the diversity of microbial responses to nitric oxide and agents … · 2013-12-20 · snocap...

The Diversity of Microbial Responses toNitric 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) nowoccupy a central position in contemporary medicine, physiology,biochemistry, and microbiology. In particular, NO plays importantantimicrobial defenses in innate immunity but microbes have evolvedintricate NO-sensing and defense mechanisms that are the subjects of avast literature. Unfortunately, the burgeoning NO literature has not alwaysbeen accompanied by an understanding of the intricacies and complexitiesof this radical and other reactive nitrogen species so that there existsconfusion and vagueness about which one or more species exert thereported biological effects. The biological chemistry of NO and derived/related molecules is complex, due to multiple species that can be generatedfrom NO in biological milieu and numerous possible reaction targets.Moreover, the fate and disposition of NO is always a function of itsbiological environment, which can vary significantly even within a singlecell. In this review, we consider newer aspects of the literature but, mostimportantly, consider the underlying chemistry and draw attention to thedistinctiveness of NO and its chemical cousins, nitrosonium (NOþ),

ADVANCES IN MICROBIAL PHYSIOLOGY, VOL. 59 Copyright # 2011 by Elsevier Ltd.ISSN: 0065-2911 All rights reservedDOI: 10.1016/B978-0-12-387661-4.00006-9

nitroxyl (NO�, HNO), peroxynitrite (ONOO�), nitrite (NO2�), and

nitrogen dioxide (NO2). All these species are reported to be generated inbiological systems from initial formation of NO (from nitrite, NOsynthases, or other sources) or its provision in biological experiments(typically from NO gas, S-nitrosothiols, or NO donor compounds). Themajor targets of NO and nitrosative damage (metal centers, thiols, andothers) are reviewed and emphasis is given to newer “-omic” methods ofunraveling the complex repercussions of NO and nitrogen oxide assaults.Microbial defense mechanisms, many of which are critical forpathogenicity, include the activities of hemoglobins that enzymicallydetoxify NO (to nitrate) and NO reductases and repair mechanisms (e.g.,those that reverse S-nitrosothiol formation). Microbial resistance to thesestresses is generally inducible and many diverse transcriptional regulatorsare involved—some that are secondary sensors (such as Fnr) and thosethat are “dedicated” (such as NorR, NsrR, NssR) in that theirphysiological function appears to be detecting primarily NO and thenregulating expression of genes that encode enzymes with NO as asubstrate. Although generally harmful, evidence is accumulating that NOmay have beneficial effects, as in the case of the squid-Vibrio light-organsymbiosis, where NO serves as a signal, antioxidant, and specificitydeterminant. Progress in this area will require a thorough understandingnot only of the biology but also of the underlying chemical principles.

Abbreviations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1371. Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1382. Historical Perspective . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1393. Origins of Reactive Nitrosative Species in Biology . . . . . . . . . . . . . . . . . 140

3.1. Nitrite Reduction and Denitrification . . . . . . . . . . . . . . . . . . . . . . . . . 1403.2. Nitrate-Derived Stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1413.3. NO Synthases and the Nitrosative Burst . . . . . . . . . . . . . . . . . . . . . 1423.4. Non-NOS Sources of NO in Microbes . . . . . . . . . . . . . . . . . . . . . . . 1453.5. The Combined Reactive Species Response . . . . . . . . . . . . . . . . . . 147

4. The Biological Chemistry of NO and Related Species . . . . . . . . . . . . . . 1484.1. NO, Its Redox Chemistry, and NO2 . . . . . . . . . . . . . . . . . . . . . . . . . 1484.2. The Reaction of NO with Superoxide Anion . . . . . . . . . . . . . . . . . . . 1514.3. Reaction with Metal Centers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1524.4. Products of NO Reduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1534.5. The Reactions of HNO with Biological Targets . . . . . . . . . . . . . . . . 154

5. Laboratory Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1565.1. The Use of Nitrogen Oxide Donors . . . . . . . . . . . . . . . . . . . . . . . . . . 1565.2. NO . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1575.3. S-Nitrosothiols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1595.4. Other Donors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1625.5. HNO Donors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 162

136 LESLEY A.H. BOWMAN ET AL.

5.6. Use of ONOO� . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1635.7. NO2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1645.8. NO2

� . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1665.9. Other Nitrogen Oxides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 166

6. Bacterial Responses to RNS: Effectors and Regulators . . . . . . . . . . . . . 1666.1. Targets of RNS in Microorganisms . . . . . . . . . . . . . . . . . . . . . . . . . . 1666.2. Microbial Defenses: The Microbe Strikes Back . . . . . . . . . . . . . . . . 1676.3. Microbial Globins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1686.4. NO and RNS Reductases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1746.5. Other Proteins Implicated in NO Tolerance . . . . . . . . . . . . . . . . . . . 1766.6. Beneficial Effects of NO in Microbial Symbioses . . . . . . . . . . . . . . . 1776.7. Microbial Responses to ONOO� Stress . . . . . . . . . . . . . . . . . . . . . . 178

7. Microbial Sensing of NO and Gene Regulation . . . . . . . . . . . . . . . . . . . . 1817.1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1817.2. Fnr . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1817.3. NorR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1827.4. NsrR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1827.5. Others (DOS, FixL, GCS) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183

8. Global and Systems Approaches to Understanding Responses to NOand RNS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1838.1. Methodology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1848.2. Outcomes from Global Transcriptomic Approaches . . . . . . . . . . . . 1888.3. Responses of Other Microbes to RNS . . . . . . . . . . . . . . . . . . . . . . . 1938.4. Proteomics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 195

9. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 197References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 198

ABBREVIATIONS

CPTIO 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-l-oxyl-3-oxide

EPR electron paramagnetic resonanceFNR ferredoxin-NADPþ reductaseFnr fumarate and nitrate reduction regulator, encoded by

fnr geneGSH glutathioneGSNO S-nitrosoglutathioneGSSG glutathione disulfide (oxidized glutathione)GTN glyceryltrinitrateHcy homocysteineiCAT isotope-coded affinity tagiTRAQ isobaric tags for relative and absolute quantitationNHE normal hydrogen electrode

137NITRIC OXIDE AND AGENTS OF NITROSATIVE STRESS

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 NONOateDETANONOate

diethylenetriamine NONOate

norV, norW genes involved in nitric oxide reduction and itsregulation (norR)

NOS NO synthasePTIO 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3 oxideRNS reactive nitrogen speciesSNAP S-nitroso-N-acetyl-D,L-penicillamineSNO S-nitrosothiolSNOCAP S-nitrosothiol captureSNP sodium nitroprussideSOD superoxide dismutase

1. OVERVIEW

Nitric oxide (NO) is a small and freely diffusible species once known pri-marily as a toxic component of air pollution. In physiology and biochemis-try, it was well known as a poison and ligand for heme proteins. Thediscovery of the enzymic generation of NO in mammalian systems andits cell signaling functions represents a watershed moment in the evolutionof our understanding of biological signal transduction. The importance ofNO as a molecule of real biological significance cannot, however, haveescaped the attention of any microbiologist, although the realization is rel-atively recent. To illustrate this point, consider that a multi-authored,edited book Microbial Gas Metabolism published in 1985 (Poole andDow, 1985) contained only three index entries for ‘nitric oxide’, namely,‘denitrification’, ‘mass spectrum cracking pattern’ and ‘reaction with cyto-chrome d.’ The general topics addressed by these specific terms illustratewell the dominant interests of microbiologists in NO a quarter of a centuryago—NO as a possible (but far from proven) intermediate in the microbe-catalyzed conversion of nitrate to dinitrogen, the biochemical analysis and


detection of the gas, and the use of NO as an experimental tool in thestudy of heme proteins.

In the intervening years, a large literature has grown up around NO andrelated species, fueled by the recognition that NO plays important antimi-crobial 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 notalways been accompanied by an understanding of the intricacies and com-plexities of NO and other “agents of nitrosative stress” so that there existsome confusion and vagueness about which one or more species exert thereported, interesting biological effects. There is, for example, a tendencyfor 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-ity that one is actually meaning NOþ or some other congener. There isonly one NO.

Butler and Nicholson (2003) suggest that “there are not many smallmolecules about which a whole book could be written,” although in biol-ogy, we doubt the veracity of this. Consider the gases oxygen, carbon diox-ide, methane, and nitrogen. Nevertheless, we agree that NO is certainlyone such species: several books could be, and have been, written on thisone gas. As a result, we are forced to be brief and focused when revisitingsome familiar aspects of NO in microbiology and apologize for any over-sights or omissions. Our objective in this review is not only to review neweraspects of this vast literature but, most importantly, to consider the under-lying chemistry and draw attention to the distinctive chemistry of NO andits chemical cousins, NOþ, NO�, HNO, ONOO�, NO2

�, and NO2.

2. HISTORICAL PERSPECTIVE

The modern era of NO research may be considered to begin in the 1980swhen NO was identified as the endothelium-derived relaxing factor(EDRF). This remarkable story and its culmination in a Nobel Prize andthe designation of NO as “molecule of the year” by Science in 1992 arecovered well elsewhere, especially in accounts by the laureates (Furchgott,1999; Ignarro, 1999, 2005; Murad, 1999). NO was also shown to participatein the regulation of the nervous and immune systems, and it was soon dis-covered that NO also plays a vital role in the resistance of mammalian hoststo microbial infections. Activated macrophages were shown to form nitriteand nitrate from arginine (Iyengar et al., 1987; Marletta et al., 1988)


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 intracellu-lar parasite Leishmania major in vitro by an L-arginine-dependent mecha-nism (Green et al., 1990) and mice infected with L. major developedexacerbated disease when the lesions were injected with the NOS inhibitorL-NMMA, providing the first compelling evidence for the attenuation byNO 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). Shortlyafter, we showed that NO dramatically upregulated expression of theEscherichia coli flavohemoglobin (Poole et al., 1996), and an enzymic func-tion in NO detoxification was demonstrated by Gardner et al. (1998). A glo-bin mutant was NO sensitive unambiguously demonstrating a physiologicalrole (Membrillo-Hernández et al., 1999). In murine macrophages, NO wasshown to have a role in bacterial clearance (Shiloh et al., 1999; Vazquez-Torres et al., 2000). Also, flavohemoglobin-catalyzed NO detoxification bySalmonella enterica serovar Typhimurium protected the bacterium fromNO-mediated killing in human macrophages (Stevanin et al., 2002).Flavohemoglobin (Hmp)was shown to catalyze the reaction of NOwith oxy-gen to give innocuous nitrate via a dioxygenase (Gardner et al., 1998, 2000,2006) or denitrosylase (Hausladen et al., 1998a, 2001) mechanism, andfurther gene reporter experiments showed that hmp gene transcription isactivated on exposure of bacteria to NO or nitrosating agents (Poole et al.,1996; Poole and Hughes, 2000; Gilberthorpe et al., 2007). Mutants were usedto demonstrate unequivocally the key role of flavohemoglobin in defenseagainst NO not only in vitro (Membrillo-Hernández et al., 1999) but alsoin 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 SPECIESIN 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


acidic conditions (as in the stomach) and the resulting nitrous acid willyield NO and other nitrogen oxides; the beneficial effects of acidifiednitrite in killing ingested pathogens, gastric mucosal integrity and othereffects are discussed elsewhere (Lundberg et al., 2004). Acidified nitriteis sometimes, but perhaps not ideally, used as a source of NO in bacterialexperiments in vitro (as covered elsewhere in this review).

The bacterial reduction of nitrite is a key reaction in anaerobic bacterialmetabolism and the subject of an immense literature (Potter et al., 2001;Lundberg et al., 2004). There are three classes of nitrite reduction. Inthe first (denitrification), reduction of nitrite to NO is catalyzed by eithercopper-containing NirK or cytochrome cd1 nitrite reductase, NirS. Theperiplasmic enzymes involved have been extensively characterized andare outside the scope of this contribution (for an authoritative review,see Potter et al., 2001). Second, when bacteria utilize nitrite as a terminalelectron acceptor, NADH- and siroheme-dependent reduction is the pri-mary pathway, encoded in E. coli by the nirBDCcydG operon. Third,nitrite can be reduced to ammonia by a widely distributed cytochrome cnitrite reductase Nrf (Potter et al., 2001), which can also, however, producelow levels of NO when nitrite is in excess under anoxic conditions (Corkerand Poole, 2003).

3.2. Nitrate-Derived Stress

Nitrate is often regarded mainly as a water pollutant and its presence in thediet of man is seen as an unfortunate consequence of the use of nitrogenfertilizers in agriculture. Increasingly stringent regulations to limit nitrateintake suggest that nitrate has wholly undesirable effects including forma-tion of N-nitrosamines, infantile methemoglobinemia, carcinogenesis, andpossibly teratogenesis (McKnight et al., 1999). However, nitrosamineformation is via nitrite (nitrosation chemistry) not nitrate; nitrite is the cul-prit. An alternative view is that the products of nitrate metabolismhave beneficial effects, especially in host defense. The argument is madeelsewhere (Lundberg et al., 2004) that nitrate-reducing commensal bacteriaplay a symbiotic role in mammalian nitrate reduction to nitrite, NO, andother products. Nitrate reduction to nitrite and thence to NO are the topicsof a vast literature, beyond our scope (for a review, see Lundberg, 2008).It is clear, though, that large amounts of NO and other reactive nitrogenspecies (RNS) are generated in vivo from salivary nitrite in the acidic stom-ach (Benjamin et al., 1994) and may contribute to killing of ingestedpathogens (Lundberg et al., 2004). Depending on nitrite concentration in


the saliva (McKnight et al., 1999), the NO concentration in the stomachheadspace 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 metalloenzymesthat catalyze the conversion of L-arginine (L-Arg) to L-citrulline and NOwith the consumption of NADPH and O2. They were first identified inmammals, and three forms are identified: endothelial NOS (eNOS orNOSIII), neuronal NOS (nNOS or NOSI), and inducible NOS (iNOS orNOSII) (reviewed in Alderton et al., 2001). The nitrosative burst, triggeredby pathogenic agonists and inflammatory mediators (Lowenstein et al.,1993), succeeds the oxidative burst and is mediated by NO production fol-lowing activation of iNOS. The first two isoforms are constitutivelyexpressed and are calcium dependent, whereas iNOS is stimulated to pro-duce NO at markedly higher levels than the constitutive isoforms followinginfection (Lowenstein and Padalko, 2004). iNOS is most important in amicrobial context and is known to occur in a wide variety of stimulatablecells such as macrophages, neutrophils, vascular smooth cells, and glialcells in the CNS (Bogdan, 2001). On microbial infection, the NO producedhas diverse, apparently conflicting functions; on the one hand, NO exertsantimicrobial and anti-inflammatory host defense effects and, on the other,proinflammatory and cytotoxic activities (Fang, 2004). The host defensefunction is exemplified by the effects of NO in microbial infections,whereas NO-mediated inflammation and pathogenesis are known in cer-tain diseases including arthritis, encephalitis, ulcerative colitis, and viralinfections.

