Transcriptional regulation of bacterial virulence gene expression by molecular oxygen and nitric oxide

Molecular oxygen (O2) and nitric oxide (NO) are diatomic gases that play major roles in infection. The host innate immune system generates reactive oxygen species and NO as bacteriocidal agents and both require O2 for their production. Furthermore, the ability to adapt to changes in O2 availability is crucial for many bacterial pathogens, as many niches within a host are hypoxic. Pathogenic bacteria have evolved transcriptional regulatory systems that perceive these gases and respond by reprogramming gene expression. Direct sensors possess iron-containing co-factors (iron–sulfur clusters, mononuclear iron, heme) or reactive cysteine thiols that react with O2 and/or NO. Indirect sensors perceive the physiological effects of O2 starvation. Thus, O2 and NO act as environmental cues that trigger the coordinated expression of virulence genes and metabolic adaptations necessary for survival within a host. Here, the mechanisms of signal perception by key O2- and NO-responsive bacterial transcription factors and the effects on virulence gene expression are reviewed, followed by consideration of these aspects of gene regulation in two major pathogens, Staphylococcus aureus and Mycobacterium tuberculosis.


Introduction
Molecular oxygen (O 2 ) and nitric oxide (NO) are freely diffusible diatomic gases because they are soluble in aqueous media but can partition into and cross biological membranes. Both gases have complex chemistries and play major roles in the host response to infection (Fig. 1). The NADPH oxidase (NOX) of professional phagocytes (e.g., macrophages and neutrophils) generates an "oxidative burst" by catalyzing the one electron reduction of O 2 to superoxide (O 2 ¡ ). A further one electron reduction of O 2 ¡ yields hydrogen peroxide (H 2 O 2 ), one of the products resulting from the action of superoxide dismutase (2O 2 ¡ C 2H C ! H 2 O 2 C O 2 ). In the presence of ferrous ions (Fe 2C ) H 2 O 2 undergoes Fenton chemistry to produce the hydroxyl radical (OH ). 1,2 Superoxide, H 2 O 2 , and OH are collectively termed reactive oxygen species (ROS) and are capable of damaging many cell components, including DNA, proteins and membranes, resulting in bacterial death or bacteriostasis. 1,2 The inducible nitric oxide synthase (iNOS), found in professional phagocytes, catalyzes the formation of NO from L-arginine and O 2 . 3 Nitric oxide is a reactive lipophilic radical, which reacts with metalloproteins and protein thiols. Furthermore like O 2 ¡ , NO production leads to the formation of other toxic molecules collectively termed reactive nitrogen species (RNS). The most important RNS are nitroxyl (NO ¡ ), nitrosonium (NO C ) and peroxynitrite (ONOO ¡ ); the last being formed as a result of the reaction of NO with O 2 ¡ , or from NO ¡ and O 2 . 4 These RNS modify metal cofactors, protein cysteine, methionine, and tyrosine residues, with consequent bacteriostatic and bacteriocidal effects. Not surprisingly, bacterial pathogens have evolved mechanisms to sense O 2 and NO and respond by deploying defensive mechanisms that detoxify ROS and RNS and repair oxidative and nitrosative damage to cell components. This review summarizes our current understanding of the mechanisms of O 2 and NO perception by transcription factors and by examination of selected examples illustrates how bacterial cells use this information to control virulence gene expression and host-pathogen interactions.
Yersina pestis) are facultative anaerobes capable of aerobic respiration, anaerobic respiration and fermentation. Thus, the ability to sense and respond to changes in O 2 availability is essential for the competiveness of these bacteria. Both direct and indirect O 2sensing regulatory systems have been characterized in these bacteria, with the Fumarate Nitrate Reduction regulator (FNR) protein of the model bacterium E. coli K-12 being the paradigm of a direct O 2 -responsive transcription factor (Fig. 2). [5][6][7] FNR is a member of the cyclic-AMP receptor protein family of transcription regulators. Under anaerobic conditions, FNR is activated by incorporation of an iron-sulfur cluster ([4Fe-4S]) coordinated by four essential cysteine residues (Cys-20, 23, 29, and 122), located within the N-terminal sensory domain of the protein. 8,9 Iron-sulfur clusters are widespread, redox-active, biological structures composed of iron and sulfide that are most commonly held in proteins by four cysteine residue thiolates that act as coordinating ligands. [5][6][7] The [4Fe-4S] 2C cluster acquired by FNR is one of the most common forms of iron-sulfur cluster. The [4Fe-4S] 2C cluster is a cube made up of two interpenetrating tetrahedra of iron (two Fe 3C and two Fe 2C ) and sulfide ions held by the essential cysteine residues of FNR interacting with the iron atoms at the vertices of the cube. The second common form of iron-sulfur cluster is the planar [2Fe-2S] 2C cluster, consisting of a [Fe 2 -(m 2 -S) 2 ] rhomb (rhombus) of two Fe 3C and two sulfide ions (both sulfide ions bridge two iron atoms hence the m 2 -S designation), again most often coordinated by four cysteine residues. The [4Fe-4S] 2C and [2Fe-2S] 2C clusters can be inter-converted, sometimes via a [3Fe-4S] 1C intermediate. Inter-conversion of the cubic [4Fe-4S] and planar [2Fe-2S] clusters drives protein conformational changes that are mediated by the need to re-orientate the ligating cysteine residues to accommodate the change in the geometry of the iron-sulfur cluster (Fig. 2). Although both [4Fe-4S] and [2Fe-2S] clusters are found in several of the regulatory proteins discussed in this review, stable [3Fe-4S] clusters have thus far not been associated with regulatory activity, but such clusters are often involved in electron-transfer proteins (as are [4Fe-4S] and [2Fe-2S] clusters).
