155

NH2 Protein S Sulfinamide

O H+ H Rearrangement

H O N OH N Protein Protein S S RSH N-Hydroxysulfenamide

Protein S SR + Disulfide NH2OH

Figure 4 Reaction of HNO with thiols.

is capable of coordinating a variety of metals and/or metalloproteins. Heme-containing proteins are currently the most highly studied among possible metalloprotein targets (Farmer and Sulc, 2005). HNO is capable of binding both ferrous and ferric heme proteins. For example, reaction of HNO with ferrous myoglobin results in the formation of a stable MbFeII–HNO complex (Sulc et al., 2004) (Reaction 12). Reaction of HNO with ferric myoglobin results in the formation of a stable ferrous- nitrosyl MbFeII–NO complex (Doyle et al., 1988) (Reaction 13).

MbFeII þ HNO ! MbFeII NðHÞO ð12Þ

MbFeIII þ HNO ! MbFeII NO þ Hþ ð13Þ Although most previous biological studies involving nitroxyl presumably involve the protonated HNO species, it should be noted that formation of the anion, 3NO , will not result in immediate HNO generation due to the spin restriction to protonation (remember that the pKa of HNO is 11.4). Therefore, if 3NO is generated in a biological system, the chemistry of this species will be prevalent. As discussed briefly above, 3NO is a strong reduc- 3 tant and should reduce/coordinate metals. Also, NO will react with O2,ina process that is isoelectronic with Reaction 5, giving ONOO (Reaction 14).

3 NO þ O2 ! ONOO ð14Þ 156 LESLEY A.H. BOWMAN ET AL.

The above discussion of the nitrogen oxides and the associated chemical descriptions is not meant to be comprehensive but considered to serve as a starting point for understanding the diversity and complexity of biological nitrogen oxide chemistry. For more complete descriptions of these and other aspects of nitrogen oxide chemistry, readers are encouraged to find one of the available reviews (e.g., Wink and Mitchell, 1998; McCleverty, 2004; Hughes, 2008; Thomas et al., 2008).

5. LABORATORY METHODS

Working with many of the nitrogen oxides described above can be accomplished using either authentic compounds, or in many cases, it is more convenient to use donor species. Herein, we discuss briefly aspects of working with the nitrogen oxides that appear to be of the most current interest—NO, NO2,N2O3,NO2 , HNO, and ONOO . For several nitrogen oxide species discussed below, the use of donor compounds is prevalent and even necessary. There are several important factors that need to be considered when using donors, however. Thus, prior to a discussion of the individual donors, a general comment on the use of donors is warranted.

5.1. The Use of Nitrogen Oxide Donors

With the use of any donor species, there are at least four important considerations that need to be accounted for when interpreting the experimental results (e.g., Fukuto et al., 2008): (1) It must be determined that the biological actions of the donor are due to the species released (i.e., NO) and not due to the donor itself; (2) it is important to understand that the biological activity can be due to donor coproducts (i.e., other species generated alongside the species of interest); (3) there is the possibility that impurities in the donor may be the active species; and (4) for donors that require biological activation, there is the possibility that the activation process itself (i.e., oxidation or reduction) can be at least partially responsible for the activity. One way to control most of the above-mentioned possibilities is to use structurally distinct donors from different compound classes (and with different mechanisms of release) and to incorporate control experiments using fully decomposed donors. Since donors from vary- ing classes are structurally distinct, their syntheses and mechanisms of release are also distinct. Thus, it is highly likely that the only thing they will NITRIC OXIDE AND AGENTS OF NITROSATIVE STRESS 157 have in common is the release of the species of interest. Therefore, if similar biological activity is observed with the varied donors, it is likely that the donated species (i.e., NO) is responsible. Controlling for possible activity associated with impurities and/or coproducts can be accomplished by simply decomposing the donor and testing the decomposed donor solution. Since NO is a fleeting species in aqueous aerobic solution (decomposes to NO2 ), allowing time for both donor decomposition and NO degradation to NO2 is necessary. For example, Dukelow et al. (2002) showed that exhausted DETA NONOate had the same growth inhibitory effect toward Pseudomonas aeruginosa in vitro as DETA NONOate, showing that NO was not responsible for the antibacterial effects observed (Dukelow et al., 2002). In contrast, investigators examining the ability of S. enterica to mount an acid tolerance response in the presence of RNS revealed that spermine NONOate sensitizes cells to acid stress, a feat dependent upon NO release, as the parent compound spermine did not elicit a reduction in cell viability (Bourret et al., 2008).

5.2. NO

Although NO can be purchased in the gaseous form in a pressurized tank (Aga and Hughes, 2008), this is often not convenient to use in many biological studies since gas-handling systems need to be in place and determining the concentrations of NO in solutions made by passing NO gas through aqueous mixtures is not trivial. One solution to providing the gas involves the use of a gas-permeable membrane (Silastic) immersed in the culture and through which gas mixtures containing NO are continuously passed. Dose rates of NO delivery are directly proportional to the length of the immersed tubing (Tamir et al., 1993). This method has been used to study induction of the E. coli SoxRS regulon and was found to be more effective than bolus additions of NO (Nunoshiba et al., 1995), while also mimicking better the continuous fluxes of the gas that occur in macrophages. In one recent development, Skinn et al. (2011) fabricated a small (65 ml) stirred reactor that incorporates a flat porous membrane for NO delivery (sitting below a stirrer) and a loop of gas-permeable tubing for O2 delivery. In trials using a 10% NO mixture and a buffer that was initially air-equilibrated, constant rates of NO2 (i.e., the end product of NO oxidation accumulation) were observed (53 mM/h). Such a system has great potential in microbial physiology experiments but have not yet been reported. Other means for exposing microbial cultures to NO include incubating Petri dishes in a con- stantly replenished atmosphere containing 10% air, 960 ppm NO, and the 158 LESLEY A.H. BOWMAN ET AL. balance as nitrogen. The additional presence of the redox cycling agent phenazine methosulfate appeared necessary for consistent NO-dependent killing but the basis for this observation seems obscure (Gardner et al., 1998). In other papers, a three-way valve was used to deliver mixtures of O2,N2, and NO, and gas mixtures were passed through a trap containing NaOH pellets to remove NO2 and higher oxides of nitrogen formed prior to entering reaction vessels (Gardner et al., 1997). A laboratory method for generating NO by the self-decomposition through disproportionation of nitrous acid is described in useful detail by Aga and Hughes (2008). However, most experimentalists examining the biology of NO utilize NO donors. There are many NO donors available and their use and chemistry has been reviewed previously (Wang et al., 2002; Miller and Megson, 2007; Aga and Hughes, 2008). Currently, one of the most utilized class of NO donors are the diazeniumdiolates (also referred to as “NONOates”) (e.g., Keefer, 2003) (Reaction 15).

þ R2N½ NONO þ H ! R2NH þ 2NO ð15Þ

Their utility is based on the wide variety of donors available with vary- ing NO release rates and the fact that NO release is spontaneous at physiological pH (i.e., does not require bioactivation). The varied NO release rates for the diazeniumdiolates have been attributed to a competition between several protonation sites on the molecule, of which only one leads to NO generation (Dutton et al., 2004a). The half-lives of these compounds range from 1 min to several hours. All of these factors make this class of NO donor highly preferable for biological studies. The only coproduct for the diazeniumdiolates is a secondary amine, which can be controlled by testing either the amine itself or the decomposed donor. In some studies mixtures or cocktails of NO donors with complementary properties may be used. For example, we have used a mixture of NOC-5 and NOC-7 in studies of E. coli to maintain a sustained output of NO in studies of hmp gene transcription (Cruz-Ramos et al.,2002). NO release from NOC-5 (half-life of 25 min at 37 C) was combined with NO release from NOC-7 (half-life of 5 min at 37 C) to provide NO release over a period of 1 h or more. In practice, NO loss through biological or nonbiological routes limited the presence of NO in cultures to about 30 min (Cruz-Ramos et al., 2002). Sodium nitroprusside (SNP, Na2Fe(CN)5NO) is a clinically relevant and commonly used donor of NO. In spite of its clinical utility, the mechanism of NO release from SNP in biological systems is not completely established and likely to be complex (Wang et al., 2002; Miller and Megson, 2007). However, it is known that SNP will not spontaneously release NO in a NITRIC OXIDE AND AGENTS OF NITROSATIVE STRESS 159 biological system. Release of NO from SNP requires either light or reductive metabolism. These factors make it difficult to accurately predict the levels of NO generated from SNP and/or compare experiments where SNP is used since light exposure and the degree of reductive metabolism can differ significantly between experimental systems. Moreover, SNP contains five cyanide ligands which can also be released and which may also have biological activity. In in vivo systems, the release of cyanide from biologically active levels of SNP is typically not a problem (at least in the short term) since NO is such a potent vasorelaxant that the levels of released cyanide are tolerated. However, considering all of these factors (possible photochemical release, the requirement for reductive metabolism, the release of cyanide) as well as others (i.e., the addition of iron), the use of SNP as an NO donor in a research setting is not optimum.

5.3. S-Nitrosothiols

Another often-used class of NO donor is S-nitrosothiols (SNOs). Although SNO species are clearly biologically relevant (e.g., Hess et al., 2005), they can do much more than simply release NO. The release of NO from SNO compounds is not spontaneous and requires light or reducing metals þ (i.e., Cu1 ) (Singh et al., 1996) (Reactions 16 and 17).

RS NOðÞ!þlight RS þ NO ð16Þ

þ þ RS NO þ Cu1 ! RS þ NO þ Cu2 ð17Þ Moreover, SNO compounds can react with other thiols to give, instead, þ HNO (Reaction 18) or transfer the equivalent of nitrosonium ion (NO )to another thiol (Reaction 19) (Wong et al., 1998):

0 RS NO þ R0SH ! RSSR þ HNO ð18Þ

RS NO þ R0SH ! RSH þ R0S NO ð19Þ The possibility of all of this chemistry occurring in a biological system makes mechanistic interpretation of experiments difficult. Thus, in studies where only the administration of NO is desired, SNO species are not ideal. Controls are clearly needed but the design of these experiments is not always straightforward. For example, GSNO is widely used as a convenient S-nitrosothiol and it might be imagined that glutathione would be a useful control molecule. However, GSH is not an ideal control molecule since it may not be the product of GSNO metabolism (see below). Nevertheless, 160 LESLEY A.H. BOWMAN ET AL. where GSH has been used (as well as GSSG) it has been shown to be relatively ineffective at eliciting upregulation of GSNO-inducible genes in C. jejuni (Monk et al., 2008), yet GSH (5 mM) is toxic to M. tuberculosis (Venketaraman et al., 2005). The evidence to date suggests that, when GSNO is added to bacterial cultures, intracellular outcomes are preceded by enzymic transformations of GSNO. The mechanisms of communication between extracellular and intracellular pools of SNOs are poorly understood but transport mechanisms involving cell-surface protein disulfide isomerases (Zai et al., 1999; Ramachandran et al., 2001), g-glutamyl transpeptidase (De Groote et al., 1995; Hogg et al., 1997), or anion exchangers (Pawloski et al., 2001) þ have been proposed. GSNO is proposed to transfer NO to outer membrane thiols in Bacillus (Morris and Hansen, 1981) but other studies suggest that active transport of the compound is required for toxicity. In S. enterica, GSNO (0.5 mM) is bacteriostatic but GSNO is not itself transported into the cell. Highly GSNO-resistant mutants were isolated from a MudJ transposon library and the insertions shown to be in dppA and dppD (De Groote et al., 1995). These genes are part of an operon encoding dipeptide permease, an ABC-family transporter responsible for L-dipeptide import. It is therefore suggested that a periplasmic transpeptidase encoded by the ggt gene removes the g-glutamyl moiety. Indeed, ggt mutants of E. coli (De Groote et al., 1995) and M. tuberculosis (Dayaram et al., 2006) are also GSNO resistant. In E. coli, the residual dipeptide, S-nitroso-L-cysteinylglycine, is then transported inward using the Dpp-encoded dipeptide permease. Figure 5 summarizes the proposed mechanisms. A similar mechanism appears to operate in E. coli since regulatory perturbations inducible by GSNO are dependent on the presence of the Dpp system (Jarboe et al., 2008). In other words, the toxic agent in the cytoplasm is not GSNO but the nitrosated dipeptide. In M. bovis, the oligopeptide permease operon (oppBCDA)is implicated in GSNO transport (Green et al., 2000). Mutation of the oppD gene encoding the ATPase component of this binding protein-dependent transport system elicits resistance to 4 mM GSH in the external medium, a concentration that inhibits the wild-type strain. Importantly, similar results were found with 0.5 mM GSNO, which is bactericidal for the wild-type strain but not the oppD mutant. The resistance of the mutant to GSH is due to diminished import of the thiol, as shown by transport studies using [3H]GSH. In view of the finding that, in S. enterica, it is trans- þ port of the dipeptide that carries the NO group (De Groote et al., 1995), Green et al. (2000) tested whether the opp system in M. bovis transports NITRIC OXIDE AND AGENTS OF NITROSATIVE STRESS 161