These enzymes are homodimeric with each monomer containing an N-terminal oxygenase domain with binding sites for heme, L-arginine, andtetrahydrobiopterin (H4B) and a C-terminal reductase domain, which hasbinding sites for FMN, FAD, and NADPH (Stuehr et al., 2009). The reduc-tase domain transfers electrons sourced from NADPH via flavin carriers toheme in the oxygenase domain. This drives the oxidation of L-arginine inthe presence of O2 to NO, citrulline, and NADPþ (Daff, 2010). Electronflow is strictly between the reductase domain of one monomer and theoxygenase domain of the other (Siddhanta et al., 1996).

As iNOS is confined to the cytoplasm, NO must diffuse to thephagosome to react with internalized microorganisms. Unlike O2

�


generation, the onset of NO production is late in the defensive process,starting around 8 h postinfection as demonstrated in murine macrophageschallenged with S. enterica (Eriksson et al., 2003). NO makes a significantcontribution to the host’s innate immune response, exemplified by a recentstudy revealing that macrophages derived from murine bone marrow gen-erate an approximately 2-log reduction in C. jejuni viability relative toiNOS mutants over a 24-h period (Iovine et al., 2008).

In a recent example of the intricacies of iNOS function, a newbornmouse model (Mittal et al., 2010) of meningitis revealed that infection withE. coli K1 resulted in iNOS expression in the brain. iNOS�/� mice, how-ever, were resistant to infection and showed normal brain morphologyand a reduced inflammatory response. An iNOS inhibitor (ami-noguanidine) also prevented meningitis and brain damage, and further,peritoneal macrophages and polymorphonuclear leukocytes from suchmutant mice showed enhanced killing of bacteria. These data suggest thatNO from iNOS is actually beneficial for E. coli K1 survival in the macro-phage 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 pro-karyotic NOS enzymes (Crane et al., 2010) are in many ways similar totheir mammalian counterparts, catalyzing the conversion of L-Arg to NOvia the intermediate No-hydroxy-L-arginine (NOHA), but the role(s) ofthe 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 evidencefor NOS-like proteins came from genome mining just over 10 years ago,revealing bacterial ORFs with high sequence similarity to mammalianNOS. Most recent data suggest that bacterial and mammalian NOSenzymes have similar reactivities with almost identical catalytic active sites.This has greatly facilitated research into the core features of all NOSproteins because many bacterial NOS are readily expressed in E. coliand provide protein for crystallographic, various spectroscopic, and kineticstudies.

The main differences between NOS of mammals and bacteria reside incofactor specificity and the nature of the reductase partners for theseenzymes (Crane et al., 2010). Not all bacterial NOS contain the pterincofactor (tetrahydrobiopterin, H4B) associated with mammalian NOS.


Tab

le1

Bacterial

NO

syntha

sesa.

Biologicalfunction

Red

uctase

Com

men

tsReferen

ceb

Nocardiaspecies

First

repo

rtof

bacterial

NOS,

butno

obviou

sNOS

gene

homolog

ue

Che

nan

dRosazza

(199

4,19

95)

Lactoba

cillu

sferm

entum

Noob

viou

sNOSge

neho

molog

ueMoritaet

al.(199

7)

Salm

onella

enterica

Typ

himurium

Noob

viou

sNOSge

neho

molog

ueCho

iet

al.(200

0)

Stap

hylococcus

aureus

NO

orNOSprotectcells

from

H2O

2

NOSho

molog

ueHon

get

al.(200

3);

Gusarov

andNud

ler

(200

5)Deino

coccus

radiod

uran

s1.

Rea

ctionwithtryp

toph

antR

NA

synthe

tase

IIto

form

4-nitro-Trp-tRNA

Trp,inv

olve

din

proteinor

second

ary

metab

olitesynthe

sis?

2.Respo

nseto

UV

radiation

expo

sure,w

ithNO

upregu

lating

tran

scriptionof

grow

thfactor?

Activewithsurrog

ate

mam

malianredu

ctase

Ada

ket

al.(200

2b);

Bud

dhaet

al.(200

4);

Patel

etal.(200

9)

Bacillus

subtilis,

Bacillus

anthracis

1.NO

orNOSprotects

cells

from

H2O

22.

Protectionag

ainst

antibiotics(see

text)

Ded

icated

redu

ctaseno

trequ

ired

(?)

First

crystalstructureof

aba

cterialNOS

Gusarov

andNud

ler

(200

5);S

hatalin

etal.

(200

8);G

usarov

etal.

(200

9)Streptom

yces

species,S.

turgidiscabies

NOSen

code

don

pathog

enicityisland

associated

withpo

tato

scab

disease.

NOS

invo

lved

inthax

tomin

(tox

in)

prod

uction

(see

text)

Noob

viou

sredu

ctase

partne

rproteins

encode

dby

NOS

pathog

enicityisland

Kerset

al.(200

4)

Sorang

ium

cellu

losum

Cov

alen

tlybo

und

unique

redu

ctase

Aga

pieet

al.(200

9)

a NOSs

have

also

been

repo

rted

ineu

karyoticmicrobe

s,no

tablytheprotozoa

Entam

oeba

histolyticaan

dTox

oplasm

ago

ndii,

butne

ithe

rge

nomecontains

anNOSho

molog

ue(C

rane

etal.,20

10).

bFor

furthe

rde

tails,see

Crane

etal.(20

10).

In the latter enzymes, H4B delivers electrons to the active site for oxygenactivation in two steps. In the first, oxygen bound to heme is activated bya single electron transfer, and in the second (final) stage of the reaction,NO is liberated. The mechanism of action of the mammalian enzymes isbeyond 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 dis-sociation 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 tomore reactive species raising the intriguing possibility that synthesis ofthese alternative species (HNO, NO�) is the true biological function, asdiscussed below. Nonetheless, NO does appear to be the major productin at least three cases (Johnson et al., 2008; Shatalin et al., 2008; Patelet al., 2009).

A case of special interest in the context of the present review is that ofStreptomyces turgidiscabies, where the NOS is encoded on a pathogenicityisland associated with potato scab disease (Crane et al., 2010). NOS hasbeen 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 biosyn-thetic nitration reaction (Johnson et al., 2008). Indeed, a feeding study with15N-Arg showed that the thaxtomin nitro group nitrogen does originatefrom the terminal guanidinium nitrogen of Arg, strongly implicating anNOS activity. Importantly, however, NO will not itself react directly tonitrate substrates such as the tryptophanyl moiety of thaxtomin, whereasoxidation products of NO (NOþ, NO2

þ, ONOO�, NO2; see Section 4)are known to nitrate aromatic groups (Hughes, 2008).

What are the potential targets of endogenously generated NO andrelated reactive species in bacteria? The examples in Table 2 include tran-scription factors, biosynthetic enzymes, metalloproteins, and kinases. How-ever, the true biological roles of NOS-derived NO in microbes remainelusive. The fact that NOS enzymes are restricted to certain species andgenera 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 acomponent of the host’s innate immune response to infection. However, anumber of papers have demonstrated that bacteria also have the capacityto synthesize NO. This ability was first demonstrated by Hollocher andothers: bacteria grown anaerobically with nitrate produced NO from nitriteand the NO was detected by nitrosation of 2,3-diaminophthalene.


Tab

le2

Bacterial

targetsof

NO

andnitrosativestress.

Targe

tBasis

Exa

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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 recentlydescribed ability of mammalian neuroglobin to generate NO from nitrite,which is suggested to be a primordial hypoxia- and redox-regulated sourceof NO for physiological functions (Tiso et al., 2011). The generation of NOby Salmonella has been demonstrated not only in vitro but also insideinfected cancer cells and in implanted tumors in live mice using mic-rosensors. In strains mutated in hmp and norV, the bacteria generatedmore NO and killed cancer cells more effectively in a nitrate-dependentmanner (Barak et al., 2010).

3.5. The Combined Reactive Species Response

ROS and RNS act in concert, exemplified by murine macrophages doublyimmunodeficient in iNOS and NAPDH oxidase (Phox) that werecompletely 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 (Pacelliet al., 1995). Additionally, NO and O2

� can rapidly react to form the highlytoxic species peroxynitrite (ONOO-) (Reaction 5), which will be discussed inSection 4.2. On account of diffusion constraints, particularly the negativecharge on O2

�, ONOO� generation tends to colocalize with NADPH oxi-dase. Peroxynitrite is often stated to be the principal nitrating agent for tyro-sine residues in proteins, yet the yield in vitro of nitrated tyrosine at pH 7.4is less when O2

� and NO are cogenerated than when a bolus of ONOO� isgiven. Goldstein et al. (2000) demonstrated that maximal nitrosation of tyro-sine occurred when NO and O2

� fluxes were equal. Although ONOO� isformed in vivo from these two radicals, and the temporal concurrence ofthese precursors, as is found at inflammatory sites, favors ONOO� forma-tion in vivo, ONOO� itself is not likely to be the nitrating species. It is gen-erally 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 tyrosineto the tyrosyl radical. Subsequent addition of NO2 to the tyrosyl radical thengives nitrotyrosine (e.g., Gunaydin and Houk, 2009). Thus, this mechanismof tyrosine nitration is highly dependent on the presence of NO2, and sinceNO2 can be formed from peroxynitrite-independent pathways, tyrosinenitration is not necessarily a marker for ONOO� formation.


4. THE BIOLOGICAL CHEMISTRY OF NO ANDRELATED SPECIES

4.1. NO, Its Redox Chemistry, and NO2

The biological chemistry of NO and derived/related molecules is poten-tially complex due to a multitude of species that can be generated fromNO in a biological milieu and the multiple possible reaction targetsassociated with these derived species (for an overview, see Lehnert andScheidt, 2010). Moreover, the fate and disposition of NO is always a func-tion of its biochemical environment, which can vary significantly evenwithin a single cell. The redox relationship between NO and related/derived nitrogen oxides is given in Fig. 1 and all of the species shown havebeen reported to be generated in biological systems from initial formationof NO. Herein will be described the chemical properties of NO andderived agents important to their biological functions and effects.

The biological utility of NO is based on its unique chemistry. First andforemost, NO has an unpaired electron and is, therefore, considered tobe a free radical (for the sake of simplicity, we have adopted the definitionof Halliwell and Gutteridge (2007)) for the term “free radical” which is anyspecies that can exist independently (i.e., free) and contains one or moreunpaired electron. The presence of an unpaired electron is immediatelyevident from the Lewis dot depiction for NO (Fig. 2). However, theunpaired electron is not associated solely with the nitrogen atom of NO(as indicated by the Lewis structure), but rather is delocalized throughout

NO+e−

−e−HNO

+e−

−e−H2NO

+e−

−e− NH2OH+2e−

−2e−NH3(+ H2O)

+e−

−e−NO+

H2O

NO2−+ 2H+

+e−

−e−NO2

−e−

H2O

+e−

−H2O

NO3− + 2H+

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.


the molecule in a p* antibonding orbital as indicated by the molecularorbital diagram (Fig. 3).

As discussed below, the free radical nature of NO as well as the orbitallocation on the unpaired electron are important factors in the biologicalchemistry of NO. Although the term free radical is often associated withextreme reactivity (e.g., strong oxidant), this is not the case with NO.The lack of oxidizing capability of NO is evidenced by its relatively lowreduction potential (E� ¼�0.55 V vs. NHE for the NO,Hþ/HNO couple;Bartberger et al., 2002; Shafirovich and Lymar, 2002). For comparison,the hydroxyl radical (HO�, a prototypical strong one-electron oxidant)has a reduction potential of 2.3 V versus NHE for the HO,Hþ/H2O coupleat pH 7 (Sawyer, 1991). Consistent with the idea that NO is a poorone-electron oxidant, NO is also poor at H-atom abstraction since thebond formed from this reaction (the H��NO bond) is weak, approximately47 kcal/mol (Dixon, 1996). Again, for comparison, when HO� abstracts ahydrogen atom, the strength of the bond made (H��OH) is very strong,119 kcal/mol. Thus, NO is generally difficult to reduce and, therefore, poorat initiating radical chemistry via oxidation pathways. However, NO will

N O

2p

2p

2s

2s

σ∗

σ∗

σ

σ

π∗

π

Figure 3 Molecular orbital diagram for NO. Schematic depiction of molecularorbitals shown.


rapidly react with existing radicals. As such, NO has been found to be apotent antioxidant capable of quenching otherwise deleterious/oxidizingradical chemistry (Rubbo et al., 1995). NO also reacts with dioxygen(which has two unpaired electrons and so, according to the definitionabove, is considered a free radical or, in this case, a diradical). The reactionof NO with O2 results in the generation of nitrogen dioxide (NO2)(Reaction 1).

2NOþO2 ! 2NO2 ð1ÞLike 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, reactionof NO with O2 takes two relatively weak oxidants (the reduction potentialfor the O2/O2

� couple is �0.33 V (Sawyer, 1991)) and forms a reasonableone-electron oxidant. NO2 is known to oxidize a variety of biologically rel-evant functional groups such as cysteine thiols, tyrosines, and polyunsatu-rated fatty acids, just to name a few. In the absence of NO2-reactivespecies (i.e., reductants), the fate of NO2 generated via the reaction ofNO with O2 is to react with another NO (since they are both radicals) togive dinitrogen trioxide (N2O3) (Reaction 2).

NO2 þNO (+ N2O3 ð2ÞN2O3 (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 analo-gous to the reaction of H2O, these nucleophiles can be nitrosated by N2O3

(Reaction 4).

N2O3 þH2O (+ 2NO2� þ 2Hþ ð3Þ

N2O3 þNuc� ! Nuc�NOþNO2� ð4Þ

Thus, the combination of Reactions 1, 2, and 4 can result in theNO-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 inthe biological literature, yet it must be stressed that they have distinctmeanings. S-nitrosation is a mechanism whereby NOþ-like species mediatean attack upon thiol side groups, whereas S-nitrosylation is a mechanisticallyambiguous term for any process that results in the generation of an SNO.