The acquisition of a [4Fe-4S] cluster by FNR results in conformational changes that reduce inter-subunit electrostatic repulsion, permitting homodimer formation, thereby enabling the C-terminal DNA-binding domain to recognize specific binding sites within target promoters. [10][11][12][13] In E. coli K-12, FNR binds to 207 sites across the chromosome, most of which are associated with genes involved in anaerobic metabolism. 13 In the presence of O 2 , the FNR [4Fe-4S] 2C cluster is converted into a [2Fe-2S] 2C form. 14,15 This conversion results in FNR dimer dissociation, such that FNR neither binds DNA nor regulates gene expression. [16][17][18][19] The [2Fe-2S] 2C cluster of FNR slowly degrades to form cluster-free (apo-) protein in the presence of O 2 in vitro and in vivo. [20][21][22] The apoprotein formed by cluster disassembly is capable of incorporating a new iron-sulfur cluster. 23  , hydrogen peroxide (H 2 O 2 ) and the hydroxyl radical ( OH). Nitric oxide (NO) is generated by the action of host inducible nitric oxide synthase (iNOS) (and by some bacteria that possess nitric oxide synthase). Nitric oxide is a reactive free radical and is a source of reactive nitrogen species such as nitroxyl (NO ¡ ), nitrosonium (NO C ), and peroxynitrite (ONOO ¡ ), which is formed by reaction of NO with O 2 ¡ , or NO ¡ and O 2 , and peroxynitrous acid (OONOH).
Nitric oxide reacts with thiol groups to modify activity by the formation of S-nitrosylated proteins (RSNO  20,24,27,28 FNR is likely to be important for virulence of pathogens that encounter changes in O 2 availability. In these cases, the absence of O 2 sensed by FNR is thought to act as an environmental cue to reprogram metabolism, by activating genes required for anaerobic respiration (e.g., those encoding nitrate and nitrite reductases), fermentation (e.g., pyruvate formatelyase, alcohol dehydrogenase) and trigger virulence gene expression during host colonization and infection. Accordingly, Bordetella pertussis, Neisseria meningitidis, Pseudomonas aeruginosa, and Salmonella enterica serovar Typhimurium (S. Typhimurium) FNR proteins were required for optimal growth and survival in vivo. [29][30][31][32] Moreover, a proteomic analysis of Shigella dysenteriae type 1 supported the importance of a switch from aerobic respiration in vitro to anaerobic catabolism in vivo. 33 As well as controlling the ability of many bacterial pathogens to adapt their metabolism to the hypoxic and anoxic niches within a host, FNR also contributes to regulating toxin production and effector protein secretion. Several strains of E. coli, Salmonella, and Shigella possess a cytolysin known as HlyE or ClyA. [34][35][36][37][38][39] In E. coli, hlyE transcription is activated from a complex FNR-dependent class II promoter and HlyE activity is detected under anaerobic growth conditions. [40][41][42][43][44][45] For these enteric bacteria, oxygen starvation could signal entry into a host and prompt expression of the HlyE cytolysin. In Salmonella Typhi, the causative agent of typhoid fever, hlyE mutants exhibited impaired invasion of human epithelial (HEp-2) cells and heterologous hlyE expression in Salmonella Typhimurium enhanced colonization of the spleen and liver in a mouse model of infection. 46 The Bacillus cereus, non-hemolytic enterotoxin (Nhe) is a member of the HlyE family of pore-forming toxins and expression of nhe is under the control of the B. cereus FNR; however this control appears to be unresponsive to O 2 -availability. 47,48 Although the B. cereus FNR has an O 2 -responsive [4Fe-4S] cluster, the cluster does not appear to be important for DNA-binding at the nhe promoter (there is evidence for monomeric apo-FNR binding) or for interaction with the redoxresponsive regulator ResD (see below). [48][49][50] Oxygen-sensing by the Shigella FNR protein has been shown to play a role in coordinating the function of a Type III secretion system (T3SS) that is important for virulence. In the anaerobic lumen of the gastrointestinal tract, FNR primes the bacterium for invasion by activating expression of the T3SS needles, while repressing the expression of spa32 and spa33, which regulate the function of the T3SS. 51 Thus, the T3SS is built and ready to function as soon as spa32 and spa33 expression is triggered. As the Shigella approach the gut mucosa, they experience an increase in O 2 availability, arising from the proximity to the capillary networks located in the villi. These micro-aerobic conditions result in FNR inactivation, by the mechanism discussed above, and the consequent de-repression of spa32 and spa33 allows invasion plasmid antigen secretion via the now functional T3SS precisely at its site of action. 51 In P. aeruginosa the FNR protein (known as ANR) is a component of a regulatory network involving NarL and RsmAYZ that regulates the T3SS in response to host cells, low calcium and low O 2 (Fig. 3). 52 Moreover, the activity of ANR was stimulated under aerobic conditions by catabolism of choline and glycine-betaine that was generated from the breakdown of host membrane/lung surfactant phosphatidylcholine by hemolytic phospholipase C (PlcH), illustrating the complex relationships between O 2 availability, FNR activity, metabolism, and virulence gene expression. 32

