The cbb₃-type cytochrome c oxidase is phylogenetically the most distant member of the heme–copper oxidase superfamily and exclusively found in bacteria (Pitcher and Watmough, 2004). The X-ray structure of the enzyme from Pseudomonas stutzeri was reported recently (Buschmann et al., 2010). This type of enzyme is known to have very high affinity for oxygen and low proton-translocation efficiency. The K_m values for oxygen were determined to be 7 and 40 nM for the enzymes from Bradyrhizobium japonicum and Campylobacter jejuni, respectively (Preisig et al., 1996; Jackson et al., 2007). The cbb₃ oxidase is known to be induced under low oxygen conditions in many bacteria, such as Paracoccus denitrificans, R. sphaeroides, and R. capsulatus (Mouncey and Kaplan, 1998; Otten et al., 2001; Swem and Bauer, 2002). In the symbiotic nitrogen fixation bacterium B. japonicum, the cbb₃ oxidase is essential for nitrogen fixation in hypoxic root nodules, in which the oxygen concentration is extremely low (∼20 nM; Preisig et al., 1993, 1996). The cbb₃ oxidase is also known to be functioning in the obligately microaerophilic bacteria Helicobacter pylori and C. jejuni (Nagata et al., 1996; Jackson et al., 2007). From these observations, the cbb₃ oxidase is recognized as the major player under the low oxygen environments.

The cbb₃ oxidase is also known to have a repressive role in the PrrBA-dependent expression of the photosynthesis genes in R. sphaeroides (O'Gara et al., 1998; Oh and Kaplan, 1999, 2000). The photosynthesis genes are usually induced under low oxygen or anaerobic conditions in non-oxygenic photosynthetic bacteria. However, disruption of the cbb₃ oxidase genes or inhibition of the cbb₃ oxidase activity results in expression of the photosynthesis genes even under aerobic conditions in R. sphaeroides. It has been proposed that the cbb₃ oxidase senses the electron flow through the respiratory chain and generates an inhibitory signal that prevents the activation of PrrBA (Oh et al., 2004).

The cbb₃ oxidase is encoded by a tetracistronic operon ccoNOQP (fixNOQP). The ccoN (fixN) product is the catalytic subunit that contains the binuclear center consisting of a high spin heme b₃ and Cu_B. The ccoO (fixO) and ccoP (fixP) genes encode transmembrane monoheme and diheme cytochrome c subunits, respectively. The gene product of ccoQ (fixQ) is thought to be required for stabilization of the core complex of the cbb₃-type oxidases (Zufferey et al., 1996; Oh and Kaplan, 2002; Peters et al., 2008).

Two complete sets of the genes encoding the cbb₃ cytochrome oxidases (Cbb3-1 and Cbb3-2) are tandemly clustered in the genome of P. aeruginosa (Stover et al., 2000). Cbb3-1 and Cbb3-2 are encoded by the ccoN1O1Q1P1 genes (PA1552–1554) and the ccoN2O2Q2P2 genes (PA1555–1557), respectively. The small ccoQ1 and ccoQ2 genes were not annotated in the genome previously (Stover et al., 2000). Tandem repeats of the genes for two cbb₃-type cytochrome oxidases are also found in the genomes of other pseudomonads, such as Pseudomonas putida, Pseudomonas fluorescens, and P. stutzeri. In addition to the two complete sets of the cco gene clusters, two orphan ccoN-like genes (PA1856 and PA4133), which are highly similar to the main subunit genes of the cbb₃ oxidases, have been identified in the P. aeruginosa genome (Stover et al., 2000). Both of these ccoN-like genes are followed by small genes (PA1855 and PA4134) that have similarity with the ccoQ genes. These extra ccoNQ-like gene clusters might have complementary functions for aerobic respiration.