Thus, the term nitrosyl does not describe a mechanism but instead refers toNO bound to a metal, for example. It needs to be noted that the biologicalaccessibility of this nitrosation chemistry is likely to be rare or, at the veryleast, very restricted due to the reaction kinetics. As shown in Reaction 1,two equivalents of NO are required for the O2-dependent generation ofNO2. Thus, the kinetics of this reaction has a second-order dependence onNO (Ford et al., 1993). This second-order dependence indicates that this pro-cess is significant only at high concentrations of NO. Since both NO and O2

favorably partition into membranes where concentrations of both are likelyto be significantly higher than in the aqueous compartments of cells, it hasbeen proposed that nitrosation chemistry of the type described above mayhave special relevance in lipophilic/membrane environments (Liu et al.,1998). As an example of the effect that the second-order dependence onNO can have, for purely aqueous, aerobic (assuming 200 mM O2) solutionsof NO, a 10 mM solution will degrade to one half its original concentrationin approximately 1 minute, whereas a 10 nM solution of NO will degradeto half its concentration in over 70 h (note that the term “half-life” was notused 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 reduceddioxygen species superoxide (O2

�), generating, initially, peroxynitrite(ONOO�) (Reaction 5).

NOþO2� ! ONOO� ð5Þ

There are numerous excellent reviews on the chemistry, biology, and(patho)physiology of this reaction and ONOO� (see, e.g., Pryor andSquadrito, 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 physio-logical pH, ONOO� will eventually decompose to nitrate (NO3

�). Thisrearrangement occurs presumably via ONOOH and involves the genera-tion of oxidizing intermediates (e.g., Gunaydin and Houk, 2008). Indeed,ONOO�/ONOOH is capable of performing oxidation chemistry on, forexample, thiols (i.e., cysteine), phenols (i.e., tyrosine), and other biologi-cally relevant reducing species. A primary fate of ONOO� in manybiological systems is reaction with carbon dioxide (CO2) giving, initially,nitrosoperoxycarbonate (ONOOCOO�) (Reaction 6). Rearrangement of


this species generates nitrocarbonate (Reaction 7), which then hydrolyzesto give NO3

� and carbonate (CO32�).

ONOO� þ CO2 ! ONOO� CðOÞO� ð6Þ

ONOO� CðOÞO� ! O2NO� CO2� ð7Þ

As with ONOOH decomposition, the rearrangement of nitrosoperoxy-carbonate to nitrocarbonate involves reactive intermediates that are alsocapable of oxidizing a number of biological molecules (e.g., Gunaydinand 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 toform NO3

�. In both oxymyoglobin and oxyhemoglobin, the bounddioxygen has significant O2

� character due to extensive donation ofelectrons from the metal to the bound O2. Thus, reaction of NO withthe heme-bound O2

� is analogous to the reaction of NO with “free”O2

� (Reactions 8 and 9), although recent studies indicate no intermediacy ofONOO� on the millisecond time scale in this chemistry (Yukl et al., 2009).

MbFeII þO2 ! MbFeII �O2 $ MbFeIII �O2�� ð8Þ

MbFeII �O2 $ MbFeIII �O2�� þNO ! MbFeIII þNO3

� ð9ÞAlong with its reaction with dioxygen and dioxygen-derived species, NO

also binds metals. Most notable in biological systems is the reaction of NOwith hemeproteins (e.g., Ford, 2010), although NO can bind other nonhememetalloproteins as well. Unlike O2 and CO, which will only bind to ferrous(Fe2þ) hemes, NO is capable of binding both ferric (Fe3þ) and ferroushemeproteins (provided there is an open or exchangeable coordination site).The reactionwith ferrous hemes results in a species that favors a 5-coordinate,square pyramidal geometry. Indeed, a major site of action of NO in mamma-lian systems is the hemeprotein soluble guanylate cyclase (sGC) which bindsNO via its ferrous heme leading to the formation of a 5-coordinate ferrousnitrosyl that is presumably responsible for enzyme activation (although theprocess may be more complicated) (Poulos, 2006). Significantly, when O2

and CO bind most ferrous hemes, a 6-coordinate geometry is preferredmaking NO a unique ligand among these small molecule diatomic signaling


species (Traylor and Sharma, 1992). Binding of NO to a ferric heme leads tothe generation of a nitrosyl complex whereby the NO ligand becomeselectrophilic (possesses nitrosonium (NOþ) character). Attack of the boundNO by a nucleophile (such as water, thiols, etc.) then leads to reduction ofthe ferric iron to ferrous and generation of a nitrosated species (Reaction 10).

FeIII �NO $ FeII �NOþ� �þNuc�H ! FeII þNuc�NOþHþ ð10ÞThis process is referred to as reductive nitrosation whereby nitrosation of

water (generating NO2�) and thiols (generating nitrosothiols) can readily

occur. Significantly, nitrosation via thesemetal catalyzed processes is not sec-ond order in NO and therefore is not as kinetically restricted as the autoxida-tion 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 tobe catalytic.

As mentioned above, NO2� can be generated via the autoxidation of

NO (Reactions 1–3) or reductive nitrosation (Reaction 10, where thenucleophile is water). Significantly, Reactions 3 and 2 are reversible,indicating that NO can be formed from high concentrations of NO2

� underacidic conditions. NO2

� can also be reduced directly to make NO underappropriate conditions. The one-electron reduction of NO2

� to NO ishighly proton dependent and can be very favorable since the reductionpotential for the NO2

�,Hþ/NO couple is 0.98 V, versus NHE.

4.4. Products of NO Reduction

Thus far, the discussion of nitrogen oxide chemistry has concentrated onspecies that are oxidized relative to NO (i.e., NO2, N2O3, NO2

�, ONOO�,etc.). Indeed, in mammalian systems, oxidation appears to be the major fateof NO. However, reduction of NO is well established in prokaryotes. One-electron reduction of NO generates nitroxyl (NO�/HNO). As mentionedpreviously, the reduction potential of the NO,Hþ/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 reviewedelsewhere (e.g., (Fukuto et al., 2005; Miranda, 2005)). The pKa of HNO hasbeen determined to be 11.4 (Shafirovich and Lymar, 2002), indicating thatHNO is the predominant species at pH 7. A unique aspect of the acid–basechemistry of HNO is that the two equilibrium partners do not have the same


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

HNO (+ 3NO� þHþ ð11ÞThus, 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 observedsince other reactions are faster than protonation–deprotonation.

Both the one- and two-electron standard reduction potentials for theHNO,Hþ/H2NO� and HNO,2Hþ/NH2OH couples are reported to beapproximately 0.7 and 0.9 V versus NHE, respectively (the two-electronprocess, the HNO,2Hþ/NH2OH couple, at pH 7, has a reduction potentialof approximately 0.3 V vs. NHE) (Shafirovich and Lymar, 2002; Duttonet al., 2005). These values predict that HNO could be easily reduced inbiological systems. Nitroxyl itself, however, can be very reducing. Thereported reduction potential for the NO/3NO� couple is �0.81 V versusNHE, indicating that 3NO� can be a very potent reducing agent (Bartbergeret al., 2002). As mentioned earlier, the N��H bond dissociation energy forH��NO is also only approximately 47 kcal/mol. This low bond strengthpredicts 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 thiolsand thiolproteins. Both experimental and theoretical work indicates thatHNO is highly thiolphilic (Doyle et al., 1988; Bartberger et al., 2001).Attack of a nucleophilic thiol at the electrophilic nitrogen atom of HNOresults in the formation of a fleeting N-hydroxysulfenamide (Fig. 4).

The N-hydroxysulfenamide intermediate has two possible fates. In thepresence of excess or vicinal thiols, the N-hydroxysulfenamide reacts toform a disulfide and hydroxylamine. A competing rearrangement can alsooccur resulting in the formation of a sulfinamide. Significantly, in biologicalsystems, disulfides are generally considered to be readily reduced back tothe corresponding thiols, whereas sulfinamides are likely to be resistantto reduction. Thus, reaction of HNO with thiols can result in eitherreversible or irreversible modifications.

Another likely class of biological targets for HNO is metals. Akin toother small molecule metal ligands (e.g., O2, NO, CO, H2S, CN

�), HNO


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

MbFeII þHNO ! MbFeII �NðHÞO ð12Þ

MbFeIII þHNO ! MbFeII �NOþHþ ð13ÞAlthough most previous biological studies involving nitroxyl presumably

involve the protonated HNO species, it should be noted that formation ofthe anion, 3NO�, will not result in immediate HNO generation due to thespin restriction to protonation (remember that the pKa of HNO is 11.4).Therefore, if 3NO� is generated in a biological system, the chemistry of thisspecies will be prevalent. As discussed briefly above, 3NO� is a strong reduc-tant and should reduce/coordinatemetals. Also, 3NO�will react withO2, in aprocess that is isoelectronic with Reaction 5, giving ONOO� (Reaction 14).

3NO� þO2 ! ONOO� ð14Þ

Protein S

HN

O

H+

Protein S

N

H

OH

Protein S

NH2

O

Protein S SR+

NH2OH

Sulfinamide

Disulfide

Rearrangement

RSHN-Hydroxysulfenamide

Figure 4 Reaction of HNO with thiols.


The above discussion of the nitrogen oxides and the associated chemicaldescriptions is not meant to be comprehensive but considered to serve as astarting point for understanding the diversity and complexity of biologicalnitrogen oxide chemistry. For more complete descriptions of these andother aspects of nitrogen oxide chemistry, readers are encouraged to findone 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 accom-plished using either authentic compounds, or in many cases, it is moreconvenient to use donor species. Herein, we discuss briefly aspects ofworking with the nitrogen oxides that appear to be of the most currentinterest—NO, NO2, N2O3, NO2

�, HNO, and ONOO�. For several nitro-gen oxide species discussed below, the use of donor compounds is prevalentand even necessary. There are several important factors that need to beconsidered when using donors, however. Thus, prior to a discussion of theindividual 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 con-siderations that need to be accounted for when interpreting the experimen-tal results (e.g., Fukuto et al., 2008): (1) It must be determined that thebiological 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 thebiological activity can be due to donor coproducts (i.e., other species gen-erated alongside the species of interest); (3) there is the possibility thatimpurities in the donor may be the active species; and (4) for donors thatrequire biological activation, there is the possibility that the activation pro-cess itself (i.e., oxidation or reduction) can be at least partially responsiblefor the activity. One way to control most of the above-mentionedpossibilities is to use structurally distinct donors from different compoundclasses (and with different mechanisms of release) and to incorporate con-trol experiments using fully decomposed donors. Since donors from vary-ing classes are structurally distinct, their syntheses and mechanisms ofrelease are also distinct. Thus, it is highly likely that the only thing they will


have in common is the release of the species of interest. Therefore, if simi-lar biological activity is observed with the varied donors, it is likely that thedonated species (i.e., NO) is responsible. Controlling for possible activityassociated with impurities and/or coproducts can be accomplished by sim-ply decomposing the donor and testing the decomposed donor solution.Since NO is a fleeting species in aqueous aerobic solution (decomposesto NO2

�), allowing time for both donor decomposition and NO degrada-tion to NO2

� is necessary. For example, Dukelow et al. (2002) showed thatexhausted DETA NONOate had the same growth inhibitory effect towardPseudomonas aeruginosa in vitro as DETA NONOate, showing that NOwas not responsible for the antibacterial effects observed (Dukelow et al.,2002). In contrast, investigators examining the ability of S. enterica tomount an acid tolerance response in the presence of RNS revealed thatspermine NONOate sensitizes cells to acid stress, a feat dependent uponNO release, as the parent compound spermine did not elicit a reductionin 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 manybiological studies since gas-handling systems need to be in place and deter-mining the concentrations of NO in solutions made by passing NO gasthrough aqueous mixtures is not trivial. One solution to providing the gasinvolves the use of a gas-permeablemembrane (Silastic) immersed in the cul-ture and throughwhich gasmixtures containingNOare continuously passed.Dose rates of NO delivery are directly proportional to the length of theimmersed tubing (Tamir et al., 1993). This method has been used to studyinduction of the E. coli SoxRS regulon and was found to be more effectivethan bolus additions of NO (Nunoshiba et al., 1995), while also mimickingbetter the continuous fluxes of the gas that occur in macrophages. In onerecent development, Skinn et al. (2011) fabricated a small (65 ml) stirredreactor that incorporates a flat porous membrane for NO delivery (sittingbelow a stirrer) and a loop of gas-permeable tubing for O2 delivery. In trialsusing a 10%NOmixture and a buffer that was initially air-equilibrated, con-stant rates of NO2

� (i.e., the end product of NO oxidation accumulation)were observed (53 mM/h). Such a system has great potential in microbialphysiology experiments but have not yet been reported. Other means forexposing microbial cultures to NO include incubating Petri dishes in a con-stantly replenished atmosphere containing 10% air, 960 ppm NO, and the


balance as nitrogen. The additional presence of the redox cycling agentphenazine methosulfate appeared necessary for consistent NO-dependentkilling but the basis for this observation seems obscure (Gardner et al.,1998). In other papers, a three-way valve was used to deliver mixtures ofO2, N2, and NO, and gas mixtures were passed through a trap containingNaOH pellets to remove NO2 and higher oxides of nitrogen formed priorto entering reaction vessels (Gardner et al., 1997). A laboratory method forgenerating NO by the self-decomposition through disproportionation ofnitrous acid is described in useful detail by Aga and Hughes (2008).

However, most experimentalists examining the biology of NO utilizeNO donors. There are many NO donors available and their use and chem-istry has been reviewed previously (Wang et al., 2002; Miller and Megson,2007; Aga and Hughes, 2008). Currently, one of the most utilized class ofNO donors are the diazeniumdiolates (also referred to as “NONOates”)(e.g., Keefer, 2003) (Reaction 15).

R2N NONO½ �� þHþ ! R2NHþ 2NO ð15ÞTheir 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 physi-ological pH (i.e., does not require bioactivation). The varied NO releaserates for the diazeniumdiolates have been attributed to a competitionbetween several protonation sites on the molecule, of which only one leadsto NO generation (Dutton et al., 2004a). The half-lives of these compoundsrange from 1 min to several hours. All of these factors make this class ofNO donor highly preferable for biological studies. The only coproductfor the diazeniumdiolates is a secondary amine, which can be controlledby testing either the amine itself or the decomposed donor.