ArcBA, a Two-Component System That Senses O 2 Indirectly
The ArcBA (Aerobic respiratory control) two-component system is an indirect sensor of O 2 availability. ArcBA generally acts as a global regulator; it has been shown to control the expression of >175 genes in E. coli K-12, 392 genes in S. Typhimurium, 58 genes in the pig pathogen Actinobacillus pleuropneumoniae, but only 24 genes in Haemophilis influenzae. In all cases the core of the ArcBA regulon consists of genes associated with central metabolic and respiratory functions, such as those encoding enzymes of the Krebs cycle (e.g., acnA, gltA, icd, fumA, mdh, and sdhCDAB-sucA-D in E. coli) and the aerobic electron transport chain (e.g., appCB, cydAB, cyoA-E, and nuoA-N in E. coli) and thus, as with FNR, dysregulation of these key aspects of bacterial physiology is likely to lead to attenuation in the infective capacity of a pathogen. The absence of O 2 results in reduction of components of the aerobic electron transport chain, including the quinone pool. The membranebound sensor, ArcB, responds to the redox state of the quinone pool via the oxidation state of two cysteine residues (in E. coli K-12, Cys-180, and Cys-241) located in a cytoplasmic PAS domain, such that in the absence of O 2 the ArcB dimer undergoes autophosphorylation. [53][54][55] Phosphoryl transfer from ArcB to the cytoplasmic regulator ArcA promotes ArcA oligomerization and DNAbinding to activate or repress the expression of target genes. In the presence of O 2 , the ArcB dimer acquires two intersubunit disulfide bonds via interaction with the quinone pool, thereby inhibiting kinase activity and promoting ArcA dephosphorylation. As noted above, in most cases, ArcBA has been shown to be a global regulator of functions associated with central metabolism and fermentation, and thus dysregulation of these key physiological activities must contribute to the observed attenuation of arcBA mutants of Klebsiella pneumoniae in the colonization of gastrointestinal tract, and of Shigella flexneri plaque formation. 56,57 ArcBA controls resistance to ROS and RNS in the highly virulent S. enterica serovar Enteritidis SE2472 strain, but the arcBA mutant was not attenuated in a mouse model of infection. 58 However, conjugal transfer of the Salmonella virulence plasmid pSLT occurs at high frequency in the gastrointestinal tract and is dependent on ArcBA. 59 In addition, ArcA has been shown to be a significant player in the regulation of: genes that are important for complement evasion in Haemophilus influenzae; the production of cholera toxin in Vibrio cholerae via regulation of toxT; and colonization of the porcine respiratory tract in A. pleuropneumoniae. [60][61][62][63]

NO Resistance-Professional NO Sensors
Given the prominent role played by NO and RNS in the innate immune response to bacterial infection it is not surprising that pathogenic bacteria have evolved elaborate mechanisms to sense NO and respond to its presence through systems that detoxify NO and repair the damage caused by RNS. Although NO is an inhibitor of many heme enzymes that bind O 2 , some terminal oxidases are capable of contributing to NO detoxification by reduction of NO (Fig. 1). Moreover, some nitrite reductases, such as NrfA, can also reduce (detoxify) NO (see below; Fig. 1). Recently, a metabolomic screen to identify the effects of NO on the metabolism of V. cholerae revealed that NnrS is an NO-induced protein, which protects iron-sulfur proteins and the cellular iron-pool by lowering the production of dinitrosyliron complexes particularly under anaerobic conditions. 64 However, the best characterized NO detoxification systems are the enzymes flavohemoglobin (Hmp) and flavorubredoxin (NorV). 4 Hmp is primarily an NO dioxygenase, converting NO to NO 3 ¡ , although it has limited anoxic NO denitrosylase activity producing NO -(nitroxyl), which leads to the formation of N 2 O. 65 Disruption of the hmp gene in S. Typhimurium severely impaired survival in macrophages. Uropathogenic E. coli hmp mutants were attenuated in a mouse urinary tract infection model, but a P. aeruginosa hmp mutant was not attenuated in a silk worm model. [66][67][68][69] Hmp has an "on board" reductase system to supply electrons to the heme at the active site, but other bacterial globins that have been implicated in NO detoxification, such as those in Campylobacter (the Cgb globin) and Mycobacterium (the HbN globin) species, appear to lack a dedicated partner reductase, suggesting that turnover of NO by these proteins might be low, or that they are promiscuous, exploiting several cellular sources of reducing power. 70,71 Nevertheless, the single domain hemoglobin (Cgb) of Campylobacter jejuni imparts NO resistance and expression of the cgb gene was induced upon exposure of the bacteria to RNS. 72,73 A prominent anaerobic/hypoxic NO detoxification system in E. coli K-12 is NorV (along with its dedicated reductase NorW), which catalyzes the reduction of NO to NO ¡ (and ultimately N 2 O). 74 Inactivation of norV (by truncation of the gene, which occurs in some natural isolates) of enterohemorrhagic E. coli O157 revealed an important role for the intact norV gene in macrophage survival and was thus considered to be a direct virulence determinant. 75 Recently, a new class of NO reductase (represented by the Hp0013 protein) has been recognized in Helicobacter pylori. 76 The H. pylori hp0013 mutant is more sensitive to NO and is defective in colonization of the stomachs of mice. 76 Bacteria that are capable of denitrification, (i.e., the stepwise reduction of nitrate to nitrogen gas via nitrite, NO and nitrous oxide; Fig. 1), possess NO reductase enzymes that catalyze the formation of N 2 O from NO. 76,77 Abolishing this activity impairs the virulence of P. aeruginosa. 69 A further route to NO detoxification under anoxic conditions is via the action of cytochrome c nitrite reductase (NrfA), which although it has a high K m for NO has a high turnover rate and alongside NorV accounts for most of the anaerobic NO reductase activity in S. Typhimurium. 78,79 As well as detoxification of NO, bacteria also respond by inducing mechanisms to repair damaged cell components. Although little information is available on these processes, it appears that the YtfE protein contributes to the repair of nitrosylated iron-sulfur clusters and Ogt has a role in DNA repair in E. coli. It has been suggested that the NO-regulated hcphcr, yeaR, and yoaG gene products have as yet uncharacterized roles in repairing NO damage. 80 The bacterial responses to NO discussed above are mostly regulated at the level of transcription by NO-responsive transcription factors, some of which are considered below.