A. Salmonella enterica B. Mycobacterium bovis

Glu-CysNO-Gly (GSNO)

Outer membrane

GGT Glu-CysNO-Gly SBP CysNO-Gly Periplasmic space Glu

Dpp Opp Inner membrane

CysNO-Gly GSNO

Transnitrosation reactions

Figure 5 Transport mechanisms for SNOs in bacteria leading to intracellular transnitrosation reactions. (A) In S. enterica, GSNO passively enters the periplasm, where a transpeptidase (GGT) removes the g-glutamyl moiety (De Groote et al., 1995). The residual dipeptide, S-nitroso-L-cysteinylglycine, is then transported inward using the Dpp-encoded dipeptide permease. (B) In M. bovis, the oligopeptide permease (Opp) affects GSNO transport (Green et al., 2000). A periplasmic substrate-binding protein (SBP) is a product of the same operon. dipeptides, but none of the tested components of GSH (L-Cys-Gly, L-Cys, Gly) showed toxicity toward this bacterium. Note that in mammalian systems, the cellular impact of GSNO or S-nitroso-N-acetyl-D,L-penicillamine (SNAP) is influenced by the availability of cysteine or cystine. Indeed, the degradation of GSNO is absolutely dependent on extracellular cystine, which is reduced to cysteine, which in turn reacts with GSNO to form S-nitrosocysteine (CysNO) in a transnitrosation reaction. CysNO is then imported by the amino acid transport system L-AT (Zhang and Hogg, 2004). 162 LESLEY A.H. BOWMAN ET AL.

5.4. Other Donors

There are several clinically used NO donors that have also been utilized as NO donors in biological experiments. Organic nitrate esters such as glyceryltrinitrate (GTN) and the iron–nitrosyl compound sodium nitroprusside (SNP) are used as sources of NO in a clinical setting or in microbiology (Joannou et al., 1998; Murray et al., 1998; Lloyd et al., 2003). However, both require reductive bioactivation, and like RSNO species, both are capable of other chemistries (see Section 5.2).

5.5. HNO Donors

Recent findings of possible pharmacological applications for HNO have stimulated the use and development of HNO donors in biological systems (Miranda, 2005; Paolocci et al., 2007; Fukuto et al., 2008). The most convenient and prevalent HNO donor is Angeli’s salt (Na2N2O3) (Reaction 20).

2 þ N2O3 þ H ! HNO þ NO2 ð20Þ Similar to the diazeniumdiolates, the release of HNO from Angeli’ssalt occurs via specific protonation on one of several possible protonation sites (Dutton et al.,2004b). The half-lives of Angeli’ssaltat25and37Care approximately 17 and 2.5 min, respectively. As shown in Reaction 20, the stoi- chiometric coproduct in Angeli’s salt decomposition is NO2 . Since NO2 is readily available, controlling its release is straightforward and easy using authentic NO2 (or a better alternative, albeit more expensive, is to examine the effects of decomposed Angeli’s salt). Thus, Angeli’s salt is a convenient and well-defined HNO donor for use in biological systems. It should be noted, however, that at acidic pH (<4), Angeli’s salt becomes an NO donor. Another previously utilized HNO donor is Piloty’s acid. Piloty’s acid is a representative of the N-hydroxylsulfonamide class of donor. The mechanism of decomposition of Piloty’s acid requires the deprotonation of a weakly acidic proton (Reaction 21), and therefore only generates HNO at a significant rate under basic conditions. ð Þ ! ð Þ ! ð Þ þ ð Þ R S O 2NHOH R S O 2 NHO R S O O HNO 21 Thus, in typical biological experiments (e.g., pH 5–7.5), Piloty’s acid is very slow at HNO release. Also, at neutral pH where Piloty’s acid decomposition is slow, autoxidation can occur in aerobic solutions, leading to NO release (Reaction 22) (Zamora et al., 1995).

ð Þ ! ð Þ ! ð Þ þ ð Þ R S O 2NHOH R S O 2NHO R S O OH NO 22 NITRIC OXIDE AND AGENTS OF NITROSATIVE STRESS 163

The slow release under typical biological conditions and the possible autoxidation makes Piloty’s acid (and derived species) less than optimum donors of HNO for many biological preparations. Recent reports indicate the utility of other HNO donors. Acyloxy nitroso compounds reported by Sha et al. (2006) require an ester hydrolysis step prior to HNO release but offer a wide array of structural diversity and diazeniumdiolates made from primary amines appear to offer structural diversity and spontaneous HNO release in vivo (Miranda et al., 2005). Significantly, the HNO-donating prodrug cyanamide (H2NCN) has been used clinically in antialcoholism therapy. Cyanamide can be oxidized to N-hydroxy cyanamide, which spontaneously decomposes to HNO and HCN (Reaction 23).

H2N CN ! HONH CN ! HNO þ HCN ð23Þ

Oxidation of cyanamide can be carried out by catalase/H2O2 (Nagasawa et al., 1990). Thus, HNO release from cyanamide requires oxidative bioactivation and simultaneous cyanide release, both of which can repre- sent complications in biological experiments. Relatively few studies can be cited to illustrate the identification of HNO as a species directly responsible for biological effects. However, a clear example is provided by work on the thiol-containing, metal-responsive yeast (Saccharomyces cerevisiae) transcription factor Ace1. Ace1 is activated by binding copper via multiple Cys thiols, resulting in the transcription of genes encoding proteins involved in copper sequestration (Shinyashiki et al.,2005). Activation of Ace1 by copper addition to cultures in the presence of various nitrogen oxides has been studied in depth. Both diethylamine NONOate (DEA/NO) and Angeli’s salt inhibited Ace1, but the inhibition by NO was oxygen dependent while the effect of the HNO donor was not (Cook et al., 2003). The results are interpreted as thiol modification by NO via the generation of nitrosating species, whereas HNO is able to react directly with protein thiols. The work also provides an example of the use of decomposed donors as controls (see Section 5.1): decomposed Angeli’ssalt was without effect and the product of HNO dimerization, nitrous oxide (N2O), was also inactive, further indicating that HNO is the active species.

5.6. Use of ONOO

Peroxynitrite has been studied extensively using both the authentic compound and via the use of donors. The most commonly utilized ONOO donors are the sydnonimines (Fig. 6). SIN-1 (R¼morpholino, Fig. 6) is a 164 LESLEY A.H. BOWMAN ET AL.

R R N N – – N NH N NH O O

Sydnonimines, R = morpholino (SIN-1)

Figure 6 General structure of sydnonimines. prototypical sydnonimine and its decomposition has been studied extensively (Bohn and Schonafinger, 1989). In aqueous, aerobic solution SIN-1 will ring open followed by one-electron oxidation by O2, generating O2 . The oxidized, ring-opened species then spontaneously releases NO. Thus, SIN-1 generates NO and O2 in a one-to-one stoichiometry. Since the reaction of NO and O2 is near diffusion controlled, aerobic SIN-1 decomposition results in formation of ONOO . Many in vitro studies have employed these generators to examine the reactivity and toxicity of ONOO , but findings have often been contradic- tory. For example, superoxide dismutase (SOD) has been shown to provide considerable protection toward SIN-1-mediated killing in E. coli, which is due to O2 scavenging, preventing ONOO formation (Brunelli et al., 1995). Conversely, it has been demonstrated that SOD actually pro- motes SIN-1 toxicity. In one study, SOD potentiated the cytotoxicity of SIN-1 toward the human hepatoma liver cell line (HepG2) by elevating H2O2 production through the catalyzed dismutation of O2 (Gergel et al., 1995). However, in another study, SOD enhanced SIN-1 killing of the parasite L. major by scavenging O2 and increasing the half-life of the true noxious species, NO (Assreuy et al., 1994). Consequently, it is apparent that when employing ONOO generators, appropriate control experiments must be conducted in order to establish whether observations are attributable to ONOO per se or its reactants.

5.7. NO2

Nitrogen dioxide is available as a compressed gas. Since NO2 is an oxidant and toxic, use of authentic gas can be problematic if proper gas handling equipment is not available. Moreover, solutions of NO2 are not stable since it will dimerize to N2O4, which hydrolyzes to give NO2 and NO3 (Reactions 24 and 25). NITRIC OXIDE AND AGENTS OF NITROSATIVE STRESS 165

2NO2 ! N2O4 ð24Þ

þ N2O4 þ H2O ! NO2 þ NO3 þ 2H ð25Þ

Thus, experiments aimed at examining specifically NO2 in a biological system are not always straightforward. In instances where the observed biological activity is thought to be a result of NO2 generation via autoxidation of NO, a convenient reagent is available for the facile conversion of NO to NO2. Nitronyl nitroxides (e.g., PTIO) or the water soluble analog carboxy PTIO (cPTIO), which are widely used research tools for scavenging NO (Akaike et al., 1993), are capable of converting NO directly to NO2 in an O2-independent manner (thus avoiding the high-order kinetics of autoxidation) (Akaike and Maeda, 1996) (Reaction 26).

PTIO þ NO ! NO2 þ PTI ð26Þ

Generation of NO2 from NO allows the possibility that N2O3 is the biologically active species since NO and NO2 react quickly to give N2O3. Dis- tinguishing between the actions of NO2 and N2O3 can be addressed by examining the effect of PTIO concentration on the biological activity (e.g., Shinyashiki et al., 2004). At low PTIO/NO ratios, the NO2 formed will have the opportunity to react with remaining NO to generate N2O3. However, at high PTIO/NO ratios, most of the NO will be converted to NO2, precluding formation of N2O3. Nitronyl nitroxides are also used to detect NO since the reaction of the EPR-active PTIO with NO generates another distinct EPR-active species (PTI) (Akaike and Maeda, 1996). Thus monitoring the conversion of PTIO to PTI via EPR can serve as a quantitative indication of the NO levels. When using the nitronyl nitroxides as research tools (either for detection/quantitation of NO or as a reagent to convert NO to NO2), it is important to realize that the ultimate products generated from the reaction of NO with PTIO depend significantly on the relative concentrations of the reactants (Goldstein et al., 2003). For example, the NO2 formed from the reaction of NO and PTIO can also react with PTIO to give NO2 and þ þ the corresponding oxoammonium cation (PTIO ). PTIO can further react with NO (in aqueous solution) to give PTIO and NO2 . Thus, quantitation of NO via PTIO is valid only when NO is in low concentration and alternative scavengers for NO2 are important considerations when PTIO is used to convert NO to NO2. Regardless, with a reasonable understanding of this chemistry, the nitronyl nitroxides are important tools for examining many aspects of nitrogen oxide chemistry and biology. 166 LESLEY A.H. BOWMAN ET AL.