In some studies mixtures or cocktails of NO donors with complementaryproperties may be used. For example, we have used a mixture of NOC-5andNOC-7 in studies ofE. coli tomaintain a sustained output of NO in studiesof hmp gene transcription (Cruz-Ramos et al., 2002). NO release fromNOC-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 provideNOrelease over a period of 1 h ormore.In practice, NO loss through biological or nonbiological routes limited thepresence of NO in cultures to about 30 min (Cruz-Ramos et al., 2002).

Sodium nitroprusside (SNP, Na2Fe(CN)5NO) is a clinically relevant andcommonly used donor of NO. In spite of its clinical utility, the mechanismof NO release from SNP in biological systems is not completely establishedand 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


biological system. Release of NO from SNP requires either light or reduc-tive metabolism. These factors make it difficult to accurately predict thelevels of NO generated from SNP and/or compare experiments whereSNP is used since light exposure and the degree of reductive metabolismcan differ significantly between experimental systems. Moreover, SNPcontains five cyanide ligands which can also be released and which mayalso have biological activity. In in vivo systems, the release of cyanide frombiologically active levels of SNP is typically not a problem (at least in theshort term) since NO is such a potent vasorelaxant that the levels ofreleased cyanide are tolerated. However, considering all of these factors(possible photochemical release, the requirement for reductive metabo-lism, 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). AlthoughSNO species are clearly biologically relevant (e.g., Hess et al., 2005), theycan do much more than simply release NO. The release of NO fromSNO compounds is not spontaneous and requires light or reducing metals(i.e., Cu1þ) (Singh et al., 1996) (Reactions 16 and 17).

RS�NO þlightð Þ ! RS� þ �NO ð16Þ

RS�NOþ Cu1þ ! RS� þNOþ Cu2þ ð17ÞMoreover, SNO compounds can react with other thiols to give, instead,

HNO (Reaction 18) or transfer the equivalent of nitrosonium ion (NOþ) toanother thiol (Reaction 19) (Wong et al., 1998):

RS�NOþR0SH ! RSSR0 þHNO ð18Þ

RS�NOþR0SH ! RSHþR0S�NO ð19ÞThe possibility of all of this chemistry occurring in a biological system

makes mechanistic interpretation of experiments difficult. Thus, in studieswhere only the administration of NO is desired, SNO species are not ideal.

Controls are clearly needed but the design of these experiments is notalways straightforward. For example, GSNO is widely used as a convenientS-nitrosothiol and it might be imagined that glutathione would be a usefulcontrol molecule. However, GSH is not an ideal control molecule since itmay not be the product of GSNO metabolism (see below). Nevertheless,


where GSH has been used (as well as GSSG) it has been shown to berelatively ineffective at eliciting upregulation of GSNO-inducible genes inC. 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 bacterialcultures, intracellular outcomes are preceded by enzymic transformationsof GSNO. The mechanisms of communication between extracellular andintracellular pools of SNOs are poorly understood but transportmechanisms involving cell-surface protein disulfide isomerases (Zai et al.,1999; Ramachandran et al., 2001), g-glutamyl transpeptidase (De Grooteet al., 1995; Hogg et al., 1997), or anion exchangers (Pawloski et al., 2001)have been proposed. GSNO is proposed to transfer NOþ to outermembrane thiols in Bacillus (Morris and Hansen, 1981) but other studiessuggest that active transport of the compound is required for toxicity.

In S. enterica, GSNO (0.5 mM) is bacteriostatic but GSNO is not itselftransported into the cell. Highly GSNO-resistant mutants were isolatedfrom a MudJ transposon library and the insertions shown to be in dppAand dppD (De Groote et al., 1995). These genes are part of an operonencoding dipeptide permease, an ABC-family transporter responsible forL-dipeptide import. It is therefore suggested that a periplasmictranspeptidase 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 residualdipeptide, S-nitroso-L-cysteinylglycine, is then transported inward usingthe Dpp-encoded dipeptide permease. Figure 5 summarizes the proposedmechanisms. A similar mechanism appears to operate in E. coli since reg-ulatory perturbations inducible by GSNO are dependent on the presenceof the Dpp system (Jarboe et al., 2008). In other words, the toxic agentin the cytoplasm is not GSNO but the nitrosated dipeptide.

In M. bovis, the oligopeptide permease operon (oppBCDA) isimplicated in GSNO transport (Green et al., 2000). Mutation of the oppDgene encoding the ATPase component of this binding protein-dependenttransport system elicits resistance to 4 mM GSH in the external medium,a concentration that inhibits the wild-type strain. Importantly, similarresults were found with 0.5 mM GSNO, which is bactericidal for thewild-type strain but not the oppD mutant. The resistance of the mutantto GSH is due to diminished import of the thiol, as shown by transportstudies using [3H]GSH. In view of the finding that, in S. enterica, it is trans-port of the dipeptide that carries the NOþ group (De Groote et al., 1995),Green et al. (2000) tested whether the opp system in M. bovis transports


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 orS-nitroso-N-acetyl-D,L-penicillamine (SNAP) is influenced by the availabilityof cysteine or cystine. Indeed, the degradation of GSNO is absolutely depen-dent on extracellular cystine, which is reduced to cysteine, which in turnreacts with GSNO to form S-nitrosocysteine (CysNO) in a transnitrosationreaction. CysNO is then imported by the amino acid transport systemL-AT (Zhang and Hogg, 2004).

Opp

A. Salmonella enterica B. Mycobacterium bovis

Glu-CysNO-Gly(GSNO)

Glu-CysNO-Gly

CysNO-Gly

CysNO-Gly

Glu

GGT

Outer membrane

Periplasmic space

Inner membrane

GSNO

SBP

Dpp

Transnitrosationreactions

Figure 5 Transport mechanisms for SNOs in bacteria leading to intracellulartransnitrosation 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 transportedinward using the Dpp-encoded dipeptide permease. (B) In M. bovis, theoligopeptide permease (Opp) affects GSNO transport (Green et al., 2000). A peri-plasmic substrate-binding protein (SBP) is a product of the same operon.


5.4. Other Donors

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

5.5. HNO Donors

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

N2O32� þHþ ! HNOþNO2

� ð20ÞSimilar to the diazeniumdiolates, the release of HNO from Angeli’s salt

occurs via specific protonation on one of several possible protonation sites(Dutton et al., 2004b). The half-lives of Angeli’s salt at 25 and 37 �C areapproximately 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 usingauthentic NO2

� (or a better alternative, albeit more expensive, is to examinethe effects of decomposed Angeli’s salt). Thus, Angeli’s salt is a convenientand well-definedHNOdonor 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 arepresentative of the N-hydroxylsulfonamide class of donor. The mecha-nism of decomposition of Piloty’s acid requires the deprotonation of aweakly acidic proton (Reaction 21), and therefore only generates HNOat a significant rate under basic conditions.

R� SðOÞ2NHOH ! R� SðOÞ2 �NHO� ! R� SðOÞO� þHNO ð21ÞThus, 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 decom-position is slow, autoxidation can occur in aerobic solutions, leading to NOrelease (Reaction 22) (Zamora et al., 1995).

R� SðOÞ2NHOH ! R� SðOÞ2NHO� ! R� SðOÞOHþNO ð22Þ


The slow release under typical biological conditions and the possibleautoxidation makes Piloty’s acid (and derived species) less than optimumdonors of HNO for many biological preparations.

Recent reports indicate the utility of other HNO donors. Acyloxynitroso compounds reported by Sha et al. (2006) require an ester hydrolysisstep prior to HNO release but offer a wide array of structural diversity anddiazeniumdiolates made from primary amines appear to offer structuraldiversity and spontaneous HNO release in vivo (Miranda et al., 2005).Significantly, the HNO-donating prodrug cyanamide (H2NCN) has beenused clinically in antialcoholism therapy. Cyanamide can be oxidized toN-hydroxy cyanamide, which spontaneously decomposes to HNO andHCN (Reaction 23).

H2N� CN ! HONH� CN ! HNO þHCN ð23ÞOxidation of cyanamide can be carried out by catalase/H2O2 (Nagasawa

et al., 1990). Thus, HNO release from cyanamide requires oxidativebioactivation 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 HNOas a species directly responsible for biological effects. However, a clearexample is provided by work on the thiol-containing, metal-responsive yeast(Saccharomyces cerevisiae) transcription factor Ace1. Ace1 is activated bybinding copper via multiple Cys thiols, resulting in the transcription of genesencoding proteins involved in copper sequestration (Shinyashiki et al., 2005).Activation of Ace1 by copper addition to cultures in the presence of variousnitrogen oxides has been studied in depth. Both diethylamine NONOate(DEA/NO) and Angeli’s salt inhibited Ace1, but the inhibition by NO wasoxygen dependent while the effect of the HNO donor was not (Cooket al., 2003). The results are interpreted as thiol modification by NO viathe generation of nitrosating species, whereas HNO is able to react directlywith protein thiols. The work also provides an example of the use ofdecomposed donors as controls (see Section 5.1): decomposed Angeli’s saltwas 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 com-pound and via the use of donors. The most commonly utilized ONOO�

donors are the sydnonimines (Fig. 6). SIN-1 (R¼morpholino, Fig. 6) is a


prototypical sydnonimine and its decomposition has been studied exten-sively (Bohn and Schonafinger, 1989). In aqueous, aerobic solution SIN-1will 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 thereaction of NO and O2

� is near diffusion controlled, aerobic SIN-1 decom-position results in formation of ONOO�.

Many in vitro studies have employed these generators to examine thereactivity and toxicity of ONOO�, but findings have often been contradic-tory. For example, superoxide dismutase (SOD) has been shown to pro-vide considerable protection toward SIN-1-mediated killing in E. coli,which is due to O2

� scavenging, preventing ONOO� formation (Brunelliet al., 1995). Conversely, it has been demonstrated that SOD actually pro-motes SIN-1 toxicity. In one study, SOD potentiated the cytotoxicity ofSIN-1 toward the human hepatoma liver cell line (HepG2) by elevatingH2O2 production through the catalyzed dismutation of O2

� (Gergel et al.,1995). However, in another study, SOD enhanced SIN-1 killing of theparasite L. major by scavenging O2

� and increasing the half-life of the truenoxious species, NO (Assreuy et al., 1994). Consequently, it is apparentthat when employing ONOO� generators, appropriate controlexperiments must be conducted in order to establish whether observationsare attributable to ONOO� per se or its reactants.

5.7. NO2

Nitrogen dioxide is available as a compressed gas. Since NO2 is an oxidantand toxic, use of authentic gas can be problematic if proper gas handlingequipment is not available. Moreover, solutions of NO2 are not stable sinceit will dimerize to N2O4, which hydrolyzes to give NO2

� and NO3�

(Reactions 24 and 25).

N

N

ONH

R

N

N

ONH

R

Sydnonimines, R = morpholino (SIN-1)

–

–

Figure 6 General structure of sydnonimines.


2NO2 ! N2O4 ð24Þ

N2O4 þH2O ! NO2� þNO3

� þ 2Hþ ð25ÞThus, experiments aimed at examining specifically NO2 in a biological

system are not always straightforward. In instances where the observedbiological activity is thought to be a result of NO2 generation via autoxida-tion of NO, a convenient reagent is available for the facile conversion ofNO to NO2. Nitronyl nitroxides (e.g., PTIO) or the water soluble analogcarboxy PTIO (cPTIO), which are widely used research tools for scaveng-ing NO (Akaike et al., 1993), are capable of converting NO directly to NO2

in an O2-independent manner (thus avoiding the high-order kinetics ofautoxidation) (Akaike and Maeda, 1996) (Reaction 26).

PTIO þNO ! NO2 þ PTI ð26ÞGeneration of NO2 from NO allows the possibility that N2O3 is the bio-

logically active species since NO and NO2 react quickly to give N2O3. Dis-tinguishing between the actions of NO2 and N2O3 can be addressed byexamining the effect of PTIO concentration on the biological activity(e.g., Shinyashiki et al., 2004). At low PTIO/NO ratios, the NO2 formedwill have the opportunity to react with remaining NO to generate N2O3.However, at high PTIO/NO ratios, most of the NO will be converted toNO2, precluding formation of N2O3.

Nitronyl nitroxides are also used to detect NO since the reaction of theEPR-active PTIO with NO generates another distinct EPR-active species(PTI) (Akaike and Maeda, 1996). Thus monitoring the conversion ofPTIO to PTI via EPR can serve as a quantitative indication of the NOlevels. When using the nitronyl nitroxides as research tools (either fordetection/quantitation of NO or as a reagent to convert NO to NO2), it isimportant to realize that the ultimate products generated from the reactionof NO with PTIO depend significantly on the relative concentrations of thereactants (Goldstein et al., 2003). For example, the NO2 formed from thereaction of NO and PTIO can also react with PTIO to give NO2

� andthe corresponding oxoammonium cation (PTIOþ). PTIOþ can furtherreact with NO (in aqueous solution) to give PTIO and NO2

�. Thus, quan-titation of NO via PTIO is valid only when NO is in low concentration andalternative scavengers for NO2 are important considerations when PTIO isused to convert NO to NO2. Regardless, with a reasonable understandingof this chemistry, the nitronyl nitroxides are important tools for examiningmany aspects of nitrogen oxide chemistry and biology.


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 importantto note, however, that high concentrations of NO2

� (especially under acidicconditions) can generateN2O3 (the anhydride of nitrous acid, HONO)whichis also a source of NO andNO2. Despite this, many studies use, as a matter ofconvenience, 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 inexperiments aimed at determining the mode of action of NO and RNS. Inone report, killing of Mycobacterium ulcerans, which causes ulcerative skindisease, was reported within 20 min but the concentration of nitrite was veryhigh (40 mM) (Phillips et al., 2004). More recently, it has been reported thatnrfA and ytfEmutants were sensitive to “NO donors” but the reagents usedwere 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 inaqueous solutions. An overview of the relationship between the nitrogencompounds discussed in Sections 4 and 5 is presented as Fig. 7.

6. BACTERIAL RESPONSES TO RNS: EFFECTORSAND REGULATORS

6.1. Targets of RNS in Microorganisms

It is frequently stated that NO is a highly reactive gas and must interactwith 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 asrestricted as those of another “gasotransmitter,” carbon monoxide (CO)(Davidge et al., 2009a). The direct cellular effects of NO are incompletelyunderstood because of the complexity of NO chemistry introduced inSection 4. Nevertheless, various biomolecules are targeted by NO andthe resulting RNS. Some examples from this vast literature are listed inTable 2 and a detailed analysis is presented in Stamler et al. (2001).