NsrR
NsrR is a member of the Rrf2 family of transcription factors and is found in most b and g proteobacteria, notable exceptions in the current context being the Pasteurellaceae, Pseudomonadales, and V. cholerae. 81,82 The E. coli NsrR protein controls the expression of >60 genes, including hmp. 83 The NsrR regulon of S. Typhimurium overlapped that of E. coli and several of the gene products were shown to be important for growth during nitrosative stress (i.e., the stress/ damage imposed on a biological system by exposure to NO and its congeners derived from the initial reaction of NO with superoxide). 84 NsrR from Neisseria gonorrhoeae, the causative agent of the sexually transmitted disease gonorrhea, is a [2Fe-2S] protein with three conserved C-terminal Cys residues that act as cluster ligands; the identity of the fourth coordinating residue is unknown although a conserved His residue has been suggested to fulfill this role. 85 DNA-binding by N. gonorrhoeae NsrR was abolished by exposure to NO, presumably due to nitrosylation of the iron-sulfur cluster. 85 The Streptomyces coelicolor NsrR protein possesses an O 2 -stable [2Fe-2S] cluster that reacts with NO to yield a dinitrosyliron complex and this form of the protein could not bind to target DNA. 86 Thus, the S. coelicolor and N. gonorrhoeae NsrR proteins have similar properties. However, although the DNA-binding activity of the NsrR protein of the non-pathogen B. subtilis was sensitive to NO, this protein apparently possesses a [4Fe-4S] cluster. 87,88 Hence, there is some uncertainty about the nature of the NsrR iron-sulfur cluster and therefore the mechanism by which NO modulates the transcriptional activity of NsrR.

NorR
NorR is a s 54 -dependent transcriptional regulator with an N-terminal GAF domain, an AAA C ATPase domain and a C-terminal helix-turn-helix DNA-binding domain (Fig. 4). GAF is a common small-molecule binding domain that is distantly related to another ligand binding domain PAS (see ArcA above). In NorR the GAF domain houses a non-heme iron center that reversibly binds NO. 89,90 In the absence of NO the GAF domain sequesters the AAA C domain, inhibiting ATPase activity and productive interaction with s 54 -RNA polymerase. The nonheme iron is thought to be hexa-coordinate and ligated by 5 amino acids (Arg-75, Asp-96, Asp-99, Cys-113, and Asp-131). Reaction with NO results in the formation of a mononitrosyl iron complex and the concomitant liberation of the AAA C domain allowing the AAA C domain to make productive interactions with the s 54 subunit of RNA polymerase and activate transcription of norVW, encoding the NorVW NO reductase. 91 The norVW promoter has three tandem enhancer sites that are essential for NorR ATPase activity. 92 In E. coli the norVW operon is the only known target for NorR, but in P. aeruginosa and V. cholerae, which lack NsrR, NorR activates hmp expression, and in a mouse prolonged colonization model a V. cholerae norR mutant was attenuated. 93

NssR
The major cause of gastroenteritis in developed countries is chicken contaminated with Campylobacter species. After ingestion, the bacteria are exposed to NO and other RNS generated by the host immune system and from acidification of the nitrite in saliva and the nitrite generated by the reduction of dietary nitrate. In C. jejuni the cyclic-AMP receptor protein (CRP) family regulator NssR controls the expression of a small regulon, including those encoding two globins Cgb and Ctb (see above). 72 NssR acts as a positive regulator of both cgb and ctb under nitrosative stress conditions, but high-affinity DNA-binding by NssR was unaffected by NO, suggesting that NssRmediated activation of cgb and ctb occurs downstream of DNA-binding. 94 The mechanism by which NssR senses the presence of NO is unknown, although it has been noted that the protein has a single cysteine that could be a target for nitrosylation or one or more tyrosine residues might be nitrated by peroxynitrite. 94

FNR
The E. coli FNR [4Fe-4S] cluster reacts not only with O 2 (see above) but also with NO (Fig. 2). [95][96][97] Reaction with NO is extremely rapid, multiphasic and results in the formation of a proteinbound nitrosylated ironsulfur cluster that resembles a pair of Roussin's red esters. 97 Reaction with NO inhibits FNR DNA-binding activity in vitro and FNR-dependent transcription in vivo. 95,97 Thus, in addition to its well-established role as an O 2 -responsive regulator of anaerobic functions, the inactivation of FNR by NO was suggested to be a final safeguard against NO toxicity by switching off transcription of genes involved in nitrate and nitrite respiration, thereby minimizing endogenous NO production when the dedicated NO-responsive regulators and detoxification systems are overwhelmed. 97