The cco1 genes for Cbb3-1 were constitutively expressed at a high level even under high oxygen conditions and slightly downregulated at the stationary phase (Comolli and Donohue, 2004; Alvarez-Ortega and Harwood, 2007; Kawakami et al., 2010). Cbb3-1 seemed to be the dominant oxidase, especially under the normal aerobic growth conditions at exponential phase in LB medium. Disruption of the cco1 genes slightly affected the aerobic growth (Comolli and Donohue, 2004; Alvarez-Ortega and Harwood, 2007). Mutation of the cco1 genes also showed a small colony phenotype (Schurek et al., 2008). In contrast, the cco2 genes for Cbb3-2 were upregulated under low oxygen conditions or at the stationary phase. The expression level of Cbb3-2 surpassed that of Cbb3-1 under those induced conditions (Kawakami et al., 2010). ANR is involved in the transcriptional activation of the cco2 genes under low oxygen conditions (Comolli and Donohue, 2004; Kawakami et al., 2010). The induction of the cco2 genes at the stationary phase likely occurred because the oxygen concentration in the medium was depleted to the level that induces the activation by ANR. RoxSR is also involved in the induction of the cco2 genes (Kawakami et al., 2010). A similar RoxSR-dependent regulation was also reported in the case of cco1 (corresponding to cco2 of P. aeruginosa) in P. putida (Fernández-Piñar et al., 2008). A cco1 cco2 double mutant of P. aeruginosa showed very slow growth under a 2% oxygen condition and failed to grow under a 0.4% oxygen condition (Alvarez-Ortega and Harwood, 2007). These results indicate that Cbb3-1 plays a primary role in aerobic growth irrespective of oxygen concentration and that Cbb3-2 plays a compensatory or supplementary role under the oxygen-depleted conditions.

The aa₃-type cytochrome c oxidase is a major member of the heme–copper oxidase superfamily and is distributed in a wide range of bacteria. This type of heme–copper oxidase enzyme is closely related to the mitochondrial terminal oxidase and has a high proton-pumping activity. The aa₃-type oxidases have low affinity for oxygen and usually play a dominant role under high oxygen conditions in many bacteria, such as P. denitrificans, B. japonicum, R. sphaeroides, and Bacillus subtilis (Bosma et al., 1987; Gabel and Maier, 1993; Flory and Donohue, 1997; Winstedt and von Wachenfeldt, 2000; Arai et al., 2008).

In P. aeruginosa, the aa₃ oxidase (Aa3) is encoded by the coxBA-PA0107-coIII (coxC) gene cluster (PA0105–0108; Stover et al., 2000). The coxA, coxB, and coIII (coxC) genes encode subunits I, II, and III, respectively. Subunit I carries the heme a₃−Cu_B binuclear catalytic center. Subunit II has a binding site of Cu_A, which is the electron transfer site with cytochrome c. PA0107 encodes a putative cytochrome c oxidase assembly protein involved in the insertion of copper into subunit I. The expression level of the cox genes is kept very low under normal laboratory growth conditions even at high oxygen tension, although these genes are slightly upregulated at the stationary phase in P. aeruginosa (Schuster et al., 2004; Alvarez-Ortega and Harwood, 2007). We recently found that the cox genes were significantly induced under starvation of carbon, nitrogen, or iron (Kawakami et al., 2010). The cox promoter was found to be dependent on a stationary phase sigma factor RpoS and repressed by RoxSR (Schuster et al., 2004; Kawakami et al., 2010). At the stationary phase, RpoS is highly expressed and activates the cox promoter, but the promoter is simultaneously repressed by RoxSR, which is expected to be active in the hypoxic high-cell-density stationary phase culture. Therefore, the expression level of the cox genes might be kept low under any nutrient-rich conditions. The cco1 cco2 cio cyo quadruple mutant, which is deficient in the four terminal oxidase gene clusters other than the cox gene cluster, was not able to grow under aerobic conditions. However, suppressor mutants that are able to grow aerobically emerged after aerobic incubation for several weeks (unpublished data). The cox structural genes had no mutation, but the roxS gene was inactivated and the RpoS-dependent promoter region of the cox genes was altered in the suppressor mutants, confirming the tight regulation of the cox genes by RoxSR and RpoS. The respiratory chain terminated by Aa3 has the highest ability to create a proton gradient across the membrane (the H⁺/O ratio is 6) among the five terminal oxidases of P. aeruginosa (unpublished data). Therefore, it is reasonable that Aa3 is utilized only under starvation conditions for efficient energy production. Utilization of Aa3 might be advantageous for proliferation in natural oligotrophic environments. However, if Aa3 were highly expressed under the nutrient-rich laboratory growth conditions, its high proton-pumping activity might cause an imbalance of energy and redox homeostasis in P. aeruginosa cells.