6.2. Microbial Defenses: The Microbe Strikes Back

Many mechanisms are known to confer NO resistance in bacteria, such asflavohemoglobin-catalyzed NO detoxification in enteric bacteria andnumerous other species as well as other globins. Confusing the literature,however, is that many experimental studies to unravel the details of NOresistance are performed with proxies for NO, often SNOs and especiallyGSNO. It is important to recognize that most of the enzymic detoxificationsystems studied, as far we can judge from current experiments, are first andforemost concerned with NO detoxification, not with SNO detoxification.The best understood of these, flavohemoglobin, is an NO-detoxifyingenzyme and has no significant activity against SNOs. Although the earliestpapers on flavohemoglobin regulation (Poole et al., 1996) and enzymicfunction (Gardner et al., 1998) were conducted with “real” NO, many later

NOL-Arginine O2

e−

O2− e−, 2H+

H2O2e−, H+

HO . + H2O

NO2

2nd orderin [NO]

N2O3

Nitrosationchemistry

("NO+"-donor)

1e− oxidant

−e−

H2O

NO2 −+ H+

−e−

ONOO−

ONOOH

H+

Powerful 1e−

oxidant

−e−, H2O

NO3−+ 2H+

−e−

RSH

RS-NO + H+

2e−

oxidant

Small %

Small %

Majorreaction

e−, H+

H2O

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


studies have substituted these agents. Hausladen et al. (1998a) provide aclear distinction between NO metabolism and SNO metabolism. It wasknown that SNO decomposition by E. coli generates nitrite and nitrateand that NO could arise from the former via chemistry described above.A quest for NO decomposition showed that although the NO-metabolizingactivity 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 developingfield: regulation of gene expression by NO and RNS and the functionalidentification of resistance mechanisms are being studied in numerous bac-teria, as well as fungi and protozoa. Here we focus on a small number ofsystems that have emerged as paradigms for future study and bring toreaders’ attention selected new papers that illustrate novel or particularlyinteresting examples.

6.3. Microbial Globins

An exciting development in biological NO biochemistry over the past15 years has been the realization that a major mechanism for NO resis-tance and detoxification in bacteria and eukaryotic microbes is hemoglo-bin-mediated NO chemistry. Microbial globins have consequently beenextensively 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 typicallydefined by reactivity toward small ligands, such as O2, CO, and NO, whichbind to the heme distal site. Reactivity with, and biological activity in rela-tion to, each of these ligands has been reported for various microbialglobins. 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-terminaldomain with binding sites for flavin adenine dinucleotide (FAD) andnicotinamide adenine dinucleotide (phosphate) [NAD(P)H]. Widely


Tab

le3

Exa

mples

ofproteins

implicated

intoleranceto

NO

andnitrosativestress

viaconsum

ptionof

NO

orS-nitrosothiols.

Class

Protein

Organ

ism(s)

Fun

ction/reaction

scatalyzed

Com

men

tsReferen

cea

1.Globins

Myo

glob

in,

hemog

lobin

Highe

ran

imals

Transient

form

ationof

peroxy

nitrite-

glob

in;q

uantitative

form

ationof

nitrate

witho

utnitrationof

glob

in

Nitrite

redu

ction;

physiologicalrole

inNO

tolerance?

NO

scav

enging

?OONO

�

scav

enging

?

Bruno

ri(200

1);

Floge

let

al.(200

1);

Hen

dgen

-Cotta

etal.

(200

8);A

scen

ziet

al.

(200

9)

Truncated

glob

in(H

bN)

Mycob

acterium

species

Con

versionof

NO

tonitrate?

NO-ind

ucible

NO

uptake

Paw

aria

etal.(20

07);

Martr

etal.(200

8);

Lam

aet

al.(200

9)Vitreoscillaglob

in(V

gb)

Vitreoscillasp.

NO

consum

ption

Mecha

nism

unkn

own;

glob

inconfersgrow

thtoleranceto

SNP

Kallio

etal.(200

7);

Freyet

al.(201

1)

Sing

le-dom

ain

glob

in(C

gb)

Cam

pyloba

cter

jejuni,C.coli

Con

fers

enha

nced

resistan

ceto

NO

andnitrosating

agen

ts

Mecha

nism

presum

edto

be“NO

diox

ygen

ase”

conv

erting

NO

tonitrate

Mon

ket

al.(200

8);

Shep

herd

etal.

(201

0a,2

011);S

mith

etal.(201

1)

Flavo

hemog

lobin

(Hmp)

E.coli,

Salm

onella,

B.subtilis,

Erw

inia,man

yothe

rs

Enzym

icde

toxification

ofNO

byconv

ersion

tonitrate

hmpex

pression

upregu

latedby

NO

andnitrosatingag

ents;

mutan

tsareNO

sensitive

Stev

anin

etal.

(200

7);L

aver

etal.

(201

0);S

vensson

etal.(201

0);W

ang

etal.(201

0b)

(con

tinued)

Tab

le3

(con

tinued)

Class

Protein

Organ

ism(s)

Fun

ction/reaction

scatalyzed

Com

men

tsReferen

cea

2. Red

uctases

Flavo

rubred

oxin

(NorVW

)E.coli,

Salm

onella

NO

redu

ctionan

dde

toxification

Upreg

ulated

inrespon

seto

NO

and

RNS

Millset

al.(200

5,20

08);Pullanet

al.

(200

7)Cytochrom

ec

nitriteredu

ctase

(NrfA)

E.coli,C.jejun

iNO

redu

ctionan

dde

toxification

Nrf

mutan

tstrains

show

high

erNO

sensitivity

Pittm

anet

al.(200

7);

Millset

al.(200

8);

vanW

onde

renet

al.

(200

8);E

insle(201

1)SN

O-lya

se;G

SH-

depe

nden

tform

alde

hyde

dehy

drog

enase

E.coli,

yeast,

mam

malian

cells

GSN

Oor

SNO

redu

ctase

Con

trolscellu

larleve

lsof

S-nitrosothiolsan

dS-nitrosylated

proteins

Fosteret

al.(200

9b);

Tav

ares

etal.(200

9)

Cytochrom

ean

dqu

inol

oxidases

Mitocho

ndria

ofhigh

erorga

nism

s,ba

cteria

NO

redu

ctionat

Cu B

Activitymay

berestricted

toox

idases

inhe

me-Cu B

family

;ph

ysiological

sign

ifican

ceun

clea

r

Butleret

al.(200

2);

Borisov

etal.(200

9)

3.Others

Cytochrom

ec0

(CycP)

Rho

doba

cter

capsulatus

NO

redu

ctase,

form

ingN

2OCycPmutan

tsare

hype

rsen

sitive

tonitrosothiolsan

dNO

Stev

anin

etal.

(200

5);H

eurlier

etal.(200

8)

a Illu

strative

only;the

mostrecent

pape

rsarecitedin

mostcases.

distributed in bacteria and lower eukaryotes, flavohemoglobins conferprotection from NO and nitrosative stresses by direct consumption ofNO (Poole and Hughes, 2000). Indeed, flavohemoglobins appear to haveno direct role in the metabolism of oxygen or other gaseous ligandsand such proteins have not been reported in higher animals.Flavohemoglobins oxidize NAD(P)H and transfer an electron to theN-terminal heme domain via a noncovalently bound FAD in the reduc-tase (or FNR, ferredoxin-NADP reductase-like) domain. Reduced hemecatalyzes the reaction between NO and O2 generating nitrate; that is,Hmp acts as an NO-detoxifying enzyme. There remains controversy overthe reaction mechanism at the heme: either NO (denitrosylase mecha-nism) (Hausladen et al., 1998b, 2001) or O2 (dioxygenase mechanism)(Gardner et al., 1998, 2000, 2006) has been claimed to bind first to theheme.

Flavohemoglobins are critical for pathogenicity in some species; forexample, E. coli and Salmonella mutants lacking Hmp are compromisedfor survival in mouse and human macrophages (Stevanin et al., 2002,2007). In the plant pathogen Erwinia chrysanthemi, HmpX not onlyprotects against nitrosative stress but also attenuates host hypersensitivereaction during infection by intercepting NO produced by the plant forthe execution of the hypersensitive cell death program (Favey et al.,1995; Boccara et al., 2005). In accordance with the role of Hmp in limitingNO-related toxicity, expression of the protein occurs only when NO ispresent in the cell environment. Indeed, hmp gene expression is tightlyregulated at the transcriptional level by NO-responsive transcriptionfactors, notably NsrR and Fnr (Spiro, 2007). NO regulation of gene expres-sion is discussed more fully below (Section 7). Tight control of Hmpsynthesis and function appears critical since constitutive Hmp expressionand function in E. coli in the absence of NO generates oxidative stressby virtue of oxygen reduction by the heme to superoxide anion (Pooleet al., 1997; Wu et al., 2004). Similarly, constitutive expression of Hmp inSalmonella 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, presum-ably because NO diverts Hmp function to nitrate formation, rather thangeneration of oxidative stress, which exacerbates the stress caused byONOO�. The flavohemoglobins from diverse bacteria have protectivefunctions against RNS, including Ralstonia eutropha, Bacillus subtilis,P. aeruginosa, Deinococcus radiodurans, S. enterica, and Klebsiellapneumoniae (Frey et al., 2002). Note, however, that in this study the agentof RNS used was SNP (1 mM), an unfortunate choice.


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 myoglobinbut no separate C-terminal reductase domain. This class is typified by theglobin of Vitreoscilla (named Vgb, VtHb, or Vhb), an obligate aerobicbacterium that grows in low oxygen environments (Webster, 1987). This glo-bin was the first bacterial hemoglobin to be crystallized, and the 3D structure(of the ferric homodimer) conforms to the classical globin fold (Bolognesiet al., 1999). This protein has been implicated in redox chemistry and NOdetoxification in vivo, but the mechanism by which the protein is rereducedafter 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 apotential site of interaction with the putative FAD/NADH reductase partner.Considerable interest has been directed at Vgb because of its possible role infacilitating oxygen transport and metabolism and the consequent biotechno-logical implications (Frey et al., 2011). Interestingly, a chimeric protein com-prising the Vitreoscilla hemoglobin and a flavoreductase domain from aflavohemoglobin (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, pathogenicbacterium C. jejuni, which is exposed to NO and other nitrosating speciesduring host infection (Iovine et al., 2008; Tarantino et al., 2009). Thissingle-domain globin, Cgb, is dramatically upregulated by the transcriptionfactor NssR in response to nitrosative stress (Elvers et al., 2005; Monket al., 2008; Smith et al., 2011). Cgb has been shown to detoxify NO andpossess a peroxidase-like heme-binding cleft. In marked contrast toVitreoscilla Vgb, there is no evidence to date that Cgb functions in oxygendelivery. Cgb can provide an electronic “push” from the proximal ligandand an electronic “pull” from the distal binding pocket, creating a favor-able environment for the isomerization of a putative ONOO� intermediatein the NO dioxygenase reaction (Shepherd et al., 2010a). We have recentlydemonstrated that the mechanism of NO detoxification is unlikely to pro-ceed via the formation of an oxyferryl (Fe(IV)¼¼O) species but that NOinteracts 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 themost recently discovered and appear widely distributed in bacteria,


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 offlavohemoglobins, trHbs adopt a 2-over-2 a-helical structure and are typ-ically 20 residues shorter than 3-over-3 globins. Sequence analysis ofmore 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 trHbgroups I and II, although the type-III trHb from C. jejuni, Ctb, hasrecently been structurally and kinetically characterized (Wainwrightet 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) remainsenigmatic: although its expression is elevated on exposure to NO andRNS, 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 anNO-sensitive phenotype (Wainwright et al., 2005). Recently we havesuggested (Smith et al., 2011) that binding of NO or oxygen (Ctb havingan especially high affinity for the latter) may serve to modulate the intra-cellular availability of NO for NssR activation. Further work is requiredto define a clear function; indeed, the same is true for many bacterialglobins. Work conducted on M. bovis revealed that trHbn stoichiometri-cally oxidizes NO to NO3

� and protects aerobic respiration from NOinhibition (Ouellett et al., 2002).

It is speculated that single-domain and truncated globins associate withhost reductases as a source of electrons for NO or O2 reduction. A recentstudy conducted on a mouse neuroglobin (Ngb) that plays a role inneuroprotection showed that NADH:flavorubredoxin oxidoreductasefrom E. coli could reduce the globin in the presence of NADH as theelectron donor (Giuffre et al., 2008). Despite early reports (Jakob et al.,1992) of a reductase purified from Vitreoscilla that reduced theVitreoscilla hemoglobin (Vgb, Vhb) in vitro, this aspect of the mode ofaction of NO-detoxifying globins remains obscure. It appears that manyglobins could, in principle, serve an NO scavenging function if providedwith a mechanism for heme iron re-reduction such as, in vitro, ferre-doxin-NADP reductase (E. coli) (Smagghe et al., 2008). The reductionmechanism may, however, involve not a specific, cognate reductase butthe general reducing environment of the bacterial cytoplasm governed,for example, by the GSH pool. A similar idea has been proposed for thereduction of bacterial NOS enzymes that lack a reductase domain(Gusarov et al., 2008).


6.4. NO and RNS Reductases

6.4.1. NO Reductases

In E. coli, NorR (see Section 7.3) activates the transcription of the norVWgenes encoding a flavorubredoxin and an associated flavoprotein, respec-tively, which together have NADH-dependent NO reductase activity(Gardner et al., 2003). Confusingly, these proteins have also been referredto 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 themost extensively characterized reductase that detoxifies NO under anaero-bic and microaerobic conditions (Gardner and Gardner, 2002; Gardneret al., 2002; Gomes et al., 2002). NorV is an oxygen-sensitive flavor-ubredoxin 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 norVmutant exhibits impaired growth in the presence of NO and is sensitivealso to SNP (Hutchings et al., 2002). E. coli also possesses a dedicatedNAD(P)H flavorubredoxin oxidoreductase, NorW, whose function isbelieved to be rereduction of the NorV protein.