5.8. NO2

Nitrite is commercially available as a stable salt. Generally, it is easily han- dled and amenable to direct use in biological experiments. It is important to note, however, that high concentrations of NO2 (especially under acidic conditions) can generate N2O3 (the anhydride of nitrous acid, HONO) which is also a source of NO and NO2. Despite this, many studies use, as a matter of convenience, acidified nitrite as an agent of nitrosative stress (see, e.g., Mendez et al., 1999; Kim et al., 2003; Mukhopadhyay et al., 2004; Iovine et al., 2008). However, these conditions are far from ideal in experiments aimed at determining the mode of action of NO and RNS. In one report, killing of Mycobacterium ulcerans, which causes ulcerative skin disease, was reported within 20 min but the concentration of nitrite was very high (40 mM) (Phillips et al., 2004). More recently, it has been reported that nrfA and ytfE mutants were sensitive to “NO donors” but the reagents used were actually GSNO and acidified nitrite (10 mM) (Harrington et al., 2009).

5.9. Other Nitrogen Oxides

Other nitrogen oxides of possible interest include NO3 and NH2OH, both of which are commercially available and fairly easy to work with in aqueous solutions. An overview of the relationship between the nitrogen compounds discussed in Sections 4 and 5 is presented as Fig. 7.

6. BACTERIAL RESPONSES TO RNS: EFFECTORS AND REGULATORS

6.1. Targets of RNS in Microorganisms

It is frequently stated that NO is a highly reactive gas and must interact with numerous and diverse biological targets. In fact, as we outline in Sec- tion 4.1, NO is not especially reactive but it is true that its targets are not as restricted as those of another “gasotransmitter,” carbon monoxide (CO) (Davidge et al., 2009a). The direct cellular effects of NO are incompletely understood because of the complexity of NO chemistry introduced in Section 4. Nevertheless, various biomolecules are targeted by NO and the resulting RNS. Some examples from this vast literature are listed in Table 2 and a detailed analysis is presented in Stamler et al. (2001). NITRIC OXIDE AND AGENTS OF NITROSATIVE STRESS 167

− 2e Powerful 1e− oxidant oxidant RS-NO + H+ e−, 2H+ e−, H+ − − H O . −e O2 2 2 HO + H2O RSH − − + e e , H

L-Arginine NO O2 H2O −e− N O H2O − 2 3 ONOO Small % 2nd order NO− +H+ 2 in [NO] Nitrosation + −e− H chemistry ("NO+"-donor) NO ONOOH − − 2 e , H2O Small % − + NO3 +2H − 1e oxidant Major reaction

Figure 7 The biological and integrative chemistry and fate of NO- and O2- derived species. Of particular importance is the generation of the one-electron oxidants NO2 and HO and nitrosating species N2O3. This figure is not meant to be comprehensive, but rather merely serves to illustrate the integrated nature of NO and O2 chemistry in biological systems. Not shown here is the possible (and likely important) role of biological redox metals in this system.

6.2. Microbial Defenses: The Microbe Strikes Back

Many mechanisms are known to confer NO resistance in bacteria, such as flavohemoglobin-catalyzed NO detoxification in enteric bacteria and numerous other species as well as other globins. Confusing the literature, however, is that many experimental studies to unravel the details of NO resistance are performed with proxies for NO, often SNOs and especially GSNO. It is important to recognize that most of the enzymic detoxification systems studied, as far we can judge from current experiments, are first and foremost concerned with NO detoxification, not with SNO detoxification. The best understood of these, flavohemoglobin, is an NO-detoxifying enzyme and has no significant activity against SNOs. Although the earliest papers on flavohemoglobin regulation (Poole et al., 1996) and enzymic function (Gardner et al., 1998) were conducted with “real” NO, many later 168 LESLEY A.H. BOWMAN ET AL. studies have substituted these agents. Hausladen et al. (1998a) provide a clear distinction between NO metabolism and SNO metabolism. It was known that SNO decomposition by E. coli generates nitrite and nitrate and that NO could arise from the former via chemistry described above. A quest for NO decomposition showed that although the NO-metabolizing activity of E. coli was stimulated by treatment of the culture with CysNO, the SNO-lyase activity was not. The major diverse mechanisms identified were reviewed previously (Poole, 2005b) and are updated in Table 3. This is a rapidly developing field: regulation of gene expression by NO and RNS and the functional identification of resistance mechanisms are being studied in numerous bacteria, as well as fungi and protozoa. Here we focus on a small number of systems that have emerged as paradigms for future study and bring to readers’ attention selected new papers that illustrate novel or particularly interesting examples.

6.3. Microbial Globins

An exciting development in biological NO biochemistry over the past 15 years has been the realization that a major mechanism for NO resistance and detoxification in bacteria and eukaryotic microbes is hemoglobin-mediated NO chemistry. Microbial globins have consequently been extensively studied and reviewed (Poole and Hughes, 2000; Frey et al., 2002; Wittenberg et al., 2002; Frey and Kallio, 2003; Wu et al., 2003; Poole, 2005b; Vinogradov et al., 2006; Lu et al., 2008). Globin function is typically defined by reactivity toward small ligands, such as O2, CO, and NO, which bind to the heme distal site. Reactivity with, and biological activity in rela- tion to, each of these ligands has been reported for various microbial globins. Here we focus only on NO.

6.3.1. Flavohemoglobins

Three classes of bacterial globin are recognized, namely, the flavohemoglobins, the single-domain “myoglobin-like” globins, and the truncated globins. Members of the best understood class, the flavohemoglobins, are distin- guished by the presence of an N-terminal globin domain (a three-on- three a-helical fold similar to myoglobin) with an additional C-terminal domain with binding sites for flavin adenine dinucleotide (FAD) and nicotinamide adenine dinucleotide (phosphate) [NAD(P)H]. Widely Table 3 Examples of proteins implicated in tolerance to NO and nitrosative stress via consumption of NO or S-nitrosothiols.

Function/reactions Class Protein Organism(s) catalyzed Comments Referencea

1. Globins Myoglobin, Higher animals Transient Nitrite reduction; Brunori (2001); hemoglobin formation of physiological role in Flogel et al. (2001); peroxynitrite- NO tolerance? NO Hendgen-Cotta et al. globin; quantitative scavenging? OONO (2008); Ascenzi et al. formation of nitrate scavenging? (2009) without nitration of globin Truncated globin Mycobacterium Conversion of NO NO-inducible NO Pawaria et al. (2007); (HbN) species to nitrate? uptake Martr et al. (2008); Lama et al. (2009) Vitreoscilla globin Vitreoscilla sp. NO consumption Mechanism unknown; Kallio et al. (2007); (Vgb) globin confers growth Frey et al. (2011) tolerance to SNP Single-domain Campylobacter Confers enhanced Mechanism presumed Monk et al. (2008); globin (Cgb) jejuni, C. coli resistance to NO to be “NO Shepherd et al. and nitrosating dioxygenase” (2010a, 2011); Smith agents converting NO to et al. (2011) nitrate Flavohemoglobin E. coli, Enzymic hmp expression Stevanin et al. (Hmp) Salmonella, detoxification of upregulated by NO (2007); Laver et al. B. subtilis, NO by conversion and nitrosating agents; (2010); Svensson Erwinia, many to nitrate mutants are NO et al. (2010); Wang others sensitive et al. (2010b)

(continued) Table 3 (continued)

Function/reactions Class Protein Organism(s) catalyzed Comments Referencea

2. Flavorubredoxin E. coli, NO reduction and Upregulated in Mills et al. (2005, Reductases (NorVW) Salmonella detoxification response to NO and 2008); Pullan et al. RNS (2007) Cytochrome c E. coli, C. jejuni NO reduction and Nrf mutant strains Pittman et al. (2007); nitrite reductase detoxification show higher NO Mills et al. (2008); (NrfA) sensitivity van Wonderen et al. (2008); Einsle (2011) SNO-lyase; GSH- E. coli, yeast, GSNO or SNO Controls cellular levels Foster et al. (2009b); dependent mammalian reductase of S-nitrosothiols and Tavares et al. (2009) formaldehyde cells S-nitrosylated proteins dehydrogenase Cytochrome and Mitochondria NO reduction at Activity may be Butler et al. (2002); quinol oxidases of higher CuB restricted to oxidases Borisov et al. (2009) organisms, in heme-CuB family; bacteria physiological significance unclear 3. Others Cytochrome c0 Rhodobacter NO reductase, CycP mutants are Stevanin et al. (CycP) capsulatus forming N2O hypersensitive to (2005); Heurlier nitrosothiols and NO et al. (2008) aIllustrative only; the most recent papers are cited in most cases. NITRIC OXIDE AND AGENTS OF NITROSATIVE STRESS 171 distributed in bacteria and lower eukaryotes, flavohemoglobins confer protection from NO and nitrosative stresses by direct consumption of NO (Poole and Hughes, 2000). Indeed, flavohemoglobins appear to have no direct role in the metabolism of oxygen or other gaseous ligands and such proteins have not been reported in higher animals. Flavohemoglobins oxidize NAD(P)H and transfer an electron to the N-terminal heme domain via a noncovalently bound FAD in the reductase (or FNR, ferredoxin-NADP reductase-like) domain. Reduced heme catalyzes the reaction between NO and O2 generating nitrate; that is, Hmp acts as an NO-detoxifying enzyme. There remains controversy over the reaction mechanism at the heme: either NO (denitrosylase mechanism) (Hausladen et al., 1998b, 2001) or O2 (dioxygenase mechanism) (Gardner et al., 1998, 2000, 2006) has been claimed to bind first to the heme. Flavohemoglobins are critical for pathogenicity in some species; for example, E. coli and Salmonella mutants lacking Hmp are compromised for survival in mouse and human macrophages (Stevanin et al., 2002, 2007). In the plant pathogen Erwinia chrysanthemi, HmpX not only protects against nitrosative stress but also attenuates host hypersensitive reaction during infection by intercepting NO produced by the plant for the execution of the hypersensitive cell death program (Favey et al., 1995; Boccara et al., 2005). In accordance with the role of Hmp in limiting NO-related toxicity, expression of the protein occurs only when NO is present in the cell environment. Indeed, hmp gene expression is tightly regulated at the transcriptional level by NO-responsive transcription factors, notably NsrR and Fnr (Spiro, 2007). NO regulation of gene expression is discussed more fully below (Section 7). Tight control of Hmp synthesis and function appears critical since constitutive Hmp expression and function in E. coli in the absence of NO generates oxidative stress by virtue of oxygen reduction by the heme to superoxide anion (Poole et al., 1997; Wu et al., 2004). Similarly, constitutive expression of Hmp in Salmonella renders cells hypersensitive to paraquat and H2O2 (Gilberthorpe et al., 2007), as well as ONOO (McLean et al., 2010b); remarkably, the toxicity of ONOO is in part alleviated by NO, presumably because NO diverts Hmp function to nitrate formation, rather than generation of oxidative stress, which exacerbates the stress caused by ONOO . The flavohemoglobins from diverse bacteria have protective functions against RNS, including Ralstonia eutropha, Bacillus subtilis, P. aeruginosa, Deinococcus radiodurans, S. enterica, and Klebsiella pneumoniae (Frey et al., 2002). Note, however, that in this study the agent of RNS used was SNP (1 mM), an unfortunate choice. 172 LESLEY A.H. BOWMAN ET AL.