The bo₃-type quinol oxidase is also a member of the heme–copper oxidase superfamily. This type of the terminal oxidase is homologous to the aa₃-type cytochrome oxidase but contains heme b and heme o. It lacks the Cu_A-binding site, which is involved in the interaction with cytochrome c, and receives electrons from ubiquinol. The bo₃ oxidase of Escherichia coli is known to have a low affinity for oxygen and functions under high oxygen conditions (Cotter et al., 1990; D'Mello et al., 1995). It is encoded by the cyoABCDE genes in E. coli (Nakamura et al., 1997). The cyoA, cyoB, and cyoC genes encode the subunits corresponding to the subunits II, I, and III of the aa₃-type cytochrome oxidase, respectively. The cyoD gene encodes subunit IV, which is proposed to assist the Cu_B-binding to subunit I during biosynthesis or assembly of the oxidase complex (Saiki et al., 1996). The cyoE encodes a protoheme IX farnesyltransferase (heme o synthetase), which is required for production of heme o from heme b (Saiki et al., 1992; Mogi et al., 1994).

The cyoABCDE genes (PA1317–1321) of the P. aeruginosa bo₃ oxidase (Cyo) are highly homologous with the corresponding genes of E. coli. Cyo was identified as a quinol oxidase and shown to have a high K_m value for oxygen by using the cco1 cco2 cox cio quadruple mutant (unpublished data). The H⁺/O ratio of Cyo was speculated to be 4, which was comparable to that of the bo₃ oxidase from E. coli (Puustinen et al., 1989). The cyo genes were downregulated at the stationary phase or under the low oxygen conditions in P. aeruginosa cells (Alvarez-Ortega and Harwood, 2007; Kawakami et al., 2010). Considering that the affinity for oxygen is low, Cyo might function under high oxygen conditions as in the case of E. coli. However, because the expression level of the cyo genes was significantly lower than that of the cco1 genes without stresses, Cyo might make a minor contribution to the cell growth under normal laboratory growth conditions. The cyo genes were found to be significantly induced by iron starvation or in the presence of a nitric oxide (NO)-generating reagent, S-nitrosoglutathione (GSNO; Ochsner et al., 2002; Kawakami et al., 2010). Cyo is likely responsible for respiration under iron-limiting conditions and the expression of the cyo genes is regulated by the transcriptional regulator Fur (ferric uptake regulator), which is known to bind to DNA in the presence of Fe²⁺ and represses the iron-regulated promoters (Vasil and Ochsner, 1999; Vasil, 2007). Because the predicted number of iron atoms sequestered in the Cyo complex is lower than those of the other terminal oxidase complexes and the respiratory complex terminated by Cyo does not require the iron-containing cytochrome bc₁ complex and soluble cytochrome c (Fujiwara et al., 1992; Thöny-Meyer, 1997), the demand for iron might be lower when Cyo is utilized. The cyo genes were upregulated by GSNO, but the role of Cyo in nitrosative stress resistance is not certain. Because Fur is known to lose its DNA-binding ability in the presence of NO (D'Autréaux et al., 2002), the upregulation by GSNO might be merely due to the inactivation of Fur by NO. An obligately aerobic bacterium, P. putida, also has five sets of terminal oxidases corresponding to those of P. aeruginosa (Ugidos et al., 2008). In P. putida, inactivation of the cyo genes relieves the catabolite repression of the phenol- or alkane-degradation genes (Petruschka et al., 2001; Dinamarca et al., 2002). The cyo mutation also leads to a significant change in the transcriptome profile, and the absence of Cyo in P. putida was compensated for by upregulation of CIO and one of the cbb₃ oxidases corresponding to Cbb3-2 of P. aeruginosa (Morales et al., 2006). In contrast to P. aeruginosa, Cyo might make a major contribution to the aerobic growth of this obligately aerobic bacterium, although it remains to be investigated whether inactivation of Cyo has a significant influence in P. aeruginosa.