Expression of E. coli NorV and NorW is regulated at the transcriptionallevel, via the regulator NorR, by a variety of NO-related species includingGSNO (Mukhopadhyay et al., 2004; Flatley et al., 2005), NO (from a saturatedsolution of the gas) (Justino et al., 2005), and NO released from NOC-5 andNOC-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 aheme-based sensor (Gardner, 2005), but instead NoR is activated by the for-mation of a mono-nitrosyl iron center located in the GAF domain (i.e., adomain related to cyclicGMP-regulated cyclic nucleotide phosphodiesterases,adenylyl cyclase and FhlA) of the protein (D’Autreaux et al., 2005). It hasbeen suggested that NorR responds exclusively to NO (Spiro, 2006), sincetreatment 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 vivoby upregulation of norVW during growth with GSNO (e.g., Flatley et al.,2005; Pullan et al., 2007). NorR is a critical sensor ofNO-related stress not onlyin E. coli but also in R. eutropha and P. aeruginosa (Spiro, 2007).

Periplasmic cytochrome c nitrite reductase, nrfA, is expressed by manyenteric pathogens including E. coli and S. enterica under microaerobic oranaerobic conditions where electron acceptors are limited. The protein cata-lyzes a six-electron reduction of NO2

� to ammonium (NH4þ) with NO as a

proposed intermediate (Simon, 2002). Indeed, we have shown that NrfA is


responsible for much of the NO evolution that occurs in vitro when anaero-bically grown cells are treated with high concentrations of nitrite (Corkerand Poole, 2003) (see below).However, the role of NrfA inNObiochemistryis controversial. It has also been known for some time that NrfA can drive afive-electron reduction of exogenous NO and a physiological relevance ofthis function has been proposed in NO consumption (Poock et al., 2002).Wild-type cells cultured anaerobically consumed 300 nmol of NO per mgof protein per min, whereas NO reduction was negligible in nrfA mutants.Additionally, growth of nrfA mutants was completely attenuated followingthe addition of 150 mM NO, whereas wild-type growth was completelyunperturbed (Poock et al., 2002). A recent comprehensive study of the NOdetoxification machinery of Salmonella has revealed roles for all threeproteins so far invoked in anaerobic NO tolerance (Mills et al., 2008). A suiteof double and triple mutants with deletions in hmp, norV, and nrfAwas usedto conclude that the NO reductase NorV and the nitrite reductase NrfA arerequired additively for NO tolerance under anaerobic fermentativeconditions as well as under anaerobic respiratory (glycerol with fumarateand nitrate) conditions. NO solutions (40 mM final concentration) were used(Mills et al., 2008). A minor role for Hmp was also demonstrated, consistentwith the low activity of this protein in anoxic NO consumption (Kim et al.,1999). It is proposed that the periplasmic location of NrfA enables NOreduction prior to cell entry while any NO that does enter the cell ismetabolized by cytoplasmic NorV (Mills et al., 2008). A constitutivelyexpressed NrfA has also been described inC. jejui, which is proposed to pro-vide 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-relatedmolecules, including S-nitrosothiols (SNOs). SNO formation is consideredto be a form of post-translational protein modification of importance innumerous 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 dehydrogenaseproteins (AdhC) were identified as exhibiting GSNO reductase activityin N. meningitidis, H. influenza, and S. pneumoniae (Kidd et al., 2007;Potter et al., 2007; Stroeher et al., 2007). Such enzymes may have importantfunctions in alleviating stress imposed by nitrosative species. In yeast, the


combined effects of flavohemoglobin and GSNO reductase (consumingNO and GSNO, respectively) modulate SNO levels. Nitrosative stressmay be mediated principally by the S-nitrosylation of a particular subsetof protein targets (Foster et al., 2009b). The levels of low molecular weightSNOs may be diminished not only by SNO-lyases but also by the activitiesof NO-consuming enzymes. Thus, the meningococcal NO reductase NorBincreases the rate of SNO (GSNO) degradation in vitro and SNO forma-tion in murine macrophages is reduced by infection with NorB-expressingmeningococci 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 theprevention of new SNO formation that is critical. These mechanisms maycontribute to bacterial pathogenesis since S-nitrosylation is criticallyinvolved in, for example, apoptosis, signaling cascades involving neuronalNOS (Jaffrey et al., 2001), and regulation of gene expression.

The reduction of GSNO has also been demonstrated by thenitroreductase, 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, thethioredoxin system has a demonstrated role in GSNO reduction, as shownby the accelerated breakdown of this nitrosating species in vitro bythioredoxin/thioredoxin reductase isolated from both E. coli (Nikitovicand Holmgren, 1996) and M. tuberculosis (Attarian et al., 2009). Furtherto this, Helicobacter pylori strains deficient in either of its two thioredoxinproteins have been shown to be more sensitive to GSNO and SNP than theisogenic wild-type strain, suggesting that the thioredoxin system may beinvolved in SNO depletion (Comtois et al., 2003).

6.5. Other Proteins Implicated in NO Tolerance

Mutational and transcriptomic approaches have revealed additionalproteins with known or inferred roles in protection from NO or RNS.Rather than speculate here on the possible roles of the numerous genesidentified as upregulated by these stresses, we focus on a subset that havereceived more careful consideration.

A transcriptomic analysis of anaerobically grown E. coli treated withNO (50 mM, from a solution of the gas) revealed upregulation not onlyof hmp, the norVW operon (Sections 6.3.1 and 6.4.1), and genes of inter-mediary metabolism, but also of ytfE (see below) and the gene for aLysR-type regulator, yidZ (Justino et al., 2005). Mutation of either generendered cells hypersusceptible to NO.


The ytfE gene has subsequently been shown to be regulated by NO orother RNS in several other studies and is one of a very small group ofgenes that is consistently and massively upregulated on bacterial exposureto NO and nitrosative stress. It is widely accepted that the YtfE proteinplays 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 ofstrains carrying a “clean” ytfE mutation confirms a role in iron–sulfur clus-ter 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 dinucleariron center having m-peroxy and m-carboxylate bridging ligands and six Hisresidues coordinating the irons (Todorovic et al., 2008). Homologues ofYtfE occur in numerous bacteria (Overton et al., 2008). In Haemophilusinfluenzae, for example, ytfE mutants are sensitive to agents of nitrosativestress, 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 macro-phage assaults, an effect that was abrogated by an inhibitor of NOS. Inter-estingly, in this organism, activation of ytfE is dependent on positivecontrol by Fnr (see Section 7.2), despite the fact that Fnr is itself aniron–sulfur cluster protein, whereas in E. coli, ytfE expression is negativelyregulated by Fnr although the effect may be indirect (Justino et al., 2006).

Several transcriptomic studies suggest a role in NO responses of the hcpand hcr genes, encoding the hybrid cluster protein (an iron–sulfur protein)and its cognate reductase. These proteins may form a hydroxylamine oxi-doreductase that converts NH2OH to ammonia (Wolfe et al., 2002; Cabelloet 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 certainmicrobial lifestyles, particularly biofilm formation and symbioses (for areview, see Wang and Ruby, 2011). The first report of a bacterial NOresponse in symbiosis was that of Meilhoc and others who showed thatSinorhizobiummeliloti upregulates>100 genes in response toNO, includinghmp, and that an hmpmutant showed decreased nitrogen fixation in planta.It is proposed that this detoxification system overcomes the inhibitory effectsof NO on nitrogen fixation during symbiosis (Meilhoc et al., 2010).


In one well-studied example, the squid-Vibrio light-organ symbiosis, NOserves as a signal, antioxidant and specificity determinant (Wang andRuby, 2011). In the early stages of the symbiosis, NOS is active and thisenzyme and NO are detectable in vesicles of the mucus secretion whereVibrio fischeri cells aggregate. Experiments with NO scavengers demon-strate that this NO influences specificity of the interaction since NOremoval permits nonsymbiotic Vibrio species to form hyperaggregates.As V. fischeri penetrates the deep crypts of the light organ, the bacteriumexperiences higher levels of NO and colonization irreversibly reduces theNO and NOS levels there. The host therefore uses NO to sense andrespond to the correct symbiont.

The genetic and biochemical evidence suggests that V. fischeri possessesan 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 ironin the form of hemin (Wang et al., 2010a). To manage the NO levels towhich V. fischeri is exposed, it possesses several NO detoxification systemsincluding 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 aspectsof 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 fromiNOS combine to generate ONOO�, which exerts greater toxicity thaneither 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 whetherONOO� is a major antimicrobial species in this scenario. Peroxynitritemay also arise from the activity of a single enzyme: exposure of murinemacrophages to Bacillus anthracis endospores upregulates NOS2. Thisisozyme generates not only NO but also superoxide, and the anticipatedproduct, 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


range of pathogens, including E. coli (Zhu et al., 1992; Brunelli et al., 1995),H. pylori (Tecder-Unal et al., 2008), and Trypanosoma cruzi (Alvarezet al., 2004). However, evidence is increasing that pathogens possess sys-tems that directly detoxify ONOO�, allowing them to evade this speciesand thrive. In Salmonella typhi, an rpoS mutant is more susceptible toONOO� than a wild-type strain but the molecular basis of this is at presentobscure (Alam et al., 2006). Detailed examples will be discussed below.

Peroxiredoxins are typically associated with the reduction of H2O2 andorganic hydroperoxides and are widespread throughout prokaryotic andeukaryotic systems (Poole, 2005a). The peroxiredoxin alkylhydroperoxidereductase subunit C (AhpC) isolated from S. enterica enabled catalyticbreakdown of ONOO� to NO2

� with a second-order rate constant of1.51�106 M�1 s�1. This proceeds via oxidation of a cysteine residue towardtheN-terminus of the protein, and catalytic turnover is achieved by reductionof AhpC by the flavoprotein AhpF. Catalysis was shown to be efficientin protecting plasmid DNA from single-strand breaks (Bryk et al., 2000).Peroxiredoxins with peroxynitritase activity, enzymes with the ability tobreak down ONOO�, have also been identified in other microbes, includingS. cerevisiae. This eukaryote possesses two peroxiredoxins, thioredoxinperoxidase I and II (Tsa1 and Tsa2), which share 86% identity at the aminoacid level. These proteins catalyze the breakdown of ONOO�with reportedsecond-order rate constants in the region of 105 M�1 s�1 but lack a dedicatedreductase 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 cata-lytic turnover of ONOO�. Classically, these heme-containing enzymesare characterized by their bifunctional capacity to break down H2O2 andorganic peroxides (Claiborne and Fridovich, 1979). However, the cata-lase-peroxidase KatG from M. tuberculosis (Wengenack et al., 1999) andS. enterica (McLean et al., 2010a) have also demonstrated peroxynitritaseactivities with reported second-order rate constants of 1.4�105 M�1 s�1

and 4.2�104 M�1 s�1, respectively. Following incubation with ONOOH,Wengenack et al. (1999) demonstrate an initial reduction in the Soret bandof 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 path-ogenic organisms that exhibit peroxynitritase activity. In light of theperoxynitritase activities of certain peroxiredoxins and catalase-peroxidases, it would appear that these enzymes have the capacity todetoxify a repertoire of reactive species, making them potentially powerfulvirulence factors.


Tab

le4

Second

-order

rate

constantsof

proteins

withpe

roxy

nitritaseactivities.

Protein

Organ

ism/Sou

rce

Tem

perature

(�C)/pH

10�5�Se

cond

-order

rate

constant

(M�1s�

1 )Referen

ce

KatG

E.coli

25/7.40

0.33

L.B

owman

andS.

McL

ean,

unpu

blishe

dfind

ing

KatG

S.enterica

25/7.40

0.42

McL

eanet

al.(201

0a)

KatG

M.tuberculosis

37/7.40

1.40

Wen

gena

cket

al.(199

9)Ahp

CH.py

lori

RT/6.75

12.10

Bryket

al.(200

0)Ahp

CM.tuberculosis

RT/6.75

13.30

Bryket

al.(200

0)Ahp

CS.

enterica

RT/6.75

15.10

Bryket

al.(200

0)Thiored

oxin

peroxida

seII

S.cerevisiae

25/7.40

5.10

Ogu

sucu

etal.(200

7)

Thiored

oxin

peroxida

seI

S.cerevisiae

25/7.40

7.40

Ogu

sucu

etal.(200

7)

Trypa

redo

xin

peroxida

seT.cruz

i37/7.40

7.20

Trujillo

etal.(200

4)

Trypa

redo

xin

peroxida

seT.brucei

37/7.40

9.00

Trujillo

etal.(200

4)

RT—room

tempe

rature.

7. MICROBIAL SENSING OF NO AND GENE REGULATION

7.1. Introduction

Since the discovery that expression of the flavohemoglobin Hmp of E. coliis upregulated by NO and that this protein constitutes an effective NOdetoxification enzyme, there has been an explosion of interest in the molec-ular mechanisms underpinning the regulation of this gene and the manyothers now implicated in NO and RNS detoxification. Excellent reviewshave recently appeared (Spiro, 2006, 2007), and the reader is referred tothose 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), andNsrR (Bodenmiller and Spiro, 2006). Spiro usefully distinguishes betweenthose that are secondary sensors and those that are “dedicated” in that theirphysiological function appears to be detecting primarily NO and thenregulating expression of genes that encode enzymes with NO as a substrate.The archetype is NsrR, which appears in enterobacteria to be the majorregulator for NO-detoxifying proteins like flavohemoglobin. In addition tosensing NO in solution, these regulators may also sense the small levels ofNO that may emanate from SNOs. Here we present only a few commentson some of the major regulators, emphasizing the sensing of NO. NO sensorsare also known in higher organisms such as the mammalian circadian proteinCLOCK (Lukat-Rodgers et al., 2010).

7.2. Fnr

Fnr is an example of the large Fnr-CRP family of transcriptional regulatorswidely distributed in bacteria (Green et al., 2001). The family also includesthe CO sensor CooA and the first clear example of an NO sensor, namely,the NnrR protein of Rhodobacter sphaeroides (Tosques et al., 1996). Thereis strong evidence that it is NO that is sensed by NnrR and other membersof the family (Spiro, 2007). The first clue to the involvement of Fnr in hmpregulation was the finding that a lysogenic hmp–lacZ reporter constructwas more highly expressed in an fnr mutant than in the parent strain(Poole et al., 1996), indicating that Fnr represses hmp transcription andconsistent with the presence of an Fnr binding site close to the �10sequence 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


dinitrosyl-iron-dithiol (DNIC) complexes (for a review, see Tonzetichet 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 thatcontrols a small regulon; some of the genes therein, notably that encodingthe hemoglobin Cgb, are known to be directly involved in NO detoxifica-tion. 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 havebeen unable to date to demonstrate any effect of NO or GSNO on DNAbinding by the purified NssR protein (Smith et al., 2011).