6.3.2. Single-Domain 3/3 Globins

The second class of bacterial globins comprises the single-domain globins. These also exhibit a three-on-three a-helical fold similar to myoglobin but no separate C-terminal reductase domain. This class is typified by the globin of Vitreoscilla (named Vgb, VtHb, or Vhb), an obligate aerobic bacterium that grows in low oxygen environments (Webster, 1987). This globin was the first bacterial hemoglobin to be crystallized, and the 3D structure (of the ferric homodimer) conforms to the classical globin fold (Bolognesi et al., 1999). This protein has been implicated in redox chemistry and NO detoxification in vivo, but the mechanism by which the protein is rereduced after a catalytic cycle is obscure (see Section 6.3.3). The disordered CD region (i.e., the vicinity of the C and D helices) in the crystal structure of Vgb is a potential site of interaction with the putative FAD/NADH reductase partner. Considerable interest has been directed at Vgb because of its possible role in facilitating oxygen transport and metabolism and the consequent biotechnological implications (Frey et al., 2011). Interestingly, a chimeric protein com- prising the Vitreoscilla hemoglobin and a flavoreductase domain from a flavohemoglobin (Fhp) relieves nitrosative stress in E. coli (Frey et al.,2002). A more comprehensive molecular genetic view of bacterial non- flavohemoglobins is offered by the microaerophilic, foodborne, pathogenic bacterium C. jejuni, which is exposed to NO and other nitrosating species during host infection (Iovine et al., 2008; Tarantino et al., 2009). This single-domain globin, Cgb, is dramatically upregulated by the transcription factor NssR in response to nitrosative stress (Elvers et al., 2005; Monk et al., 2008; Smith et al., 2011). Cgb has been shown to detoxify NO and possess a peroxidase-like heme-binding cleft. In marked contrast to Vitreoscilla Vgb, there is no evidence to date that Cgb functions in oxygen delivery. Cgb can provide an electronic “push” from the proximal ligand and an electronic “pull” from the distal binding pocket, creating a favorable environment for the isomerization of a putative ONOO intermediate in the NO dioxygenase reaction (Shepherd et al., 2010a). We have recently demonstrated that the mechanism of NO detoxification is unlikely to pro- ceed via the formation of an oxyferryl (Fe(IV)¼¼O) species but that NO interacts with the Fe(III) and Fe(II) species (Shepherd et al., 2011).

6.3.3. Truncated Globins

The third class of globins comprises the truncated proteins, which are the most recently discovered and appear widely distributed in bacteria, NITRIC OXIDE AND AGENTS OF NITROSATIVE STRESS 173 microbial eukaryotes, and plants (Wittenberg et al., 2002; Milani et al., 2003; Ascenzi et al., 2007). Instead of the classical 3-over-3 a-helical sand- wich motif adopted by single-domain globins and the heme domains of flavohemoglobins, trHbs adopt a 2-over-2 a-helical structure and are typically 20 residues shorter than 3-over-3 globins. Sequence analysis of more than 200 trHbs indicates that they can be divided into three groups: I, II, and III (sometimes referred to as N, O, and P, respectively) (Wittenberg et al., 2002). Most studies on trHbs have focused on trHb groups I and II, although the type-III trHb from C. jejuni, Ctb, has recently been structurally and kinetically characterized (Wainwright et al., 2005, 2006; Nardini et al., 2006, Lu et al., 2007b, 2008; Bolli et al., 2008). The function of this globin (named Ctb but also trHbP) remains enigmatic: although its expression is elevated on exposure to NO and RNS, via the action of the NO-responsive regulator NssR (Elvers et al., 2005; Monk et al., 2008), mutation of the ctb gene does not give an NO-sensitive phenotype (Wainwright et al., 2005). Recently we have suggested (Smith et al., 2011) that binding of NO or oxygen (Ctb having an especially high affinity for the latter) may serve to modulate the intracellular availability of NO for NssR activation. Further work is required to define a clear function; indeed, the same is true for many bacterial globins. Work conducted on M. bovis revealed that trHbn stoichiometri- cally oxidizes NO to NO3 and protects aerobic respiration from NO inhibition (Ouellett et al., 2002). It is speculated that single-domain and truncated globins associate with host reductases as a source of electrons for NO or O2 reduction. A recent study conducted on a mouse neuroglobin (Ngb) that plays a role in neuroprotection showed that NADH:flavorubredoxin oxidoreductase from E. coli could reduce the globin in the presence of NADH as the electron donor (Giuffre et al., 2008). Despite early reports (Jakob et al., 1992) of a reductase purified from Vitreoscilla that reduced the Vitreoscilla hemoglobin (Vgb, Vhb) in vitro, this aspect of the mode of action of NO-detoxifying globins remains obscure. It appears that many globins could, in principle, serve an NO scavenging function if provided with a mechanism for heme iron re-reduction such as, in vitro,ferredoxin-NADP reductase (E. coli)(Smaggheet al., 2008). The reduction mechanism may, however, involve not a specific, cognate reductase but the general reducing environment of the bacterial cytoplasm governed, for example, by the GSH pool. A similar idea has been proposed for the reduction of bacterial NOS enzymes that lack a reductase domain (Gusarov et al.,2008). 174 LESLEY A.H. BOWMAN ET AL.

6.4. NO and RNS Reductases

6.4.1. NO Reductases

In E. coli, NorR (see Section 7.3) activates the transcription of the norVW genes encoding a flavorubredoxin and an associated flavoprotein, respectively, which together have NADH-dependent NO reductase activity (Gardner et al., 2003). Confusingly, these proteins have also been referred to as FlRd and FlRd-red (flavorubredoxin reductase) (Gomes et al., 2002), names that do not relate to any E. coli gene. E. coli NorV is perhaps the most extensively characterized reductase that detoxifies NO under anaerobic and microaerobic conditions (Gardner and Gardner, 2002; Gardner et al., 2002; Gomes et al., 2002). NorV is an oxygen-sensitive flavorubredoxin with an NO reactive di-iron center that reduces NO to (N2O) as well as oxygen to water (Gomes et al., 2002). Thus, anaerobically a norV mutant exhibits impaired growth in the presence of NO and is sensitive also to SNP (Hutchings et al., 2002). E. coli also possesses a dedicated NAD(P)H flavorubredoxin oxidoreductase, NorW, whose function is believed to be rereduction of the NorV protein. Expression of E. coli NorV and NorW is regulated at the transcriptional level, via the regulator NorR, by a variety of NO-related species including GSNO (Mukhopadhyay et al., 2004; Flatley et al.,2005), NO (from a saturated solution of the gas) (Justino et al., 2005), and NO released from NOC-5 and NOC-7 (Pullan et al., 2007). A NorR protein was first discovered in R. eutropha (for a review, see Spiro, 2007). It has been suggested that NorR is a heme-based sensor (Gardner, 2005), but instead NoR is activated by the formation of a mono-nitrosyl iron center located in the GAF domain (i.e., a domain related to cyclic GMP-regulated cyclic nucleotide phosphodiesterases, adenylyl cyclase and FhlA) of the protein (D’Autreaux et al.,2005).Ithas been suggested that NorR responds exclusively to NO (Spiro, 2006), since treatment of the ferrous NorR protein with NO leads to activation in vitro (D’Autreaux et al., 2005). However, its activity can also be inferred in vivo by upregulation of norVW during growth with GSNO (e.g., Flatley et al., 2005; Pullan et al., 2007). NorR is a critical sensor of NO-related stress not only in E. coli but also in R. eutropha and P. aeruginosa (Spiro, 2007). Periplasmic cytochrome c nitrite reductase, nrfA, is expressed by many enteric pathogens including E. coli and S. enterica under microaerobic or anaerobic conditions where electron acceptors are limited. The protein cata- þ lyzes a six-electron reduction of NO2 to ammonium (NH4 ) with NO as a proposed intermediate (Simon, 2002). Indeed, we have shown that NrfA is NITRIC OXIDE AND AGENTS OF NITROSATIVE STRESS 175 responsible for much of the NO evolution that occurs in vitro when anaerobically grown cells are treated with high concentrations of nitrite (Corker and Poole, 2003) (see below). However, the role of NrfA in NO biochemistry is controversial. It has also been known for some time that NrfA can drive a five-electron reduction of exogenous NO and a physiological relevance of this function has been proposed in NO consumption (Poock et al., 2002). Wild-type cells cultured anaerobically consumed 300 nmol of NO per mg of protein per min, whereas NO reduction was negligible in nrfA mutants. Additionally, growth of nrfA mutants was completely attenuated following the addition of 150 mM NO, whereas wild-type growth was completely unperturbed (Poock et al., 2002). A recent comprehensive study of the NO detoxification machinery of Salmonella has revealed roles for all three proteins so far invoked in anaerobic NO tolerance (Mills et al., 2008). A suite of double and triple mutants with deletions in hmp, norV, and nrfA was used to conclude that the NO reductase NorV and the nitrite reductase NrfA are required additively for NO tolerance under anaerobic fermentative conditions as well as under anaerobic respiratory (glycerol with fumarate and nitrate) conditions. NO solutions (40 mM final concentration) were used (Mills et al., 2008). A minor role for Hmp was also demonstrated, consistent with the low activity of this protein in anoxic NO consumption (Kim et al., 1999). It is proposed that the periplasmic location of NrfA enables NO reduction prior to cell entry while any NO that does enter the cell is metabolized by cytoplasmic NorV (Mills et al., 2008). A constitutively expressed NrfA has also been described in C. jejui, which is proposed to provide a basal level of NO detoxification (Pittman et al., 2007).

6.4.2. Detoxification and Metabolism of SNOs

“NO biology” involves not only NO but also a family of NO-related molecules, including S-nitrosothiols (SNOs). SNO formation is considered to be a form of post-translational protein modification of importance in numerous cell signaling events (Hess et al., 2005; Foster et al., 2009a). In one early case, an enzyme capable of metabolizing GSNO ( a “SNO- lyase”) as well as protein SNOs was identified as the glutathione- dependent formaldehyde dehydrogenase of mammals, yeast, and E. coli (Liu et al., 2001). Subsequently, several other alcohol dehydrogenase proteins (AdhC) were identified as exhibiting GSNO reductase activity in N. meningitidis, H. influenza,andS. pneumoniae (Kidd et al., 2007; Potter et al., 2007; Stroeher et al., 2007). Such enzymes may have important functions in alleviating stress imposed by nitrosative species. In yeast, the 176 LESLEY A.H. BOWMAN ET AL. combined effects of flavohemoglobin and GSNO reductase (consuming NO and GSNO, respectively) modulate SNO levels. Nitrosative stress may be mediated principally by the S-nitrosylation of a particular subset of protein targets (Foster et al., 2009b). The levels of low molecular weight SNOs may be diminished not only by SNO-lyases but also by the activities of NO-consuming enzymes. Thus, the meningococcal NO reductase NorB increases the rate of SNO (GSNO) degradation in vitro and SNO formation in murine macrophages is reduced by infection with NorB-expressing meningococci or with Hmp-expressing Salmonella or E. coli (Laver et al., 2010). It appears that it is the removal of NO by these enzymes and the prevention of new SNO formation that is critical. These mechanisms may contribute to bacterial pathogenesis since S-nitrosylation is critically involved in, for example, apoptosis, signaling cascades involving neuronal NOS (Jaffrey et al., 2001), and regulation of gene expression. The reduction of GSNO has also been demonstrated by the nitroreductase, NtrA, from Staphylococcus aureus (Tavares et al., 2009), which binds GSNO with a higher affinity than does the glutathione- dependent formaldehyde dehydrogenase of E. coli. Additionally, the thioredoxin system has a demonstrated role in GSNO reduction, as shown by the accelerated breakdown of this nitrosating species in vitro by thioredoxin/thioredoxin reductase isolated from both E. coli (Nikitovic and Holmgren, 1996) and M. tuberculosis (Attarian et al., 2009). Further to this, Helicobacter pylori strains deficient in either of its two thioredoxin proteins have been shown to be more sensitive to GSNO and SNP than the isogenic wild-type strain, suggesting that the thioredoxin system may be involved in SNO depletion (Comtois et al., 2003).