The cyanide-insensitive quinol oxidase (CIO) has been found and described from P. aeruginosa and C. jejuni (Matsushita et al., 1983; Cunningham et al., 1997; Jackson et al., 2007). While the Cbb3-1, Cbb3-2, Aa3, and Cyo oxidases belong to the heme–copper oxidase superfamily, CIO is the only copper-free terminal oxidase in P. aeruginosa. It consists of two subunits encoded by the cioAB genes, which are highly homologous to the cydAB genes for the cytochrome bd-type quinol oxidases (Cunningham et al., 1997; Jünemann, 1997; Jackson et al., 2007). The bd oxidases have high affinity for oxygen and contain low-spin heme b₅₅₈, high spin heme b₅₉₅, and heme d (D'mello et al., 1996; Jünemann, 1997). E. coli has two bd oxidases (Bekker et al., 2009), one of which is known to be induced and predominant under oxygen-limited conditions (Rice and Hempfling, 1978; Cotter et al., 1990). In the free-living nitrogen-fixing bacterium Azotobacter vinelandii, the bd oxidase rapidly consumes oxygen and protects the oxygen-sensitive nitrogenase complex (Kelly et al., 1990; Poole and Hill, 1997). This phenomenon is called respiratory protection.

Although the cioAB genes for CIO are highly homologous to the cydAB genes for the bd oxidase, CIOs from P. aeruginosa and C. jejuni lack the spectral features for heme b₅₉₅ and heme d (Matsushita et al., 1983; Cunningham and Williams, 1995; Cunningham et al., 1997; Jackson et al., 2007). It had been considered that the hemes of the bd oxidase are replaced by other unknown redox centers in CIO. However, recent analysis using the membrane vesicle of a CIO-overproducing Gluconobacter oxydans revealed that CIO carries all of the hemes b₅₅₈, b₅₉₅, and d. The absence of the spectroscopic properties was predicted to be caused by the unique ligand-binding properties of CIO (Mogi et al., 2009). A distinctive feature that could be used easily to discriminate CIO from the bd oxidase is that the conserved sequence of the periplasmic loop (Q-loop) that contains the putative quinol oxidizing site is significantly shorter in CioA than in CydA (Cunningham et al., 1997). CIO is known to have higher resistance to cyanide than Cyo (Mogi et al., 2009). In contrast to the finding that the bd oxidases have high affinity for oxygen, CIO of C. jejuni was reported to have low affinity for oxygen, with a K_m value of 0.8 μM (Jackson et al., 2007). It has been recognized that CIO of P. aeruginosa has high affinity for oxygen because the cco1 cco2 cio triple mutant, which lacks Cbb3-1, Cbb3-2, and CIO, could not grow microaerobically under a 2% O₂ concentration (Alvarez-Ortega and Harwood, 2007). However, our recent analysis using the cco1 cco2 cox cyo quadruple mutant revealed that the K_m value of CIO of P. aeruginosa for oxygen is significantly higher than those of Cbb3-1 and Cbb3-2 and comparable to those of Aa3 and Cyo, indicating that CIO of P. aeruginosa is a low affinity enzyme like CIO of C. jejuni (unpublished data).

Pseudomonas aeruginosa produces a respiratory chain inhibitor, hydrogen cyanide, at concentrations up to 300 μM under low oxygen conditions. The concentrations are high enough to inhibit the activity of the heme–copper oxidases. Therefore, CIO is believed to have a role as an electron sink under cyanogenic conditions (Cunningham and Williams, 1995; Cunningham et al., 1997; Blumer and Haas, 2000). The reason why the cco1 cco2 cio triple mutant could not grow under the 2% O₂ condition (Alvarez-Ortega and Harwood, 2007) was probably because of the inhibition of Aa3 and Cyo by the endogenous cyanide. Cyanide is reported to be the primary toxic factor responsible for the paralytic killing of Caenorhabditis elegans by P. aeruginosa (Gallagher and Manoil, 2001). CIO appeared to be required for this pathogenicity because the mutant of the cioAB genes (PA3929–3930) had lower killing activity against the nematoda (Zlosnik et al., 2006).