7.3. NorR

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

7.4. NsrR

NsrR is widely distributed in Gram-negative bacteria, but in thegammaproteobacteria, its function is assumed by NorR, which controlsexpression of the hmp gene inP. aeruginosa andVibrio cholerae (for a review,see Tucker et al., 2010). NsrR was first identified genetically in E. coli in 2005and is now considered a major regulator of the hmp, ygbA, nrfA, and ytfEgenes and others in E. coli (Mukhopadhyay et al., 2004; Rodionov et al.,2005; Bodenmiller and Spiro, 2006). InE. coli and Salmonella, NsrR repressesits regulated genes so that, in an nsrR mutant, exceptionally high levels ofHmp, for example, are made (Gilberthorpe et al., 2007), exceeding thoseobserved in a wild-type strain in the presence of 1 mMGSNO. Recent studieson threeNsrRproteins (fromStreptomyces coelicolor, B. subtilis, andNeisseriagonorrhoea), expressed in and purified from E. coli, show that NsrR is aniron–sulfur protein, although the cluster is not the same in each case (for a


review, see Tucker et al., 2010). The present picture is that NsrR proteins con-tain anO2-stable [2Fe–2S] cluster that is sensitive to NO. The discrepancies inthe literature raise the concern that, on expression in E. coli, inappropriateclusters, relative to the native bacterium, are incorporated. Alternatively,clusters in vivomay become degraded during aerobic purification. Whateverthe 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 isactivated by the response regulator component DevR of the two-componentregulatory mechanism called DevSRT (also known as DosSRT) (Dasguptaet al., 2000; Sardiwal et al., 2005). DevS andDosT each possess heme cofactorsto sense O2, being inactive when heme(II) binds the ligand. Anoxia, but alsothe presence of NO or CO, leads to activation. The stability of the Fe(II)–O2

complex is enhanced by interdomain interactions, making DevS an efficientgas sensor. The proteins autophosphorylate and then transfer a phosphategroup to the gene regulatorDevR.Recent studies (Yukl et al., 2011) show thatthe heme–O2 form of DevS reacts efficiently with NO to produce nitrate in areaction reminiscent of the dioxygenase activity of Hmp and some otherglobins. The resultant Fe(III) form of DevS is inactive but has a high affinityfor NO. The data suggest that, on exposure to NO, the inactive oxy–hemecomplex is rapidly converted to Fe(II)–NO, triggering the onset of the dor-mant phase and promoting mycobacterial survival.

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

Due to the complex nature of the interactions between microorganismsand various RNS, many studies now utilize a variety of “omic” techniquesavailable to more fully understand the targets of and responses to thesestresses. Global/systems approaches have the advantage of being able tohighlight predicted as well as unexpected and novel responses of themicrobe. These approaches are able to show us the interactions, robust-ness, and modularity of the complex microbial systems in place for thesensing and detoxification of RNS.


Both transcriptomic and proteomic techniques have been used to inves-tigate nitrosative stress in microorganisms. Computational modeling is alsoincreasingly utilized to interpret the vast amount of data generated by suchapproaches. However, care must be taken in the design of experiments sothat 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 conductedunder batch culture conditions. Here, an initial volume of media and cul-ture is added to a closed system and growth is monitored over time withno further addition or expulsion of media. In such a system, nutrients willbecome limited and metabolite levels will increase over time. In response,the cell population physiology will adapt to the changing conditions. Thesechanges are in addition to adaptations made in response to the addition ofa stressor such as NO.

However, for detailed transcriptomic and proteomic analysis, it is highlypreferable to keep all conditions constant so that gene/protein alterationscan be attributed solely to addition of the stress and not to other factorssuch as changes in growth rate or differences in nutrient levels. In continu-ous chemostat culture, fresh medium is pumped into the culture vessel andsurplus culture medium removed to maintain a constant volume. In thissystem, growth becomes limited by 1 or more nutrients in the medium.After this “steady state” is reached, growth rate is controlled by the addi-tion of fresh medium and hence fresh delivery of the limiting nutrient. Inthis way, growth rate and therefore population density can be controlled.This approach can be utilized with complex media; however, the use of adefined medium allows identification and tight control of a single limitingnutrient, frequently the carbon or nitrogen source. Defined media can alsoensure the bioavailability of all micronutrients, which could otherwise besequestered 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, workdetailed in this chapter, unless otherwise stated, will focus upon dataaccumulated using batch or continuous chemostat culture, which has beenutilized for investigation of a variety of nitrosative stresses.

An important criticism of continuous culture is the accumulation ofloss-of-function rpoS mutations, which can overtake the culture (Notley-


McRobb et al., 2002). However, further analysis found that this phenome-non does not occur in anaerobic cultures (King and Ferenci, 2005), nordoes 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 monitor-ing of cell cultures is required. This, and other evolutions, may affectthe usefulness of chemostat cultures in providing a physiologically stablepopulation for examination compared to batch culture. However, it isalmost certain that undesirable and uncontrolled changes in growth ratewill affect the outcome and interpretation of batch growths when growthinhibitory compounds are added. This is highlighted in the literature wherebatch culture experiments are more variable than those conducted undercontinuous culture conditions (Piper et al., 2002).

8.1.2. Transcriptomics

Microarray analysis is a valuable tool for investigating microbial targets ofand responses to nitrosative stress. This method of measuring globalchanges in gene expression in response to RNS has been utilized in a num-ber of studies with a variety of bacteria, including M. tuberculosis (Ohnoet 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 nitrosativestress have been most widely studied (Mukhopadhyay et al., 2004; Flatleyet 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 differencesbetween data sets utilizing differing culture conditions can be readilyidentified with very few transcriptional units being identified in common.In addition to culture conditions, it is also important to distinguish betweenthe different agents of RNS-mediated stress used as they cannot be usedinterchangeably (e.g., NOþ, NO, NO�), given that they have unique reac-tion chemistries (Hughes, 1999; Aga and Hughes, 2008) (Section 4.5) andelicit 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” studiesand the desire to obtain information on the regulatory networks that causechanges in gene transcription, new methods are required to assess and


accurately interpret data. Computational modeling allows a quantitativeestimation of the regulatory relationship between transcription factorsand genes (Sanguinetti et al., 2006).

Probabilistic state space modeling has been utilized in a number ofrecent microbial studies concerned with monitoring E. coli responses tovarious environmental changes (Partridge et al., 2007; Davidge et al.,2009b; Shepherd et al., 2010b). McLean et al. (2010c) used this modelingtechnique to highlight changes in the activity of four transcription factorsof E. coli due to ONOO� exposure (OxyR, ArgR, CysB, and PhoB) andwere also able to confirm a lack of activity of some transcription factorsthat the microarray data suggested may have been altered (e.g., FNRand IHF). A comparison of this microarray data was also made with dataobtained from exposure to H2O2 using mathematical modeling, whichallowed the similarities and differences between the two stresses to behighlighted at transcription factor level. However, as with any comparison,experimental design and continuity are of paramount importance in orderto ensure the models generated are meaningful.

8.1.4. Proteomics

The measurement of changing protein levels has classically been measuredusing techniques such as 2D gel electrophoresis or liquid chromatographyfollowed by mass spectrometry. For the former, protein samples areseparated by isoelectric point (pI) and/or molecular weight and detectedby staining. Protein levels are quantified according to staining intensityand subsequent identification performed by isolation of protein bands,sample digestion, and mass spectrometry. This technique has been widelyused for decades and is the basis for several proteomic analyses ofnitrosative stress in microbial cultures (Monk et al., 2008; Qu et al., 2009).

Early techniques focused upon the identification and characterization ofproteins; however, more recently, the focus has extended to the quantita-tive and comparative measurement of global changes of protein transcrip-tion via the use of chromatography, mass spectrometry, and bioinformatics.One technique used for investigation of the response of Desulfovibriovulgaris to nitrate stress was via a shotgun proteomic method utilizing iso-baric 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 peptidesusing mass spectrometry and allows the parallel proteome analysis of upto four samples via the labeling of primary amines with amine-specific


isobaric reagents (Thompson et al., 2003; Ross et al., 2004). Once tagged,the labeled peptides from different samples (e.g., pre- and poststress) aremixed, separated using 2D liquid chromatography and analyzed using massspectrometry and tandem mass spectrometry. The iTRAQ-tagged peptidesrelease reporter ions, the quantities of which are recorded and the peakarea used to quantitate the relative abundance of their originating proteins(Ross et al., 2004).

Approaches have also been developed to specifically identifyS-nitrosated targets across a given organism’s proteome following chal-lenge with RNS. The biotin switch method developed by Jaffrey et al.(2001) was pioneering in singling out SNO-bound proteins. It operates byblocking free cysteine thiols via S-methylthiolation followed by reductionof S-nitrosothiols with ascorbate. Thiols are subsequently labeled with asulfhydryl-specific biotinylating agent. Labeled proteins are isolated by astreptavidin pull-down assay and separated by SDS-PAGE. Bands of inter-est are cut from the gel and subjected to MALDI-TOF to uncover modifiedproteins. Jaffrey et al. (2001) used this technique to identify endogenouslyS-nitrosated proteins in a rat model by comparing data derived from awild-type mouse and an nNOS mutant. Findings revealed that targetsincluded metabolic, structural, and signaling proteins (Jaffrey et al., 2001).

Modifications to the Biotin Switch method have lead to the developmentof several novel techniques that couple labeling of NO-bound thiols to directpeptide 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 Switchmethod with the isotope-coded affinity tag (iCAT) approach. iCAT wasdeveloped to differentially label two biological samples with discrete isotopelabels that were subsequently subjected to liquid chromatography tandemmass spectrometry (LC-MS/MS) to identify and quantify differences in pro-tein expression levels. The tag comprises a thiol reactive group, a heavy orlight isotope linker and a biotin affinity tag. Labels were designed to reactwith free sulfhydryl groups that are ubiquitous in proteins, enabling widecoverage of the proteome (Gygi et al., 1999). SNOCAP, on the other hand,was developed to solely label thiol groups derived from the reduction ofS-nitrosothiols (Paige et al., 2008). In brief, free thiols are blocked andS-nitrosothiols are reduced with ascorbate to form thiols. Heavy and lightisotopically labeled thiol biotinylating agents are used to tag newly formedthiols from cell populations subjected to two different biological conditions.These samples are mixed and tryptically digested, and tagged proteins arepurified using neutravidin. The sample mixture is subsequently subjectedto LC-MS/MS, which allows for the identification of modified proteins


(without the need for gel electrophoretic separation) and enables the relativequantification of SNO sites between two different samples. Liquid chroma-tography is employed to reduce the complexity of the sample by separatingpeptides before MS analysis. The mass spectrophotometer is subsequentlyset up to measure the relative signal intensities of identical peptide pairs thatonly differ in mass by a fixed value dictated by the mass difference of theheavy and light isotope labels. Operating in MS/MS mode, the amino acidsequences of individual fragmented peptides contained in the digest mixturecan be determined. Databases are then queried to identify proteins fromwhich the sequenced peptides originated. Paige et al. (2008) successfullyemployed SNOCAP to uncover glutathione-reducible and nonreducibleproteins in cultured cells. This method was the first to enable the relativequantification of S-nitrosation on a proteome-wide scale between twodifferent 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 usingtranscriptomics. In particular, the response to NO, GSNO, and ONOO�

has been examined in the Poole laboratory utilizing the same continuousculture and defined minimal media conditions, which allows for a directcomparison of the effects of the three reactive nitrogen species.

Transcriptome profiling experiments were used to investigate the tran-scriptional basis of the response to the presence of GSNO in both aerobicand anaerobic cultures of E. coliMG1655 (Flatley et al., 2005). Aerobically,17 genes were upregulated, most notably those involved in the detoxificationof NO and methionine biosynthesis. Among the NO detoxification genesupregulated were hmp and norV, which encode an NO-consumingflavohemoglobin and flavorubredoxin, respectively (Sections 6.3 and 6.4).Additionally, the transcription of six genes involved in methionine biosyn-thesis or regulation was significantly elevated. Mutants of metN, metI,and metR exhibited growth sensitivity to GSNO, and exogenouslyprovided methionine was found to rescue this phenotype. This supports thehypothesis that GSNO nitrosates homocysteine, withdrawing it from themethionine biosynthesis pathway. Anaerobically, 10 genes were significantlyupregulated in response to GSNO, of which, norV, hcp, metB, metR, and


metF were also upregulated aerobically, suggesting that the response toGSNO is broadly similar under both conditions. These data revealed newgenes important for GSNO tolerance and demonstrated that methioninebiosynthesis is a casualty of nitrosative stress, an observation not seen inprevious studies utilizing batch culture conditions and complex media(Mukhopadhyay et al., 2004).

Further transcriptomic work aimed to compare and contrast the responsesof E. coli to the nitrosating agent GSNO with that of NO. Previous studiesinvestigated the effects of NO under both aerobic and anaerobic conditions,but these observations were made only in batch culture (Mukhopadhyayet al., 2004; Justino et al., 2005) and thus could not be legitimately comparedto data obtained in chemically defined medium under continuous cultureconditions. The resulting study (Pullan et al., 2007) made use of the samedefined minimal medium and identical continuous culture conditions as theGSNO 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-livesof 5 and 25 min, respectively, at pH 7.0 and 37 �C. The data revealed somesimilarities and several marked differences in the transcriptional responsesto these distinct nitrosative stresses. NO causes an upregulation of nitrosativestress 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 inactivationofFnrwas seen,with upregulation of Fnr-repressedgenes and downregulationof Fnr-activated genes being observed. Notably, expression of none of themetgenes was altered, suggesting that homocysteine nitrosation does not occurand so, in contrast to GSNO, methionine biosynthesis is not a target of NOstress under anoxic conditions.

Jarboe et al. (2008) extended these observations by applying networkcomponent analysis to transcriptomic data sets and showed that GSNOtargets homocysteine (Hcy) and cysteine with disruption of the methioninebiosynthesis pathway. Reaction of GSNO with Hcy and Cys resulted inaltered regulatory activity of MetJ, MetR, and CysB, activation of thestringent response, and growth inhibition. Supplementation with methio-nine 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 ear-lier reported to be an effective endogenous antagonist of GSNO-mediatedcytotoxicity (Degroote et al., 1996). Further distinction between the effectsof NO and GSNO is provided by the observation that the upregulation ofHmp via the transcriptional regulator NsrR may be demonstrated to arisefrom the submicromolar levels of NO released from GSNO, but GSNOinternalization is not required for this (Jarboe et al., 2008).