6.5. Other Proteins Implicated in NO Tolerance

Mutational and transcriptomic approaches have revealed additional proteins with known or inferred roles in protection from NO or RNS. Rather than speculate here on the possible roles of the numerous genes identified as upregulated by these stresses, we focus on a subset that have received more careful consideration. A transcriptomic analysis of anaerobically grown E. coli treated with NO (50 mM, from a solution of the gas) revealed upregulation not only of hmp, the norVW operon (Sections 6.3.1 and 6.4.1), and genes of intermediary metabolism, but also of ytfE (see below) and the gene for a LysR-type regulator, yidZ (Justino et al., 2005). Mutation of either gene rendered cells hypersusceptible to NO. NITRIC OXIDE AND AGENTS OF NITROSATIVE STRESS 177

The ytfE gene has subsequently been shown to be regulated by NO or other RNS in several other studies and is one of a very small group of genes that is consistently and massively upregulated on bacterial exposure to NO and nitrosative stress. It is widely accepted that the YtfE protein plays an important role in Fe–S cluster metabolism/repair (Justino et al., 2007) and it has been proposed that the protein should be renamed RIC (Repair of Iron Centers) (Overton et al., 2008). A recent reanalysis of strains carrying a “clean” ytfE mutation confirms a role in iron–sulfur cluster repair and also points to a role for YtfE in H2O2 resistance (Vine et al., 2010). The E. coli protein is a dimer with two Fe atoms per monomer. Spectroscopic analysis of the purified protein reveals a nonheme dinuclear iron center having m-peroxy and m-carboxylate bridging ligands and six His residues coordinating the irons (Todorovic et al., 2008). Homologues of YtfE occur in numerous bacteria (Overton et al., 2008). In Haemophilus influenzae, for example, ytfE mutants are sensitive to agents of nitrosative stress, although very high concentrations of acidified sodium nitrite (10 mM) and GSNO (5 mM) were used and described as “NO donors” by Harrington et al. (2009). A ytfE mutant was also sensitive to macrophage assaults, an effect that was abrogated by an inhibitor of NOS. Inter- estingly, in this organism, activation of ytfE is dependent on positive control by Fnr (see Section 7.2), despite the fact that Fnr is itself an iron–sulfur cluster protein, whereas in E. coli, ytfE expression is negatively regulated by Fnr although the effect may be indirect (Justino et al., 2006). Several transcriptomic studies suggest a role in NO responses of the hcp and hcr genes, encoding the hybrid cluster protein (an iron–sulfur protein) and its cognate reductase. These proteins may form a hydroxylamine oxidoreductase that converts NH2OH to ammonia (Wolfe et al., 2002; Cabello et al., 2004) but a role in resisting peroxide stress has also been proposed (Almeida et al., 2006).

6.6. Beneficial Effects of NO in Microbial Symbioses

NO is not only a toxic radical but may have beneficial effects in certain microbial lifestyles, particularly biofilm formation and symbioses (for a review, see Wang and Ruby, 2011). The first report of a bacterial NO response in symbiosis was that of Meilhoc and others who showed that Sinorhizobium meliloti upregulates >100 genes in response to NO, including hmp, and that an hmp mutant showed decreased nitrogen fixation in planta. It is proposed that this detoxification system overcomes the inhibitory effects of NO on nitrogen fixation during symbiosis (Meilhoc et al., 2010). 178 LESLEY A.H. BOWMAN ET AL.

In one well-studied example, the squid-Vibrio light-organ symbiosis, NO serves as a signal, antioxidant and specificity determinant (Wang and Ruby, 2011). In the early stages of the symbiosis, NOS is active and this enzyme and NO are detectable in vesicles of the mucus secretion where Vibrio fischeri cells aggregate. Experiments with NO scavengers demonstrate that this NO influences specificity of the interaction since NO removal permits nonsymbiotic Vibrio species to form hyperaggregates. As V. fischeri penetrates the deep crypts of the light organ, the bacterium experiences higher levels of NO and colonization irreversibly reduces the NO and NOS levels there. The host therefore uses NO to sense and respond to the correct symbiont. The genetic and biochemical evidence suggests that V. fischeri possesses an NO sensor, H-NOX (heme NO/oxygen binding; Wang et al., 2010a), which governs the response of at least 20 genes to the NO in the squid. Ten of these genes are Fur-regulated, presumably reflecting the iron- limited environment of the host and the fact that the host supplies iron in the form of hemin (Wang et al., 2010a). To manage the NO levels to which V. fischeri is exposed, it possesses several NO detoxification systems including those encoded by the hmp, norVW, and nrf genes (Wang et al., 2010b). The bacterium also contains an NO-insensitive terminal oxidase, AOX (Dunn et al., 2010; Spiro, 2010), with an unidentified redox center(s) and mode of action. This fascinating symbiosis will surely reveal new aspects of NO as a signaling molecule in modulating symbioses.

6.7. Microbial Responses to ONOO Stress

It is frequently supposed that superoxide anion from the oxidative burst (Phox or NOX2, the NADH-dependent phagocytic oxidase) and NO from iNOS combine to generate ONOO , which exerts greater toxicity than either radical alone. However, the roles of Phox and iNOS are both tempo- rally (Vazquez-Torres et al., 2000) and genetically (Craig and Slauch, 2009) separable during Salmonella infection, so it is questionable whether ONOO is a major antimicrobial species in this scenario. Peroxynitrite may also arise from the activity of a single enzyme: exposure of murine macrophages to Bacillus anthracis endospores upregulates NOS2. This isozyme generates not only NO but also superoxide, and the anticipated product, ONOO , is detectable by the dihydrorhodamine assay (peroxynitrite-mediated oxidation of dihydrorhodamine). In this case, ONOO does not appear to have microbicidal activity (Weaver et al., 2007). In other examples, ONOO demonstrates toxicity toward a broad NITRIC OXIDE AND AGENTS OF NITROSATIVE STRESS 179 range of pathogens, including E. coli (Zhu et al., 1992; Brunelli et al., 1995), H. pylori (Tecder-Unal et al., 2008), and Trypanosoma cruzi (Alvarez et al., 2004). However, evidence is increasing that pathogens possess systems that directly detoxify ONOO , allowing them to evade this species and thrive. In Salmonella typhi,anrpoS mutant is more susceptible to ONOO than a wild-type strain but the molecular basis of this is at present obscure (Alam et al., 2006). Detailed examples will be discussed below. Peroxiredoxins are typically associated with the reduction of H2O2 and organic hydroperoxides and are widespread throughout prokaryotic and eukaryotic systems (Poole, 2005a). The peroxiredoxin alkylhydroperoxide reductase subunit C (AhpC) isolated from S. enterica enabled catalytic breakdown of ONOO to NO2 with a second-order rate constant of 1.51106 M 1 s 1. This proceeds via oxidation of a cysteine residue toward the N-terminus of the protein, and catalytic turnover is achieved by reduction of AhpC by the flavoprotein AhpF. Catalysis was shown to be efficient in protecting plasmid DNA from single-strand breaks (Bryk et al., 2000). Peroxiredoxins with peroxynitritase activity, enzymes with the ability to break down ONOO , have also been identified in other microbes, including S. cerevisiae. This eukaryote possesses two peroxiredoxins, thioredoxin peroxidase I and II (Tsa1 and Tsa2), which share 86% identity at the amino acid level. These proteins catalyze the breakdown of ONOO with reported second-order rate constants in the region of 105 M 1 s 1 but lack a dedicated reductase partner. Enzymatic activities are thus recycled by the thioredoxin/ thioredoxin reductase system (Ogusucu et al., 2007). Catalase-peroxidases offer an alternative to peroxiredoxins in the catalytic turnover of ONOO . Classically, these heme-containing enzymes are characterized by their bifunctional capacity to break down H2O2 and organic peroxides (Claiborne and Fridovich, 1979). However, the catalase-peroxidase KatG from M. tuberculosis (Wengenack et al., 1999) and S. enterica (McLean et al., 2010a) have also demonstrated peroxynitritase activities with reported second-order rate constants of 1.4105 M 1 s 1 and 4.2104 M 1 s 1, respectively. Following incubation with ONOOH, Wengenack et al. (1999) demonstrate an initial reduction in the Soret band of KatG followed by recovery upon exhaustion of ONOOH. This implic- ates the involvement of heme in the catalysis of ONOO breakdown. Table 4 lists the second-order rate constants of proteins isolated from pathogenic organisms that exhibit peroxynitritase activity. In light of the peroxynitritase activities of certain peroxiredoxins and catalase- peroxidases, it would appear that these enzymes have the capacity to detoxify a repertoire of reactive species, making them potentially powerful virulence factors. Table 4 Second-order rate constants of proteins with peroxynitritase activities.

10 5 Second-order Protein Organism/Source Temperature (C)/pH rate constant (M 1 s 1) Reference

KatG E. coli 25/7.40 0.33 L. Bowman and S. McLean, unpublished finding KatG S. enterica 25/7.40 0.42 McLean et al. (2010a) KatG M. tuberculosis 37/7.40 1.40 Wengenack et al. (1999) AhpC H. pylori RT/6.75 12.10 Bryk et al. (2000) AhpC M. tuberculosis RT/6.75 13.30 Bryk et al. (2000) AhpC S. enterica RT/6.75 15.10 Bryk et al. (2000) Thioredoxin S. cerevisiae 25/7.40 5.10 Ogusucu et al. (2007) peroxidase II Thioredoxin S. cerevisiae 25/7.40 7.40 Ogusucu et al. (2007) peroxidase I Tryparedoxin T. cruzi 37/7.40 7.20 Trujillo et al. (2004) peroxidase Tryparedoxin T. brucei 37/7.40 9.00 Trujillo et al. (2004) peroxidase

RT—room temperature. NITRIC OXIDE AND AGENTS OF NITROSATIVE STRESS 181

7. MICROBIAL SENSING OF NO AND GENE REGULATION

7.1. Introduction

Since the discovery that expression of the flavohemoglobin Hmp of E. coli is upregulated by NO and that this protein constitutes an effective NO detoxification enzyme, there has been an explosion of interest in the molecular mechanisms underpinning the regulation of this gene and the many others now implicated in NO and RNS detoxification. Excellent reviews have recently appeared (Spiro, 2006, 2007), and the reader is referred to those articles for details. In brief, NO activates gene expression via SoxR (Ding and Demple, 2000), Fnr (Cruz-Ramos et al., 2002), OxyR (Kim et al., 2002), NorR (D’Autreaux et al., 2005), Fur (D’Autreaux et al., 2004), and NsrR (Bodenmiller and Spiro, 2006). Spiro usefully distinguishes between those that are secondary sensors and those that are “dedicated” in that their physiological function appears to be detecting primarily NO and then regulating expression of genes that encode enzymes with NO as a substrate. The archetype is NsrR, which appears in enterobacteria to be the major regulator for NO-detoxifying proteins like flavohemoglobin. In addition to sensing NO in solution, these regulators may also sense the small levels of NO that may emanate from SNOs. Here we present only a few comments on some of the major regulators, emphasizing the sensing of NO. NO sensors are also known in higher organisms such as the mammalian circadian protein CLOCK (Lukat-Rodgers et al., 2010).

7.2. Fnr

Fnr is an example of the large Fnr-CRP family of transcriptional regulators widely distributed in bacteria (Green et al., 2001). The family also includes the CO sensor CooA and the first clear example of an NO sensor, namely, the NnrR protein of Rhodobacter sphaeroides (Tosques et al., 1996). There is strong evidence that it is NO that is sensed by NnrR and other members of the family (Spiro, 2007). The first clue to the involvement of Fnr in hmp regulation was the finding that a lysogenic hmp–lacZ reporter construct was more highly expressed in an fnr mutant than in the parent strain (Poole et al., 1996), indicating that Fnr represses hmp transcription and consistent with the presence of an Fnr binding site close to the 10 sequence of the hmp promoter, to which Fnr binds (Cruz-Ramos et al., 2002). NO modifies the [4Fe–4S] cluster of Fnr in vitro to form 182 LESLEY A.H. BOWMAN ET AL. dinitrosyl-iron-dithiol (DNIC) complexes (for a review, see Tonzetich et al., 2010), relieving repression of hmp. The Fnr-like protein of Azotobac- ter vinelandii, CydR, is also NO sensitive (Wu et al., 2000). A further member of this family is the NssR protein of C. jejuni that controls a small regulon; some of the genes therein, notably that encoding the hemoglobin Cgb, are known to be directly involved in NO detoxification. Although NssR has been reported so far to respond only to GSNO, it is known that Cgb gene regulation is activated by NO. However, we have been unable to date to demonstrate any effect of NO or GSNO on DNA binding by the purified NssR protein (Smith et al., 2011).