The cioAB genes are upregulated at stationary phase or at very low ambient oxygen concentration (0.4–0.5%) in P. aeruginosa (Cooper et al., 2003; Alvarez-Ortega and Harwood, 2007; Kawakami et al., 2010). Respiratory chain inhibitors, cyanide and sodium nitroprusside (SNP), significantly induce the cio genes (Comolli and Donohue, 2002; Cooper et al., 2003; Kawakami et al., 2010). The cio genes are also induced by copper starvation or disruption of the senC gene (PA0114), which encodes a putative copper chaperone for the heme–copper oxidases (Frangipani et al., 2008; Frangipani and Haas, 2009). Since CIO is the only non-heme–copper oxidase in P. aeruginosa, CIO must be important for respiration under copper-restricted conditions. The cio genes are positively regulated by RoxSR and disruption of the cco1 genes for Cbb3-1 causes upregulation of the cio promoter (Comolli and Donohue, 2004). Regulation of the RoxSR activity by the electron flow through the cbb₃ oxidase might be operative in P. aeruginosa as in the case of PrrBA in R. sphaeroides (Oh and Kaplan, 2000). The expression of CIO is negatively regulated by ANR and an anr mutant has been shown to express a high level of CIO, especially under low oxygen concentrations (Cooper et al., 2003). It seems likely that ANR prevents the overproduction of CIO when it is not necessary. The stationary phase sigma factor RpoS also activates the expression of the cio genes, although its contribution is minor when ANR or RoxSR is operative (Kawakami et al., 2010). The regulation of the cio genes in P. aeruginosa is thus complicated, but CIO seems to be controlled so as to be expressed when the other terminal oxidases of the heme–copper superfamily are not functioning.

Two redox-responsive transcriptional regulators, ANR and RoxSR, and a stationary phase sigma factor RpoS play dominant roles in the control of the multiple terminal oxidases in P. aeruginosa (Figure 2). ANR functions as a global regulator for anaerobic gene expression in response to oxygen depletion (Galimand et al., 1991; Sawers, 1991; Zimmermann et al., 1991). Approximately 170 transcription units are predicted to belong to the ANR regulon (Trunk et al., 2010). ANR is an analog of E. coli FNR (fumarate nitrate reductase regulator), which senses intracellular oxygen levels by an oxygen-sensitive [4Fe–4S]²⁺ cluster bound to N-terminal Cys residues (Kiley and Beinert, 1998; Unden et al., 2002).

RoxSR is an analog of PrrBA/RegBA of purple photosynthetic bacteria, which activates expression of the photosynthesis genes under low oxygen conditions (Dubbs and Tabita, 2004; Elsen et al., 2004; Eraso et al., 2008). It has been proposed that PrrBA of R. sphaeroides receives an inhibitory signal from the electron flow through the cbb₃ oxidase because the PrrBA-dependent genes are upregulated even under aerobic conditions when the cbb₃ oxidase activity is blocked by inhibitors or mutations (O'Gara et al., 1998; Oh and Kaplan, 1999, 2000; Kim et al., 2007). The activity of RegBA of R. capsulatus and Rhodospirillum rubrum is proposed to be controlled by the redox status of ubiquinones (Grammel and Ghosh, 2008; Wu and Bauer, 2010). The sensing signal of RoxSR of P. aeruginosa is not certain at present. Mutation of the cco1 genes for Cbb3-1 caused upregulation of the RoxSR-dependent expression of CIO in P. aeruginosa PAK, suggesting the possibility that RoxSR senses the electron flow through Cbb3-1 (Comolli and Donohue, 2004). However, because Cbb3-1 is the dominant terminal oxidase under normal aerobic growth conditions, deletion of Cbb3-1 might greatly influence the redox status of the ubiquinone pool. In the case of P. putida, in which Cyo might make a major contribution under aerobic conditions, the absence of Cyo causes upregulation of CIO as well as a significant change in the transcriptome profile (Morales et al., 2006). In any case, the activity of RoxSR of Pseudomonas species is expected to be controlled, directly or indirectly, by the redox status of the respiratory chain.