E. coli K-12 samples were also investigated for their anaerobic responseto 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 anaer-obic conditions as shown by Kim et al. (1999). In addition, Fur- and FNR-mediated repression was relieved and genes responsible for iron–sulfurcluster assembly/repair were upregulated (ytfE and the isc and sufoperons), 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 transcrip-tional responses to GSNO and acidified sodium nitrite (Mukhopadhyayet al., 2004) in richmedium and batch culture conditions.Again, upregulationof the nitrosative stress detoxification genes norVW and hmpA was seen inresponse to both acidified nitrite and GSNO. This study suggested that,under these conditions, the sensing of NOwas alsomediated bymodificationof the transcription factor, Fur implicating iron limitation as a consequenceof nitrosative stress. However, involvement of the Fur regulator was not seenin later work by Flatley et al. (2005); this could be due to the choice of mediaused in each case as the bioavailability of iron in the Luria broth used byMukhopadhyay and coworkers was likely low, due to a lack of chelators inthe medium (Hughes and Poole, 1991). There was also no evidence forupregulation of the methionine biosynthesis pathway in the data derivedfrom rich medium, reflecting the potential discrepancies between data setscollected under differing experimental conditions.

Uropathogenic Escherichia coli (UPEC), responsible for many urinarytract infections in humans, encounter multiple stresses during their transitthrough the body including RNS. Recently, data have been presented toshow that UPECs preconditioned with acidified sodium nitrite were betterable to colonize the bladders of mice than nonconditioned bacteria (Boweret al., 2009). Microarray analysis of the UPEC response to acidified sodiumnitrite suggested that upregulation of NsrR-regulated genes, multiple genesinvolved in the transport and metabolism of polyamines, and other stressresponsive factors may be responsible for the competitive advantage. Thisdata suggest that the route of infection and hence the stresses encounteredby the bacterium (e.g., RNS) have a major impact upon host colonizationand 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 continuousculture conditions in order to assess the effects of this highly reactive species


and to allow direct comparison to the GSNO and NO data sets. This studyutilized a bolus addition of commercial ONOO� to the vessel and feed line.The resulting microarray data revealed that, in contrast to GSNO and NO, anumber of oxidative stress response genes were upregulated, most notablykatG and ahpCF. KatG has been proposed to act as a peroxynitritase inM. tuberculosis (Wengenack et al., 1999) as have KatG and AhpCF inS. enterica (Bryk et al., 2000) (McLean et al., 2010a). Interestingly, theexpression of none of the recognized nitrosative stress response genes wasaltered after ONOO� exposure, indicating that this reactive species doesnot 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 clas-sical nitrosative stress response genes is not surprising as proteins such asHmp and NorVW specifically detoxify NO, levels of which would not beexpected to increase during ONOO� exposure. Further, an increase inHmp expression during ONOO� stress has deleterious effects on S. enterica(McLean et al., 2010b), causing hypersensitivity to the stress. This is probablydue 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 reg-ulation of other nitrosative stress response genes including those responsiblefor nitrite detoxification (nrfA and hcp) was unaltered byONOO� exposure,suggesting that, while undoubtedly levels of nitrite will increase upon expo-sure to ONOO�, levels were either insufficient to upregulate these genesor detoxification of ONOO� is more significant than removal of the compar-atively inert nitrite. Some genes that were upregulated are of unknown func-tion and could play a role in response to the apparent S-nitrosylation of thiolsor in response to the nitration of tyrosine residues in proteins.

Other targets of ONOO� suggested by interpretation of the microarraydata included cysteine (cys) and arginine (arg) biosynthesis as well asgenes involved in iron–sulfur cluster assembly/repair (suf and isc genes),the high-affinity phosphate transport system (pst), and the (gsi) glutathioneimport system. The transcription levels of several genes encodingmembrane and transport proteins were also altered both positively andnegatively, which suggests that ONOO� reacts with proteins in themembrane 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 thanone of the above stresses and none whose transcript levels are altered byall three (Fig. 8.). Upregulated in response to GSNO and NO are genes


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 assemblyand repair, indicating a common target of these two species. The only genesignificantly altered by both GSNO and ONOO� codes for a poorlycharacterized protein, YeaJ. This protein is suggested to be a putativediguanylate cyclase due to the presence of a characteristic GGDEF motifand is responsible for synthesis of the second messenger, cyclic di-GMP.Many proteins containing the GGDEF domain also contain other domainsthat can receive signals. YeaJ contains an upstream motif suggested to bean S-nitrosation site (TDCD) (Stamler et al., 1997), which could providea signal for some of the cellular responses to RNS.

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


8.3. Responses of Other Microbes to RNS

8.3.1. C. jejuni

C. jejuni, a predominant causative agent of bacterial gastrointestinal dis-ease worldwide (Friedman et al., 2000), was found to significantlyupregulate eight genes in response to GSNO stress (Elvers et al., 2005)including cgb, which codes for a single-domain globin known to protectthe bacteria from nitrosative stress; ctb (Cj0465c), a truncated globin; andsix genes of unknown function. Further work confirmed the rapid responseof cgb upregulation as a protective measure against GSNO (Monk et al.,2008). In this study, other genes showing enhanced transcription levelswere the truncated globin ctb and a variety of heat-shock response, irontransport, and oxidative stress response genes including trxA, trxB, ahpC,and two putative oxidoreductases. Although the function of the hemoglo-bin Cgb is well established in NO detoxification (see Section 6.3.2), thefunction of the truncated globin Ctb is less clear. When ctb is mutated,no major compensatory transcriptomic adaptations are evident (Smithet al., 2011). One hypothesis is that, by binding NO or O2 avidly, Ctbdampens the response to NO under hypoxic conditions, perhaps becauseCgb 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 (Adaket al., 2002a) and also coexists with denitrifying bacteria, so it is likelyto have developed mechanisms of detoxification of both exogenouslyand endogenously formed RNS. Microarray analysis of B. subtilis expo-sure to NO-saturated solutions under aerobic conditions revealed thatthe 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 werethose of hmp and members of the Fur and PerR regulons with sB genesbeing only slightly upregulated. B. subtilis cultures exposed to SNPshowed induction of hmp as well as the sB, ResDE (Ye et al., 2000)and Rex (Larsson et al., 2005) regulons (Rogstam et al., 2007). TheResDE and Rex regulons are upregulated by changes in redox status orlowered oxygen availability.


8.3.3. M. tuberculosis

The response of M. tuberculosis to RNS has also been investigated (Ohnoet al., 2003). Microarray analysis using two separate NO donors, NOR-3and Spermine NONOate, identified the upregulation of 36 genes includingthose playing roles in small molecule metabolism (including a nitratereductase, narX, and ferredoxin, fdxA), macromolecule metabolism (sigE),and cell processes including a putative nitrite extrusion protein narK2 andgenes coding for probable transmembrane proteins. Genes downregulatedin the same study included those encoding for putative transcriptionalregulators and cell envelope/energy metabolism proteins, suggesting ametabolic downshift in response to the NO donors.

The transcriptomic response ofM. tuberculosis to macrophage attack hasfound evidence of a response to nitrosative stress (Schnappinger et al., 2003;Cappelli et al., 2006), including upregulation of RNS detoxification genesincluding alkyl hydroperoxidase (ahpC), which has peroxynitritase activity(Bryk et al., 2000), and nitrate reductase (narX), as well as other genespreviously hypothesized to alter their transcriptional activity in responseto 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 detox-ification of oxides of nitrogen. The flavohemoglobin, fhp, is most highlyupregulated; other genes coding for Nor, MoaB1, and NarK1 are alsoupregulated.

8.3.5. S. aureus

When exposed to SNAP, S. aureus genes involved in iron homeostasis,hypoxic/fermentative metabolism, the flavohemoglobin hmp and the2-component system srrAB were upregulated. SrrAB has been shown toregulate the expression of many NO-induced metabolic genes (Throupet al., 2001). When exposed to nitrite, biofilm formation is inhibited inS. aureus and a clear response to both oxidative and nitrosative stresscan be seen via increases in genes involved in DNA repair, detoxificationof ROS and RNS, and iron homeostasis (Schlag et al., 2007).


8.3.6. N. meningitidis

Activity of the NO-sensitive repressor NsrR from N. meningitidis has beenassessed by microarrays in response to Spermine NONOate (Heurlieret al., 2008). Target genes under the control of NsrR included norB (NOreductase), dnrN (repair of nitrosative damage to iron–sulfur clusters),aniA (nitrite reductase), nirV (nitrite reductase assembly protein), andmobA (possible molybdenum metabolism) and were all upregulated inresponse 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 nitrosativestress (Sebbane et al., 2006). Most striking was the 10- to 20-fold increase inexpression of hmp. Upregulation of metLRBF, nrdHIEF, ytfE, hcp, hcr,and tehB was also observed. The latter four genes and hmp are repressedin E. coli by the transcription factor NsrR (Bodenmiller and Spiro, 2006),the homologue of which (YPO0379) was downregulated 1.7- to 4-fold inthe bubo. Upregulation of the methionine biosynthesis pathway may alsoindicate a response to S-nitrosylation in buboes as identified in the transcrip-tional analysis to GSNO in E. coli (Flatley et al., 2005).

8.4. Proteomics

One study utilizing C. jejuni sought to identify both transcriptomic andproteomic alterations in response to GSNO (Monk et al., 2008). Using2D gel proteomic analysis (Holmes et al., 2005), the authors found thatlevels of the two globins, Ctb and Cgb, were enhanced at both the trans-criptomic and proteomic level as was the heat-shock protein DnaK. Someproteins 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 dif-fering timescales used for each approach; for microarray analysis, cellswere incubated for 10 min, whereas proteomic analysis utilized samplesthat had been incubated three times longer. These discrepancies couldreflect responses on differing timescales, a lack of translation or rapid pro-tein degradation.


A proteomic analysis of the response of H. pylori to the NOþ releasingmolecule SNP using 2D proteomics revealed 38 proteins with altered expres-sion levels (Qu et al., 2009). Among these were proteins involved inprocessing, antioxidation (including TrxR), general stress response, andvirulence.

The study of nitrosative stresses in microbial species using modernproteomic techniques is still in its infancy. However, quantitativeproteomic analysis has been undertaken to investigate nitrate stress inthe anaerobic sulfate-reducing bacterium D. vulgaris Hildenborough usingiTRAQ (Redding et al., 2006). Changes in the protein profile of this organ-ism were analyzed upon addition of sodium nitrate using iTRAQ labelingand tandem liquid chromatography separation coupled with mass spec-trometry detection. The authors found that the use of 103 mM sodiumnitrate produced only a mild effect upon the proteome, with proteinsinvolved in central metabolism and the sulfate reduction pathway beingunperturbed. Unsurprisingly, proteins involved in nitrate stress detoxifica-tion were increased as well as those for transport of proline, glycine-betaine,and glutamate, suggesting that nitrate stress also induced salt stress. Inaddition, levels were increased for several oxidative stress response, ABStransport system, and iron–sulfur cluster containing proteins.

As discussed previously (Section 8.1.4), not only have proteomic met-hods been applied to monitor changes in protein expression levels acrossthe proteome in response to stress, they have also been used to unravelthe S-nitrosoproteome of organisms following challenge with RNS.Recently, a novel fluorescence-based approach has been developed andtested on E. coli cell lysates incubated with GSNO to identify S-nitrosatedproteins across the proteome. Twenty modified proteins were uncovered,with functions in protein synthesis and folding, global regulation, quorumsensing, 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 29modified proteins, a large proportion of which were found to be involvedin intermediary and lipid metabolism. Proteins involved in the defenseagainst oxidative and nitrosative stresses were also nitrosated (Rheeet al., 2005). In 2007, Brandes and coworkers applied a method to identifyall reversibly modified thiols mediated by the NO donor, DEA/NO,including S-nitrosothiols, disulfide bonds, and sulfenic acids. Investigatorsuncovered 10 altered proteins, six of which were encoded by essentialgenes (Brandes et al., 2007). More recently, researchers have uncoveredfive S-nitrosated proteins in H. pylori following incubation of cell lysateswith GSNO, namely, GroEL, a chaperone and heat-shock protein; UreA,


urease alpha subunit; TsaA, alkylhydroperoxide reductase; and two codingsequences of unknown function (Qu et al., 2011).

Other techniques, such as isotope-coded affinity tags (iCATs) andS-nitrosothiol capture (SNOCAP), are currently being developed for usein studying the interaction between RNS and microbes but so far thereappears 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 elaborateand effective defense measures mounted. Certain recurrent themes areevident. Microbes (in most cases, the information relates to bacteria) areable to resist NO in their environments by a relatively small number ofdetoxification mechanisms, the best understood being globins that catalyzeNO conversion to nitrate and reductases that produce nitroxyl anion, andultimately, nitrous oxide. The species sensed that induces the expressionof these enzymes is probably the same, that is, NO, but the possibilityexists that activation of the defense response may be achieved by proteinnitrosation, for example, rather than sensing of NO by a metal center. Amajor weakness in our understanding is how bacteria sense and detoxifyother RNS, such as those commonly used in experimental studies, notably,S-nitrosothiols (especially GSNO), SNP, and acidified nitrite. In the case ofGSNO, SNO reductases are known to denitrosylate affected proteins, butthere do not appear to be any detoxification mechanisms recognized sofar that detoxify the products of SNP and acidified nitrite, but only theresultant NO. Several approaches may assist in tackling this problem. First,investigators should exercise great care when designing experiments toensure that the nitrosative stress applied is well characterized. SNP, forexample, 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 mayeven release other biologically active species (cyanide, in this case).In the case of peroxynitrite, it appears that this species should probablynot be regarded as an agent of NO-related stress, but as a species capableof nitration, nitrosylation, and oxidative stress. Second, sound principles ofmicrobial physiology should be applied in experimental design, particularlyso that culture conditions are reproducible and consistent with the use ofthe RNS species employed. Finally, much may be learned by adopting asystems or modeling approach to unraveling the complexities of the


microbial response. Since the outcomes of NO or RNS exposure are sopervasive, affecting directly and indirectly a host of cellular processes, suchapproaches have great potential but are in their infancy.

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