SoxR
The SoxRS system of enteric bacteria consists of two DNAbinding proteins, which act sequentially to regulate the transcription of >100 genes in response to redox stress caused by exposure to superoxide and/or bacteria-and plant-derived redox-cycling www.landesbioscience.com molecules, such as pyocyanin and plumbagin. [98][99][100][101] This regulon includes genes encoding proteins involved in detoxification of ROS (superoxide dismutase), repair of ROS-mediated damage (endonuclease IV), and replacement of ROS-sensitive components by resistant ones (fumarase C). Thus, the SoxRS regulon contributes to resisting the toxic effects of the macrophage oxidative burst. Although SoxR is widely distributed, SoxS is absent in non-enteric bacteria, and in these cases SoxR is responsible for regulating all members of the regulon. 102 The SoxRS and SoxR systems have been associated with fluoroquinolone resistance in Salmonella serovars, the ability of P. aeruginosa to survive in macrophages, cause systemic infections following burn wounds and cause pulmonary infections, and virulence of Vibrio vulnificus and Xanthomonas campestris. [103][104][105][106][107] SoxR is a homodimeric, MerR family protein. 107 Each monomer has a cluster of four cysteine residues (Cys-X 2 -Cys-X-Cys-X 5 -Cys) that binds a solvent-exposed [2Fe-2S] 1C center in an asymmetric electrostatic environment. 108 All forms of SoxR bind to target DNA, but it is the one-electron oxidation of the [2Fe-2S] 1C form of SoxR that generates the transcriptionally active [2Fe-2S] 2C form. Furthermore, DNA contributes to setting the sensitivity of the SoxR switch, shifting the reduction potential from ¡285 mV for SoxR in solution to C200 mV for SoxR bound to its cognate DNA. 109 Transcriptional activation occurs by remodeling the ¡35 and ¡10 promoter elements. 108 Upon activation, SoxR activates transcription of soxS, and the SoxS protein switches on expression of the SoxRS regulon. The system is switched off by a SoxR reductase, encoded by rseC and rsxABCDGE, and by proteolytic degradation of SoxS. 110,111 As well as responding to redox-cycling molecules, the E. coli SoxR [2Fe-2S] cluster reacts with NO to form a protein-bound dinitrosyl-iron complex that activates expression of soxS and hence the SoxRS regulon. [112][113][114][115] The activation of the SoxRS system by NO conferred resistance to activated macrophages and was thus considered important in virulence. 112 Thus, although the primary role of SoxR is to sense and respond to oxidative stress, it may play a significant secondary role in the response to nitrosative stress.

OxyR
OxyR is a member of the LysR family of transcription factors and is responsible for coordinating the response to peroxide stress in many bacteria. In E. coli, OxyR controls a regulon that includes the sRNA oxyS and genes encoding proteins for the detoxification of peroxides (catalase, alkylhydroperoxidase), for the repair of damaged cell components (methionine sulfoxide reductase) and protection of DNA (Dps). 116 OxyR exists as a homotetramer, with each subunit possessing two domains; an Nterminal DNA-binding domain and a C-terminal sensory domain (Fig. 5). 117 The latter contains the redox-reactive  -199), which forms a sulfenic acid (S-OH) in the presence of peroxide stress. This form of OxyR is proposed to be able to regulate gene expression, although more likely acts as an intermediate in forming the true active form, which is able to bind DNA and serve as a transcriptional regulator and contains an intra-molecular disulphide bond between Cys-199 and Cys-208. OxyR returns to its inactive form (Cys-199, SH; Cys-208, SH) by the action of glutaredoxin 1 and glutathione. (B) A secondary role of OxyR is as a nitrosative stress responder. S-nitrosylation of Cys-199, forming S-NO, leads to activation of OxyR, de-nitrosylation, forming SH, returns OxyR to its inactive form.
cysteine residue (Cys-199), which in the presence of peroxide stress forms a sulfenic acid (Cys-199, S-OH) that is apparently sufficient to activate OxyR, but there is good structural and biochemical evidence that the active form of OxyR has an intra-subunit disulfide bond linking Cys-199 and Cys-208; thus the sulfenic acid form is likely to be an intermediate in the production of the disulfide form. 117,118 Upon oxidation, OxyR recruits RNA polymerase to target promoters to activate transcription, or represses gene expression by promoter occlusion. OxyR is switched off when redox balance is restored by the action of glutaredoxin 1 (an OxyR target) and glutathione. Not surprisingly, OxyR is considered to be important in co-ordinating the response to ROS generated during the oxidative burst of macrophages and has been shown to be critical for full virulence of many bacterial pathogens. For example, OxyR has been shown to contribute to the virulence of Bacteroides fragilis, E. coli, Francisella novicida, K. pneumoniae, P. aeruginosa, Ralstonia solanacearum, X. campestris, and Y. pestis, but not Mycobacterium marinum or intestinal colonization by S. enterica. [119][120][121][122][123][124][125][126][127][128][129] In addition to its primary role in response to peroxide stress, OxyR is activated by nitrosative stress as a result of S-nitrosylation (Cys-199, S-NO); de-nitrosylation (Cys-199, SH) inactivates OxyR. 118,130 S-Nitrosylation of OxyR induced expression of a set of genes, distinct from those activated in response to oxidative stress, which limited S-nitrosylation of proteins and thereby contributed to protection from nitrosative stress. 131

Oxygen and NO Sensing in Staphylococcus aureus
Staphylococcus aureus is carried on the skin and mucosa (anterior nares) by up to 20% of the population at any one time without any harmful effects. However, it is an opportunistic pathogen that is capable of causing a range of diseases including bacteremia, chronic lung infections, endocarditis, food poisoning, meningitis, osteomyelitis, skin infections, and wound infections, and is one of the most common causes of hospital acquired infection. 132 The bacterium is a facultative anaerobe and the ability to adapt to anoxic conditions and mount a defense against host-generated NO is vital in the pathogenesis of many of these diseases. Despite this, the mechanisms that enable S. aureus to perceive and respond to changes in the availabilities O 2 and NO are poorly understood. In this section the roles of three staphylococcal two-component regulators in these processes are reviewed.