7.3. NorR

NorR is a s54-dependent enhancer binding protein. In R. eutropha, it elicits NO-triggered activation of a two-gene operon (norAB) encoding the respiratory nitrate reductase. The homologue in E. coli activates the divergently transcribed norVW operon in response to NO, SNP, GSNO, or acidified nitrite (see Section 6.4.1). These genes encode a flavorubredoxin and its cognate reductase that reduce NO to N2O. NorR is activated by formation of mono-nitrosyl complex at a mono-nuclear iron center situated in the GAF domain (D’Autreaux et al., 2005; Tucker et al., 2005). Evidence that NorR senses NO comes from experiments in vitro that demonstrate activation on treating the Fe(II) form with NO. In P. aeruginosa, NorR activates the divergent hmp-like gene fhp (Arai et al., 2005).

7.4. NsrR

NsrR is widely distributed in Gram-negative bacteria, but in the gammaproteobacteria, its function is assumed by NorR, which controls expression of the hmp gene in P. aeruginosa and Vibrio cholerae (for a review, see Tucker et al.,2010). NsrR was first identified genetically in E. coli in 2005 and is now considered a major regulator of the hmp, ygbA, nrfA, and ytfE genes and others in E. coli (Mukhopadhyay et al., 2004; Rodionov et al., 2005; Bodenmiller and Spiro, 2006). In E. coli and Salmonella, NsrR represses its regulated genes so that, in an nsrR mutant, exceptionally high levels of Hmp, for example, are made (Gilberthorpe et al., 2007), exceeding those observed in a wild-type strain in the presence of 1 mM GSNO. Recent studies on three NsrR proteins (from Streptomyces coelicolor, B. subtilis, and Neisseria gonorrhoea), expressed in and purified from E. coli,showthatNsrRisan iron–sulfur protein, although the cluster is not the same in each case (for a NITRIC OXIDE AND AGENTS OF NITROSATIVE STRESS 183 review, see Tucker et al., 2010). The present picture is that NsrR proteins contain an O2-stable [2Fe–2S] cluster that is sensitive to NO. The discrepancies in the literature raise the concern that, on expression in E. coli, inappropriate clusters, relative to the native bacterium, are incorporated. Alternatively, clusters in vivo may become degraded during aerobic purification. Whatever the answer, NsrR senses NO via an iron–sulfur cluster which is nitrosylated, leading to derepression of target genes.

7.5. Others (DOS, FixL, GCS)

M. tuberculosis survives hypoxia and NO by entering a dormant state of non- replicating persistence. The dormancy regulon that allows this transition is activated by the response regulator component DevR of the two-component regulatory mechanism called DevSRT (also known as DosSRT) (Dasgupta et al., 2000; Sardiwal et al.,2005). DevS and DosT each possess heme cofactors to sense O2, being inactive when heme(II) binds the ligand. Anoxia, but also the presence of NO or CO, leads to activation. The stability of the Fe(II)–O2 complex is enhanced by interdomain interactions, making DevS an efficient gas sensor. The proteins autophosphorylate and then transfer a phosphate group to the gene regulator DevR. Recent studies (Yukl et al., 2011) show that the heme–O2 form of DevS reacts efficiently with NO to produce nitrate in a reaction reminiscent of the dioxygenase activity of Hmp and some other globins. The resultant Fe(III) form of DevS is inactive but has a high affinity for NO. The data suggest that, on exposure to NO, the inactive oxy–heme complex is rapidly converted to Fe(II)–NO, triggering the onset of the dormant phase and promoting mycobacterial survival.

8. GLOBAL AND SYSTEMS APPROACHES TO UNDERSTANDING RESPONSES TO NO AND RNS

Due to the complex nature of the interactions between microorganisms and various RNS, many studies now utilize a variety of “omic” techniques available to more fully understand the targets of and responses to these stresses. Global/systems approaches have the advantage of being able to highlight predicted as well as unexpected and novel responses of the microbe. These approaches are able to show us the interactions, robust- ness, and modularity of the complex microbial systems in place for the sensing and detoxification of RNS. 184 LESLEY A.H. BOWMAN ET AL.

Both transcriptomic and proteomic techniques have been used to investigate nitrosative stress in microorganisms. Computational modeling is also increasingly utilized to interpret the vast amount of data generated by such approaches. However, care must be taken in the design of experiments so that interpretation is able to accurately reflect the events occurring in vivo.

8.1. Methodology

8.1.1. Culture Conditions

The majority of experiments on growing microbial cultures are conducted under batch culture conditions. Here, an initial volume of media and culture is added to a closed system and growth is monitored over time with no further addition or expulsion of media. In such a system, nutrients will become limited and metabolite levels will increase over time. In response, the cell population physiology will adapt to the changing conditions. These changes are in addition to adaptations made in response to the addition of a stressor such as NO. However, for detailed transcriptomic and proteomic analysis, it is highly preferable to keep all conditions constant so that gene/protein alterations can be attributed solely to addition of the stress and not to other factors such as changes in growth rate or differences in nutrient levels. In continuous chemostat culture, fresh medium is pumped into the culture vessel and surplus culture medium removed to maintain a constant volume. In this system, growth becomes limited by 1 or more nutrients in the medium. After this “steady state” is reached, growth rate is controlled by the addition of fresh medium and hence fresh delivery of the limiting nutrient. In this way, growth rate and therefore population density can be controlled. This approach can be utilized with complex media; however, the use of a defined medium allows identification and tight control of a single limiting nutrient, frequently the carbon or nitrogen source. Defined media can also ensure the bioavailability of all micronutrients, which could otherwise be sequestered by components of a complex medium such as Luria broth (Hughes and Poole, 1991). Other forms of continuous culture (e.g., tur- bidostat, pH auxostat) have been developed (Pirt, 1985); however, work detailed in this chapter, unless otherwise stated, will focus upon data accumulated using batch or continuous chemostat culture, which has been utilized for investigation of a variety of nitrosative stresses. An important criticism of continuous culture is the accumulation of loss-of-function rpoS mutations, which can overtake the culture (Notley- NITRIC OXIDE AND AGENTS OF NITROSATIVE STRESS 185

McRobb et al., 2002). However, further analysis found that this phenome- non does not occur in anaerobic cultures (King and Ferenci, 2005), nor does it occur, for example, in the E. coli MG1655 strain (King et al., 2004) used in many microarray studies though it is clear that strict monitoring of cell cultures is required. This, and other evolutions, may affect the usefulness of chemostat cultures in providing a physiologically stable population for examination compared to batch culture. However, it is almost certain that undesirable and uncontrolled changes in growth rate will affect the outcome and interpretation of batch growths when growth inhibitory compounds are added. This is highlighted in the literature where batch culture experiments are more variable than those conducted under continuous culture conditions (Piper et al., 2002).

8.1.2. Transcriptomics

Microarray analysis is a valuable tool for investigating microbial targets of and responses to nitrosative stress. This method of measuring global changes in gene expression in response to RNS has been utilized in a number of studies with a variety of bacteria, including M. tuberculosis (Ohno et al., 2003), B. subtilis (Moore et al., 2004; Rogstam et al., 2007), P. aeruginosa (Firoved et al., 2004), C. jejuni (Elvers et al., 2005), C. neoformans (Chow et al., 2007), S. aureus (Richardson et al., 2006; Schlag et al., 2007), and Neisseria meningitidis (Heurlier et al., 2008). How- ever, it is perhaps unsurprising that the responses of E. coli to nitrosative stress have been most widely studied (Mukhopadhyay et al., 2004; Flatley et al., 2005; Justino et al., 2005; Pullan et al., 2007; Bower et al., 2009; McLean et al., 2010c). It is in this literature that the stark differences between data sets utilizing differing culture conditions can be readily identified with very few transcriptional units being identified in common. In addition to culture conditions, it is also important to distinguish between the different agents of RNS-mediated stress used as they cannot be used þ interchangeably (e.g., NO , NO, NO ), given that they have unique reaction chemistries (Hughes, 1999; Aga and Hughes, 2008) (Section 4.5) and elicit quite different responses (Flatley et al., 2005; Pullan et al., 2007).

8.1.3. Modeling

Due to the ever-increasing amounts of data derived from “omic” studies and the desire to obtain information on the regulatory networks that cause changes in gene transcription, new methods are required to assess and 186 LESLEY A.H. BOWMAN ET AL. accurately interpret data. Computational modeling allows a quantitative estimation of the regulatory relationship between transcription factors and genes (Sanguinetti et al., 2006). Probabilistic state space modeling has been utilized in a number of recent microbial studies concerned with monitoring E. coli responses to various environmental changes (Partridge et al., 2007; Davidge et al., 2009b; Shepherd et al., 2010b). McLean et al. (2010c) used this modeling technique to highlight changes in the activity of four transcription factors of E. coli due to ONOO exposure (OxyR, ArgR, CysB, and PhoB) and were also able to confirm a lack of activity of some transcription factors that the microarray data suggested may have been altered (e.g., FNR and IHF). A comparison of this microarray data was also made with data obtained from exposure to H2O2 using mathematical modeling, which allowed the similarities and differences between the two stresses to be highlighted at transcription factor level. However, as with any comparison, experimental design and continuity are of paramount importance in order to ensure the models generated are meaningful.