SrrAB
The two-component system ResDE is required for anaerobic respiration in many gram-positive bacteria; in Staphylococci the ResDE othologs are known as SrrAB. 133 The ResE (SrrB) protein is a membrane-anchored sensor that autophosphorylates in the absence of O 2 and then transfers the phosphate to the cytoplasmic response regulator ResD (SrrA). The precise signal sensed by these systems is unknown; it is unlikely to be O 2 per se but more likely a physiological consequence of O 2 -starvation, such as changes in the redox state of the electron transport chain (see ArcBA above). Under anaerobic conditions, SrrAB downregulates agr-RNAIII, a regulatory RNA that enhances the production of secreted virulence factors such as serine protease and a-hemolysin, and inhibits the synthesis of cell-surface proteins such as protein A (Fig. 6). 134 SrrAB also downregulates synthesis of the toxic shock syndrome toxin 1 (TSST1) and enhances transcription of the ica operon resulting in increased production of extracellular polysaccharide. 135,136 A strain of S. aureus that overexpressed srrAB was attenuated in a rabbit model of endocarditis by »100-fold, presumably due to the repression of major virulence factors such as agr-RNAIII, TSST1, and protein A, and hence O 2 -sensing (probably indirectly) by SrrAB modifies the virulence of S. aureus. 136 NreABC Staphylococcus aureus can utilize O 2 , nitrate, or nitrite as a terminal electron acceptor. However, unlike the enteric bacteria in which regulation of genes required for anaerobic respiration is coordinated by the global O 2 -sensing transcription factor FNR, in the staphylococci the regulation of nitrate-nitrite respiration is assumed by the proteins encoded by the nreABC operon. 137 The NreBC proteins constitute a two-component system; however, how NreA impacts on the activity of NreBC is unknown, but NreA has a GAF domain and is thought to be involved in sensing nitrate. NreB is a cytoplasmic histidine kinase with four Cys residues located within an N-terminal PAS domain that binds a [4Fe-4S] cluster. Like FNR, the NreB iron-sulfur cluster is disassembled in the presence of O 2 , such that in the absence of O 2 the kinase activity of NreB is activated. 138,139 Thus, in the absence of O 2 NreB phosphorylates the response regulator, NreC, which is then competent for site-specific DNA-binding to activate expression of at least 40 genes including the anaerobic respiratory nar and nir operons, genes involved in nitrogen metabolism, fermentation, and biofilm formation. [138][139][140] An S. aureus narJ mutant emerged from a large-scale (6300 insertion mutants) screening experiment for strains attenuated in a mouse model of systemic infection, but this strain was similarly attenuated in vitro and hence probably has a general growth defect. 141 Thus, the evidence indicates that NreABC does not play a major role in the control of virulence gene expression in response to hypoxia but is important as a fitness factor in anoxic environments where nitrate is available, which could be relevant to infection. Accordingly, O 2 availability has been suggested to control the spatial and temporal expression of Cid, an autolysin that contributes to the provision of extracellular DNA in the biofilm matrix by controlling bacterial programmed cell death, because the cidABC operon was induced under the hypoxic conditions that exist in the interior of the tower structures in biofilms. 142 This link between NreABC regulation and biofilm growth and maturation is potentially important because S. aureus is one of the most frequent causes of biofilm-associated infection on indwelling medical implants.
AirSR A third S. aureus two-component system, AirRS (formerly YhcRS), acts as a global regulator under anoxic conditions and controls, directly or indirectly, the expression (both up-and downregulation) of >350 genes, including the Agr regulatory system (Fig. 6) and virulence factors such as capsular polysaccharide synthesis (cap5A), protein A (spa), leukotoxin (lukD), and g-hemolysin (hlgC) in the Newman strain, and nreABC (see above) as well as several metabolic genes, which could be important for virulence in the WCUH29 isolate, in which AirSR appears to be essential. 143,144 The Nterminal region of the histidine kinase AirS has a cysteine cluster (Cys-X 7 -Cys-X-Cys-X 17 -Cys) that acts as the locus for a [2Fe-2S] cluster. The AirS iron-sulfur cluster reacted relatively slowly with O 2 as demonstrated by the fact that the protein could be isolated with a [2Fe-2S] cluster under aerobic conditions; however, the cluster reacted more rapidly with hydrogen peroxide, resulting in cluster degradation. The cluster also reacted with the nitrosating agent S-nitrosoglutathione to yield a protein bound dinitrosyl-iron-dithiol complex. 143 Oxidative degradation of the [2Fe-2S] cluster to form apo-AirS or formation of the nitrosylated cluster inhibited the kinase activity of AirS and consequently interrupted transfer of phosphate to the response regulator AirR, switching off expression of the AirRS regulon. 143 The phenotypic consequences of disruption of the AirRS system are increased resistance to H 2 O 2 , vancomycin, norfloxacin, and ciprofloxacin under anaerobic conditions. 143