8.1.4. Proteomics

The measurement of changing protein levels has classically been measured using techniques such as 2D gel electrophoresis or liquid chromatography followed by mass spectrometry. For the former, protein samples are separated by isoelectric point (pI) and/or molecular weight and detected by staining. Protein levels are quantified according to staining intensity and subsequent identification performed by isolation of protein bands, sample digestion, and mass spectrometry. This technique has been widely used for decades and is the basis for several proteomic analyses of nitrosative stress in microbial cultures (Monk et al., 2008; Qu et al., 2009). Early techniques focused upon the identification and characterization of proteins; however, more recently, the focus has extended to the quantitative and comparative measurement of global changes of protein transcription via the use of chromatography, mass spectrometry, and bioinformatics. One technique used for investigation of the response of Desulfovibrio vulgaris to nitrate stress was via a shotgun proteomic method utilizing isobaric tags for relative and absolute quantitation (iTRAQ) (Redding et al., 2006). This method, commercialized by Applied Biosystems in 2004, enables the simultaneous identification and quantification of peptides using mass spectrometry and allows the parallel proteome analysis of up to four samples via the labeling of primary amines with amine-specific NITRIC OXIDE AND AGENTS OF NITROSATIVE STRESS 187 isobaric reagents (Thompson et al., 2003; Ross et al., 2004). Once tagged, the labeled peptides from different samples (e.g., pre- and poststress) are mixed, separated using 2D liquid chromatography and analyzed using mass spectrometry and tandem mass spectrometry. The iTRAQ-tagged peptides release reporter ions, the quantities of which are recorded and the peak area used to quantitate the relative abundance of their originating proteins (Ross et al., 2004). Approaches have also been developed to specifically identify S-nitrosated targets across a given organism’s proteome following challenge with RNS. The biotin switch method developed by Jaffrey et al. (2001) was pioneering in singling out SNO-bound proteins. It operates by blocking free cysteine thiols via S-methylthiolation followed by reduction of S-nitrosothiols with ascorbate. Thiols are subsequently labeled with a sulfhydryl-specific biotinylating agent. Labeled proteins are isolated by a streptavidin pull-down assay and separated by SDS-PAGE. Bands of interest are cut from the gel and subjected to MALDI-TOF to uncover modified proteins. Jaffrey et al. (2001) used this technique to identify endogenously S-nitrosated proteins in a rat model by comparing data derived from a wild-type mouse and an nNOS mutant. Findings revealed that targets included metabolic, structural, and signaling proteins (Jaffrey et al., 2001). Modifications to the Biotin Switch method have lead to the development of several novel techniques that couple labeling of NO-bound thiols to direct peptide capture methods that automate the detection of altered proteins. A prime example is the relatively novel S-nitrosothiol capture (SNOCAP) technique (Paige et al., 2008) that links attributes of the Biotin Switch method with the isotope-coded affinity tag (iCAT) approach. iCAT was developed to differentially label two biological samples with discrete isotope labels that were subsequently subjected to liquid chromatography tandem mass spectrometry (LC-MS/MS) to identify and quantify differences in protein expression levels. The tag comprises a thiol reactive group, a heavy or light isotope linker and a biotin affinity tag. Labels were designed to react with free sulfhydryl groups that are ubiquitous in proteins, enabling wide coverage of the proteome (Gygi et al., 1999). SNOCAP, on the other hand, was developed to solely label thiol groups derived from the reduction of S-nitrosothiols (Paige et al., 2008). In brief, free thiols are blocked and S-nitrosothiols are reduced with ascorbate to form thiols. Heavy and light isotopically labeled thiol biotinylating agents are used to tag newly formed thiols from cell populations subjected to two different biological conditions. These samples are mixed and tryptically digested, and tagged proteins are purified using neutravidin. The sample mixture is subsequently subjected to LC-MS/MS, which allows for the identification of modified proteins 188 LESLEY A.H. BOWMAN ET AL.

(without the need for gel electrophoretic separation) and enables the relative quantification of SNO sites between two different samples. Liquid chromatography is employed to reduce the complexity of the sample by separating peptides before MS analysis. The mass spectrophotometer is subsequently set up to measure the relative signal intensities of identical peptide pairs that only differ in mass by a fixed value dictated by the mass difference of the heavy and light isotope labels. Operating in MS/MS mode, the amino acid sequences of individual fragmented peptides contained in the digest mixture can be determined. Databases are then queried to identify proteins from which the sequenced peptides originated. Paige et al. (2008) successfully employed SNOCAP to uncover glutathione-reducible and nonreducible proteins in cultured cells. This method was the first to enable the relative quantification of S-nitrosation on a proteome-wide scale between two different samples.

8.2. Outcomes from Global Transcriptomic Approaches

8.2.1. Responses of E. coli

8.2.1.1. Responses of E. coli to NO and RNS In E. coil, a number of nitrosative stresses have been analyzed using transcriptomics. In particular, the response to NO, GSNO, and ONOO has been examined in the Poole laboratory utilizing the same continuous culture and defined minimal media conditions, which allows for a direct comparison of the effects of the three reactive nitrogen species. Transcriptome profiling experiments were used to investigate the transcriptional basis of the response to the presence of GSNO in both aerobic and anaerobic cultures of E. coli MG1655 (Flatley et al., 2005). Aerobically, 17 genes were upregulated, most notably those involved in the detoxification of NO and methionine biosynthesis. Among the NO detoxification genes upregulated were hmp and norV, which encode an NO-consuming flavohemoglobin and flavorubredoxin, respectively (Sections 6.3 and 6.4). Additionally, the transcription of six genes involved in methionine biosynthesis or regulation was significantly elevated. Mutants of metN, metI, and metR exhibited growth sensitivity to GSNO, and exogenously provided methionine was found to rescue this phenotype. This supports the hypothesis that GSNO nitrosates homocysteine, withdrawing it from the methionine biosynthesis pathway. Anaerobically, 10 genes were significantly upregulated in response to GSNO, of which, norV, hcp, metB, metR, and NITRIC OXIDE AND AGENTS OF NITROSATIVE STRESS 189 metF were also upregulated aerobically, suggesting that the response to GSNO is broadly similar under both conditions. These data revealed new genes important for GSNO tolerance and demonstrated that methionine biosynthesis is a casualty of nitrosative stress, an observation not seen in previous studies utilizing batch culture conditions and complex media (Mukhopadhyay et al., 2004). Further transcriptomic work aimed to compare and contrast the responses of E. coli to the nitrosating agent GSNO with that of NO. Previous studies investigated the effects of NO under both aerobic and anaerobic conditions, but these observations were made only in batch culture (Mukhopadhyay et al., 2004; Justino et al.,2005) and thus could not be legitimately compared to data obtained in chemically defined medium under continuous culture conditions. The resulting study (Pullan et al., 2007) made use of the same defined minimal medium and identical continuous culture conditions as the GSNO work. Addition of NO in this study utilized the addition of the NO- releasing compounds, NOC-5 and NOC-7, which release NO with half-lives of 5 and 25 min, respectively, at pH 7.0 and 37 C. The data revealed some similarities and several marked differences in the transcriptional responses to these distinct nitrosative stresses. NO causes an upregulation of nitrosative stress response genes such as hmp and norV, as in the anaerobic study. Responses appeared to be regulated by global regulators including Fnr, IscR, Fur, SoxR, NsrR, and NorR. Anaerobically, evidence for the NO inactivation of Fnr was seen, with upregulation of Fnr-repressed genes and downregulation of Fnr-activated genes being observed. Notably, expression of none of the met genes was altered, suggesting that homocysteine nitrosation does not occur and so, in contrast to GSNO, methionine biosynthesis is not a target of NO stress under anoxic conditions. Jarboe et al. (2008) extended these observations by applying network component analysis to transcriptomic data sets and showed that GSNO targets homocysteine (Hcy) and cysteine with disruption of the methionine biosynthesis pathway. Reaction of GSNO with Hcy and Cys resulted in altered regulatory activity of MetJ, MetR, and CysB, activation of the stringent response, and growth inhibition. Supplementation with methionine abrogated the GSNO effects (Pullan et al., 2007; Jarboe et al., 2008) but was without effect on NO sensitivity (Pullan et al., 2007). Hcy was earlier reported to be an effective endogenous antagonist of GSNO-mediated cytotoxicity (Degroote et al., 1996). Further distinction between the effects of NO and GSNO is provided by the observation that the upregulation of Hmp via the transcriptional regulator NsrR may be demonstrated to arise from the submicromolar levels of NO released from GSNO, but GSNO internalization is not required for this (Jarboe et al., 2008). 190 LESLEY A.H. BOWMAN ET AL.

E. coli K-12 samples were also investigated for their anaerobic response to NO gas in minimal salt medium in batch cultures (Justino et al., 2005). The authors found that norVW and hmp were significantly upregulated, highlighting the importance of Hmp for the detoxification of NO in anaerobic conditions as shown by Kim et al. (1999). In addition, Fur- and FNR- mediated repression was relieved and genes responsible for iron–sulfur cluster assembly/repair were upregulated (ytfE and the isc and suf operons), in broad agreement with the later study by Pullan et al. (2007). E. coli MG1655 was also the strain used for a study of aerobic transcriptional responses to GSNO and acidified sodium nitrite (Mukhopadhyay et al., 2004) in rich medium and batch culture conditions. Again, upregulation of the nitrosative stress detoxification genes norVW and hmpA was seen in response to both acidified nitrite and GSNO. This study suggested that, under these conditions, the sensing of NO was also mediated by modification of the transcription factor, Fur implicating iron limitation as a consequence of nitrosative stress. However, involvement of the Fur regulator was not seen in later work by Flatley et al. (2005); this could be due to the choice of media used in each case as the bioavailability of iron in the Luria broth used by Mukhopadhyay and coworkers was likely low, due to a lack of chelators in the medium (Hughes and Poole, 1991). There was also no evidence for upregulation of the methionine biosynthesis pathway in the data derived from rich medium, reflecting the potential discrepancies between data sets collected under differing experimental conditions. Uropathogenic Escherichia coli (UPEC), responsible for many urinary tract infections in humans, encounter multiple stresses during their transit through the body including RNS. Recently, data have been presented to show that UPECs preconditioned with acidified sodium nitrite were better able to colonize the bladders of mice than nonconditioned bacteria (Bower et al., 2009). Microarray analysis of the UPEC response to acidified sodium nitrite suggested that upregulation of NsrR-regulated genes, multiple genes involved in the transport and metabolism of polyamines, and other stress responsive factors may be responsible for the competitive advantage. This data suggest that the route of infection and hence the stresses encountered by the bacterium (e.g., RNS) have a major impact upon host colonization and bacterial survival.

8.2.1.2. Responses of E. coli to ONOO Transcriptomic analysis of the E. coli response to ONOO (McLean et al., 2010c) was also undertaken using defined minimal media and continuous culture conditions in order to assess the effects of this highly reactive species NITRIC OXIDE AND AGENTS OF NITROSATIVE STRESS 191 and to allow direct comparison to the GSNO and NO data sets. This study utilized a bolus addition of commercial ONOO to the vessel and feed line. The resulting microarray data revealed that, in contrast to GSNO and NO, a number of oxidative stress response genes were upregulated, most notably katG and ahpCF. KatG has been proposed to act as a peroxynitritase in M. tuberculosis (Wengenack et al., 1999) as have KatG and AhpCF in S. enterica (Bryk et al., 2000) (McLean et al., 2010a). Interestingly, the expression of none of the recognized nitrosative stress response genes was altered after ONOO exposure, indicating that this reactive species does not act as a classical nitrosative stress, as do NO and GSNO (Flatley et al., 2005; Pullan et al., 2007). However, the lack of response by some of the classical nitrosative stress response genes is not surprising as proteins such as Hmp and NorVW specifically detoxify NO, levels of which would not be expected to increase during ONOO exposure. Further, an increase in Hmp expression during ONOO stress has deleterious effects on S. enterica (McLean et al., 2010b), causing hypersensitivity to the stress. This is probably due to the Hmp-catalyzed production of superoxide in the absence of NO (Orii et al., 1992; Membrillo-Hernandez et al., 1996; Wu et al., 2004). The regulation of other nitrosative stress response genes including those responsible for nitrite detoxification (nrfA and hcp) was unaltered by ONOO exposure, suggesting that, while undoubtedly levels of nitrite will increase upon exposure to ONOO , levels were either insufficient to upregulate these genes or detoxification of ONOO is more significant than removal of the compar- atively inert nitrite. Some genes that were upregulated are of unknown function and could play a role in response to the apparent S-nitrosylation of thiols or in response to the nitration of tyrosine residues in proteins. Other targets of ONOO suggested by interpretation of the microarray data included cysteine (cys) and arginine (arg) biosynthesis as well as genes involved in iron–sulfur cluster assembly/repair (suf and isc genes), the high-affinity phosphate transport system (pst), and the (gsi) glutathione import system. The transcription levels of several genes encoding membrane and transport proteins were also altered both positively and negatively, which suggests that ONOO reacts with proteins in the membrane as well as causing lipid oxidation and nitration (Radi et al., 1991; Szabo et al., 2007) during its passage into the cell.

8.2.1.3. An emerging picture of the NO and RNS responses in E. coli There are only a small subset of genes altered in response to more than one of the above stresses and none whose transcript levels are altered by all three (Fig. 8.). Upregulated in response to GSNO and NO are genes 192 LESLEY A.H. BOWMAN ET AL.

Figure 8 The global responses of E. coli to discreet nitrosative stresses. A simplified comparative overview of E. coli transcript levels altered in response to GSNO, NO, and ONOO are represented in the Venn diagram as well as areas of common response between stresses.

involved in NO resistance and those that elicit nitrosative stress resistance (NorR and NsrR regulons, respectively). Unique to the NO and ONOO responses are those genes responsible for iron–sulfur cluster assembly and repair, indicating a common target of these two species. The only gene significantly altered by both GSNO and ONOO codes for a poorly characterized protein, YeaJ. This protein is suggested to be a putative diguanylate cyclase due to the presence of a characteristic GGDEF motif and is responsible for synthesis of the second messenger, cyclic di-GMP. Many proteins containing the GGDEF domain also contain other domains that can receive signals. YeaJ contains an upstream motif suggested to be an S-nitrosation site (TDCD) (Stamler et al., 1997), which could provide a signal for some of the cellular responses to RNS. NITRIC OXIDE AND AGENTS OF NITROSATIVE STRESS 193

8.3. Responses of Other Microbes to RNS

8.3.1. C. jejuni

C. jejuni, a predominant causative agent of bacterial gastrointestinal disease worldwide (Friedman et al., 2000), was found to significantly upregulate eight genes in response to GSNO stress (Elvers et al., 2005) including cgb, which codes for a single-domain globin known to protect the bacteria from nitrosative stress; ctb (Cj0465c), a truncated globin; and six genes of unknown function. Further work confirmed the rapid response of cgb upregulation as a protective measure against GSNO (Monk et al., 2008). In this study, other genes showing enhanced transcription levels were the truncated globin ctb and a variety of heat-shock response, iron transport, and oxidative stress response genes including trxA, trxB, ahpC, and two putative oxidoreductases. Although the function of the hemoglobin Cgb is well established in NO detoxification (see Section 6.3.2), the function of the truncated globin Ctb is less clear. When ctb is mutated, no major compensatory transcriptomic adaptations are evident (Smith et al., 2011). One hypothesis is that, by binding NO or O2 avidly, Ctb dampens the response to NO under hypoxic conditions, perhaps because Cgb function (NO detoxification) is O2 dependent (Smith et al., 2011). Further work is needed to understand the role of this truncated globin.

8.3.2. B. subtilis

The Gram-positive soil bacterium B. subtilis contains an NOS (Adak et al., 2002a) and also coexists with denitrifying bacteria, so it is likely to have developed mechanisms of detoxification of both exogenously and endogenously formed RNS. Microarray analysis of B. subtilis exposure to NO-saturated solutions under aerobic conditions revealed that the most strongly induced genes were hmp and members of the sB, Fur, and to a lesser extent, PerR regulons (Moore et al., 2004). Anaerobically, the same genes were upregulated, however; the strongest responses were those of hmp and members of the Fur and PerR regulons with sB genes being only slightly upregulated. B. subtilis cultures exposed to SNP showed induction of hmp as well as the sB, ResDE (Ye et al., 2000) and Rex (Larsson et al., 2005) regulons (Rogstam et al., 2007). The ResDE and Rex regulons are upregulated by changes in redox status or lowered oxygen availability. 194 LESLEY A.H. BOWMAN ET AL.

8.3.3. M. tuberculosis

The response of M. tuberculosis to RNS has also been investigated (Ohno et al., 2003). Microarray analysis using two separate NO donors, NOR-3 and Spermine NONOate, identified the upregulation of 36 genes including those playing roles in small molecule metabolism (including a nitrate reductase, narX, and ferredoxin, fdxA), macromolecule metabolism (sigE), and cell processes including a putative nitrite extrusion protein narK2 and genes coding for probable transmembrane proteins. Genes downregulated in the same study included those encoding for putative transcriptional regulators and cell envelope/energy metabolism proteins, suggesting a metabolic downshift in response to the NO donors. The transcriptomic response of M. tuberculosis to macrophage attack has found evidence of a response to nitrosative stress (Schnappinger et al., 2003; Cappelli et al., 2006), including upregulation of RNS detoxification genes including alkyl hydroperoxidase (ahpC), which has peroxynitritase activity (Bryk et al., 2000), and nitrate reductase (narX), as well as other genes previously hypothesized to alter their transcriptional activity in response to nitrosative stresses.

8.3.4. P. aeruginosa

P. aeruginosa upregulates a number of genes in response to GSNO stress (Firoved et al., 2004). Of these, many are directly responsible for the detoxification of oxides of nitrogen. The flavohemoglobin, fhp, is most highly upregulated; other genes coding for Nor, MoaB1, and NarK1 are also upregulated.

8.3.5. S. aureus

When exposed to SNAP, S. aureus genes involved in iron homeostasis, hypoxic/fermentative metabolism, the flavohemoglobin hmp and the 2-component system srrAB were upregulated. SrrAB has been shown to regulate the expression of many NO-induced metabolic genes (Throup et al., 2001). When exposed to nitrite, biofilm formation is inhibited in S. aureus and a clear response to both oxidative and nitrosative stress can be seen via increases in genes involved in DNA repair, detoxification of ROS and RNS, and iron homeostasis (Schlag et al., 2007). NITRIC OXIDE AND AGENTS OF NITROSATIVE STRESS 195

8.3.6. N. meningitidis

Activity of the NO-sensitive repressor NsrR from N. meningitidis has been assessed by microarrays in response to Spermine NONOate (Heurlier et al., 2008). Target genes under the control of NsrR included norB (NO reductase), dnrN (repair of nitrosative damage to iron–sulfur clusters), aniA (nitrite reductase), nirV (nitrite reductase assembly protein), and mobA (possible molybdenum metabolism) and were all upregulated in response to the NO donor. Evidence also suggests that the anaerobic- response regulator Fnr is sensitive to NO, but not to the extent of NsrR.

8.3.7. Yersinia pestis

Microarray analysis of the causative agent of the bubonic plague, Y. pestis, isolated from the buboes of rats showed a definite response to nitrosative stress (Sebbane et al., 2006). Most striking was the 10- to 20-fold increase in expression of hmp. Upregulation of metLRBF, nrdHIEF, ytfE, hcp, hcr, and tehB was also observed. The latter four genes and hmp are repressed in E. coli by the transcription factor NsrR (Bodenmiller and Spiro, 2006), the homologue of which (YPO0379) was downregulated 1.7- to 4-fold in the bubo. Upregulation of the methionine biosynthesis pathway may also indicate a response to S-nitrosylation in buboes as identified in the transcriptional analysis to GSNO in E. coli (Flatley et al., 2005).

8.4. Proteomics

One study utilizing C. jejuni sought to identify both transcriptomic and proteomic alterations in response to GSNO (Monk et al., 2008). Using 2D gel proteomic analysis (Holmes et al., 2005), the authors found that levels of the two globins, Ctb and Cgb, were enhanced at both the transcriptomic and proteomic level as was the heat-shock protein DnaK. Some proteins were upregulated that did not appear in the transcriptomic data (e.g., Cj0383c and Cj0509c) and vice versa. This could be a result of the differing timescales used for each approach; for microarray analysis, cells were incubated for 10 min, whereas proteomic analysis utilized samples that had been incubated three times longer. These discrepancies could reflect responses on differing timescales, a lack of translation or rapid protein degradation. 196 LESLEY A.H. BOWMAN ET AL.

þ A proteomic analysis of the response of H. pylori to the NO releasing molecule SNP using 2D proteomics revealed 38 proteins with altered expression levels (Qu et al., 2009). Among these were proteins involved in processing, antioxidation (including TrxR), general stress response, and virulence. The study of nitrosative stresses in microbial species using modern proteomic techniques is still in its infancy. However, quantitative proteomic analysis has been undertaken to investigate nitrate stress in the anaerobic sulfate-reducing bacterium D. vulgaris Hildenborough using iTRAQ (Redding et al., 2006). Changes in the protein profile of this organism were analyzed upon addition of sodium nitrate using iTRAQ labeling and tandem liquid chromatography separation coupled with mass spectrometry detection. The authors found that the use of 103 mM sodium nitrate produced only a mild effect upon the proteome, with proteins involved in central metabolism and the sulfate reduction pathway being unperturbed. Unsurprisingly, proteins involved in nitrate stress detoxification were increased as well as those for transport of proline, glycine-betaine, and glutamate, suggesting that nitrate stress also induced salt stress. In addition, levels were increased for several oxidative stress response, ABS transport system, and iron–sulfur cluster containing proteins. As discussed previously (Section 8.1.4), not only have proteomic methods been applied to monitor changes in protein expression levels across the proteome in response to stress, they have also been used to unravel the S-nitrosoproteome of organisms following challenge with RNS. Recently, a novel fluorescence-based approach has been developed and tested on E. coli cell lysates incubated with GSNO to identify S-nitrosated proteins across the proteome. Twenty modified proteins were uncovered, with functions in protein synthesis and folding, global regulation, quorum sensing, signal transduction, and bacterial attachment (Wiktorowicz et al., 2011). Investigators examining the S-nitrosoproteome of M. tuberculosis, produced in response to cellular challenge with NaNO2, uncovered 29 modified proteins, a large proportion of which were found to be involved in intermediary and lipid metabolism. Proteins involved in the defense against oxidative and nitrosative stresses were also nitrosated (Rhee et al., 2005). In 2007, Brandes and coworkers applied a method to identify all reversibly modified thiols mediated by the NO donor, DEA/NO, including S-nitrosothiols, disulfide bonds, and sulfenic acids. Investigators uncovered 10 altered proteins, six of which were encoded by essential genes (Brandes et al., 2007). More recently, researchers have uncovered five S-nitrosated proteins in H. pylori following incubation of cell lysates with GSNO, namely, GroEL, a chaperone and heat-shock protein; UreA, NITRIC OXIDE AND AGENTS OF NITROSATIVE STRESS 197 urease alpha subunit; TsaA, alkylhydroperoxide reductase; and two coding sequences of unknown function (Qu et al., 2011). Other techniques, such as isotope-coded affinity tags (iCATs) and S-nitrosothiol capture (SNOCAP), are currently being developed for use in studying the interaction between RNS and microbes but so far there appears to be no published work in this field.

9. CONCLUSIONS

The past 15 years have seen a remarkable transformation of our apprecia- tion of the assaults on microbes by NO and RNS and also the elaborate and effective defense measures mounted. Certain recurrent themes are evident. Microbes (in most cases, the information relates to bacteria) are able to resist NO in their environments by a relatively small number of detoxification mechanisms, the best understood being globins that catalyze NO conversion to nitrate and reductases that produce nitroxyl anion, and ultimately, nitrous oxide. The species sensed that induces the expression of these enzymes is probably the same, that is, NO, but the possibility exists that activation of the defense response may be achieved by protein nitrosation, for example, rather than sensing of NO by a metal center. A major weakness in our understanding is how bacteria sense and detoxify other RNS, such as those commonly used in experimental studies, notably, S-nitrosothiols (especially GSNO), SNP, and acidified nitrite. In the case of GSNO, SNO reductases are known to denitrosylate affected proteins, but there do not appear to be any detoxification mechanisms recognized so far that detoxify the products of SNP and acidified nitrite, but only the resultant NO. Several approaches may assist in tackling this problem. First, investigators should exercise great care when designing experiments to ensure that the nitrosative stress applied is well characterized. SNP, for example, is an agent that, although easy to obtain, will have quite unpre- dictable effects in terms of the extent and rates of NO release and may even release other biologically active species (cyanide, in this case). In the case of peroxynitrite, it appears that this species should probably not be regarded as an agent of NO-related stress, but as a species capable of nitration, nitrosylation, and oxidative stress. Second, sound principles of microbial physiology should be applied in experimental design, particularly so that culture conditions are reproducible and consistent with the use of the RNS species employed. Finally, much may be learned by adopting a systems or modeling approach to unraveling the complexities of the 198 LESLEY A.H. BOWMAN ET AL. microbial response. Since the outcomes of NO or RNS exposure are so pervasive, affecting directly and indirectly a host of cellular processes, such approaches have great potential but are in their infancy.

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