Nitric oxide responses
The relationship between S. aureus and NO is more complex than that described above for enteric pathogens because S. aureus is one of a few gram-positive bacteria that possess a nitric oxide synthase. [145][146][147] In several of these bacteria the capacity to synthesize NO has been shown to contribute to bacterial virulence, increase resistance to oxidative stress and provide protection against antibiotics, and, in the case of Streptomyces sturgidiscabies, nitration is required to activate a phytotoxin. [147][148][149] The S. aureus nitric oxide synthase also protects against killing by neutrophils, as well as being involved in the development of skin abscesses in a mouse model. [150][151][152] Staphylococcus aureus is tolerant to nitrosative stress by transcriptional reprogramming that involves at least 84 identified genes, many of which have roles in iron-homeostasis and hypoxic metabolism; the latter falling under the influence of the indirect O 2 -responsive SrrAB two-component system (see above). Consequently, an srrAB mutant exhibited enhanced sensitivity to NO and this was partially attributed to dysregulation of the NO detoxification enzyme Hmp (see above), but the divergently transcribed ldh1 gene, encoding lactate dehydrogenase was subsequently shown to be essential for virulence and maintaining redox balance during nitrosative stress. 153,154 The overlap between the response to anoxia and exposure to NO could be accounted for by NO-mediated inhibition of aerobic respiration triggering the activation of the SrrAB two-component system and consequently the role of SrrAB could be extended beyond the control of hypoxic metabolic and major virulence factor genes to include NO resistance; a combination that readily explains the attenuation of the srrAB mutant.
At the time of writing, the only S. aureus gene regulator that reacts directly with NO is the AirSR two-component system (see above). Nitrosylation of the [2Fe-2S] cluster of AirS by NO inhibits its histidine kinase activity and hence switches off the regulatory activity of AirR. 143 Further work is needed to establish whether NO is a physiological signal for the AirSR system, but at this stage it seems to be a good candidate. Figure 6. The Agr regulatory system. The agr locus consists of divergently transcribed agrBDCA and RNAIII genes. The former is driven from promoter 2 (P2) and encodes proteins that constitute the Agr quorum sensing system. The latter is driven from P3 and encodes the 26 amino acid d-hemolysin and the regulatory RNA, RNAIII. AgrC and AgrA are a two-component system that responds to accumulation of an autoinducer peptide (AIP, a tailed thiolactone ring) that is generated by processing of AgrD by the membrane-bound AgrB protein and SpsB. The accumulation of AIP in the extracellular milieu is sensed by AgrC resulting in phosphorylation and activation of AgrA. RNAIII downregulates expression of cell surface proteins and upregulates exoprotein (toxin) production.

The Response of Mycobacterium tuberculosis to O 2 and NO
Mycobacterium tuberculosis is the causative agent of tuberculosis (TB) in humans and infects up to one-third (»2 billion) of the world's population, of which 5-10% are at risk of developing active TB. 155 Fortunately, most infected individuals are essentially asymptomatic, carrying the bacteria in lung lesions, known as tubercules. Exposure to hypoxia and NO in the tubercule, reprograms M. tuberculosis gene expression to facilitate entry into a non-replicative, drug-resistant, persistent state. [155][156][157][158][159][160][161] In this state, known as latency, the bacteria survive for decades in the infected lung, before potentially emerging as an active TB infection when an individual becomes immune-compromised. [156][157][158][159] Among the environmental cues that trigger transition to dormancy within the host are hypoxia and exposure to NO. 161,162 Therefore, sensing and responding to these signals is a central feature of M. tuberculosis virulence and TB pathogenesis. The sensory mechanisms and roles of some of the key transcription regulators involved in this process are discussed below.
DosR/S/T As noted above M. tuberculosis is exposed to NO and hypoxia during the course of an infection. Adaption in response to these signals is mediated by the three-component dormancy survival regulator (DosR/S/T). The two sensor kinases, DosS and DosT possess tandem GAF domains, the first of which (GAF-A) contains a penta-coordinate ferrous-heme that interacts with NO, O 2 , and CO, followed by histidine kinase and ATPase domains. [163][164][165] Although it has been proposed that DosS is a redox sensor and DosT a hypoxia sensor, it is mostly likely that both are gas sensors. 166 The deoxy-ferrous forms of DosS and DosT autophosphorylate in the absence of O 2 or when NO (or CO) binds at the sensory heme; binding of O 2 inhibits autophosphorylation as a result of conformational changes initiated by hydrogen-bonding network involving O 2 -bound to heme and a conserved Tyr residue. 166 The inactive oxy-heme-form of DosS is readily converted to the active ferrous-NO-form in the presence of low concentrations of NO, activating the DosR regulon. 167 Phosphorylated DosS/T transfers phosphate to DosR, activating DNA-binding and initiating the dormancy gene expression program, which includes dosS. Despite their similarity, DosS and DosT appear to play distinct roles, the former acting in final phase and the latter in the initial phase of the transition to dormancy, and they exhibit some differences in ligand binding; notably that DosT traps O 2 better than DosS. 166,[168][169][170] In response to hypoxia, non-toxic concentrations of NO and adaptation to an in vitro dormant state DosR controls the expression of a common set of »50 identified genes. 162,[171][172][173][174] Among these genes are those involved in controlling the shift from aerobic to anaerobic metabolism, allowing the bacilli to survive during hypoxia-induced dormancy, and be positioned to return to replication (and thus active infection) upon re-oxygenation. [174][175][176][177] This permits the bacteria to enter dormancy, aiding survival, when conditions are unfavorable for active infection. The individual contributions of many genes induced by the Dos R/S/ T regulon in helping M. tuberculosis survival during dormancy (persistence factors), remain unclear. Nevertheless, the essential role played by the Dos system in the ability of M. tuberculosis to establish and emerge from dormancy is a major contributor to TB pathogenesis, allowing the establishment of an enormous reservoir of infection.

WhiB-like proteins
Mycobacterium tuberculosis possesses seven WhiB-like (Wbl) proteins. Wbl proteins are found exclusively in the actinomycetes and play important roles in developmental processes. 178 All Wbl proteins have four highly conserved cysteine residues, with the central two forming a CXXC motif in the majority of the members, and a weakly predicted helixturn-helix in the C-terminal region. 179 These two key features suggest that Wbl www.landesbioscience.com proteins bind a metal co-factor, which senses and responds to environmental signals to modulate DNA-binding via the C-terminal region. Accordingly, several Wbl proteins from S. coelicolor and M. tuberculosis have been identified as iron-sulfur proteins that are redox, O 2 , and/or NO-sensitive. [180][181][182][183][184][185][186][187][188] Furthermore, conditional DNA-binding activity has been demonstrated in several cases. [184][185][186] The best characterized M. tuberculosis Wbl proteins are WhiB1 and WhiB3.
The M. tuberculosis whiB1 gene is essential and the conserved cysteine residues of the encoded protein coordinate a [4Fe-4S] 2C cluster, which unlike that of FNR (see above) is stable in the presence of O 2 . 186,188 However, like FNR, the WhiB1 iron-sulfur cluster reacts rapidly with 8 molecules of NO, forming an octa-nitrosylated cluster. 188 Reaction of holo-WhiB1 with NO converts WhiB1 from a non-DNA-binding form to a form capable of binding both the whiB1 and groEL2 (encoding an essential chaperonin) promoters, and repressing transcription of both genes in vitro. 186,189 Repression of groEL2 expression by WhiB1 might contribute to inhibiting M. tuberculosis growth during the NO-induced transition to the persistent non-replicating state that is characteristic of latent tuberculosis infections. DNA-binding activity was also observed with both the oxidized (disulfide form) and reduced (dithiol form) apo-WhiB1. Thus the presence/absence and state of the iron-sulfur cluster, and the oxidation state of cysteine residues in apo-WhiB1, govern the ability of WhiB1 to bind DNA via its C-terminal region. 186,187 The full extent of the WhiB1 regulon is currently unknown, but its role as an essential, aerobic NO-sensing transcription factor implies that WhiB1 and the genes that it controls are likely to contribute to transcriptional reprogramming in the host environment. 186 WhiB3 from M. tuberculosis is the best studied of the Wbl proteins and like WhiB1, possesses a [4Fe-4S] cluster and is a DNA-binding protein that controls several aspects of virulence, including the biosynthesis of complex surface-associated virulence lipids. 185,[190][191][192] The expression of whiB3 is enhanced in macrophages and the mouse lung, indicating that M. tuberculosis regulates whiB3 expression in response to environmental signals associated with infection; this is supported by the findings that hypoxia and NO induced whiB3 expression. [193][194][195][196] Its role in virulence is clear-mice infected with a whiB3 null mutant showed increased survival. 186 Moreover, WhiB3 directly controls expression of genes involved in the biosynthesis of the secreted, immuno-modulatory lipids, poly-and di-acyltrehaloses, sulfolipids, and phthiocerol dimycocerosates that are associated with persistence and latency, as well as the storage lipid triacylglycerol (TAG). The iron-sulfur form of WhiB3 (holo-WhiB3), in both reduced and oxidized states, binds DNA very weakly, but the oxidized (disulfide form) of apo-WhiB3 exhibits strong DNA-binding activity. 192 The WhiB3 iron-sulfur cluster reacts with both O 2 and NO, probably via mechanisms resembling that for FNR and WhiB1 (see above). 185 It has been suggested that these responses indicate that under the oxidizing aerobic conditions associated with active TB infections apo-WhiB3 is transcriptionally active, whereas under the reducing hypoxic conditions associated with latency WhiB3 possesses an iron-sulfur cluster and is transcriptionally inactive. Hence WhiB3 senses the redox state of the bacterium via the presence/absence of the iron-sulfur cluster and the propensity of the cysteine residues that ligate the iron-sulfur cluster to form intramolecular disulfide bonds under oxidizing conditions. 185,191 The physiological significance of the NO-reactivity of the WhiB3 iron-sulfur cluster has yet to be established.

Conclusions
The ability to sense and respond to changes in O 2 availability and exposure to the toxic gas NO is crucial for many bacterial pathogens. Both these gases can act as environmental cues to reprogram gene expression and thereby promote the ability to grow and replicate within a host (e.g., switching from aerobic to anaerobic metabolism and synthesizing systems for NO detoxification), to activate expression of virulence factors to attack a host (e.g., T3SS, HlyE, LukD, TSST1) and, in the case of M. tuberculosis and possibly other pathogens, facilitate entry into a persistent, non-replicating state (Fig. 7). Several transcription factors involved in O 2 sensing are global regulators (e.g., ArcBA, FNR, SrrAB) controlling key aspects of central metabolism, as well as genes encoding virulence factors (Fig. 7). This suggests that these core regulators have been evolutionarily co-opted to coordinate virulence gene expression with the metabolic adaptations triggered by host-associated hypoxia. On the other hand NO-responsive transcription factors appear to play more specialized roles associated with NO detoxification and redox homeostasis (Fig. 7). However, regulators such as DosS/T/R, WhiB3, and FNR can act as sensors of both O 2 and NO, raising questions of how their respective sensory co-factors react with, and discriminate between, these similar gases to trigger different patterns of gene expression. Research targeted toward obtaining a deeper understanding of the interplay between differential signal perception and the transcriptional outputs resulting from the action of multiple regulators acting at the promoters of virulence genes should provide new paradigms in host-pathogen interaction by defining the transcriptional colloquy that is crucial in determining the outcome of an infection.

Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.

Acknowledgments
Work on O 2 -and NO-sensing in E. coli and M. tuberculosis has been supported by the United Kingdom Biotechnology and Biological Sciences Research Council with responsive mode project grants (extant grant BB/K000071/1) and the SysMO initiative (BB/I004122/1).