ANR activates the expression of Cbb3-2, which is a high affinity enzyme and predicted to be dominant under low oxygen conditions (Ray and Williams, 1997; Comolli and Donohue, 2004). ANR represses the expression of CIO (Cunningham et al., 1997; Cooper et al., 2003; Comolli and Donohue, 2004), which was recently found to be a low affinity enzyme (unpublished data). Another low affinity enzyme, Cyo, is probably repressed in an indirect manner by ANR (Kawakami et al., 2010), which is in contrast to the direct repression of Cyo by ANR in P. putida (Ugidos et al., 2008). RoxSR regulates the expression of all five terminal oxidases, though whether directly or indirectly is not certain (Comolli and Donohue, 2002; Kawakami et al., 2010). Aa3 is repressed and the other four terminal oxidases are activated by RoxSR. Some other genes related to respiratory function, such as hemB and nuoAL, are under the control of RoxSR, indicating that RoxSR plays an extensive role in the regulation of respiration in P. aeruginosa (Kawakami et al., 2010). Cbb3-2, Aa3, and CIO are upregulated at the stationary phase. RpoS plays a significant role in the expression of Aa3, but make a minor contribution to the regulation of CIO and no contribution to that of Cbb3-2 (Cooper et al., 2003; Schuster et al., 2004; Kawakami et al., 2010). RpoS is known to be necessary for survival under carbon starvation in P. aeruginosa (Jørgensen et al., 1999; Suh et al., 1999). Therefore, the induction of Aa3 under nutrient starvation conditions might be mainly the effect of the function of RpoS.

Regulation of the terminal oxidases not only by peripheral oxygen tension by ANR but also by the redox status of the respiratory chain by RoxSR might be a sophisticated mechanism for fine tuning of multiple enzymes with different characteristics, because the redox status of the respiratory components is significantly affected by nutritional conditions and respiratory stressors as well as oxygen availability. Availability of nitrogen oxides that act as alternative electron acceptors for anaerobic respiration might also influence the redox status of the respiratory chain under low oxygen conditions.

The cbb₃ oxidases are dominantly expressed and function even under the aerobic conditions in P. aeruginosa. This feature is unique to P. aeruginosa, because the cbb₃ oxidases are known to have very high affinity for oxygen and are usually repressed under high oxygen conditions in other bacterial species (Preisig et al., 1996; Mouncey and Kaplan, 1998; Otten et al., 2001; Swem and Bauer, 2002; Jackson et al., 2007). Our preliminary experiment revealed that both of the two cbb₃ oxidases of P. aeruginosa also have high affinity for oxygen (unpublished data). The low affinity enzymes are highly induced only under starvation or stressed conditions (Kawakami et al., 2010). This is in contrast to the case of the non-pathogenic obligately aerobic bacterium P. putida, which is phylogenetically close to P. aeruginosa and has the same set of five terminal oxidases, but in which Cyo might make a major contribution under aerobic conditions (Morales et al., 2006; Fernández-Piñar et al., 2008; Ugidos et al., 2008).

The dominant expression and function of the high affinity enzymes might indicate that the P. aeruginosa cells are maintained in a microaerobic state even under aerobic conditions. P. aeruginosa actively produces a microaerobic environment even at high aeration rates by reducing the transfer rate of oxygen from the gas phase into the liquid phase (Sabra et al., 2002). This phenomenon was predicted to be due to the production of biosurfactants such as rhamnolipid. P. aeruginosa strains infected in the CF airway show the mucoid phenotype by overproduction of the exopolysaccharide alginate, which is known to act as a physical barrier to oxygen entering the cell. Even the non-mucoid strain PAO1 forms a mucoid polysaccharide capsule, which might consist primarily of alginate, on the cell surface as a response to oxidative stress (Sabra et al., 2002). Alginate is also reported to restrict the diffusion of oxygen (Hassett, 1996). A nitrogen-fixing bacterium, A. vinelandii, which is closely related to Pseudomonas, is also known to produce alginate as a barrier to protect oxygen-sensitive nitrogenase from oxygen (Sabra et al., 2000).

The microaerobic physiology of P. aeruginosa might be significantly related to its pathogenesis. During pulmonary infection, P. aeruginosa is exposed to reactive oxygen species produced by the host immune cells. The physical blockage of oxygen transfer by production of the polysaccharide layer and reduction of the oxygen diffusion rate would be advantageous for P. aeruginosa to survive under such oxidative stress conditions. Moreover, release of virulence factors, such as elastase and pyocyanin, into the culture supernatant of P. aeruginosa has been reported to be enhanced by the microaerobic culture conditions (Sabra et al., 2002).

Nitrate reductase catalyzes the first step of denitrification, reduction of nitrate to nitrite. Three types of nitrate reductases, Nar, Nap, and Nas, which are localized to the cytoplasmic membrane, periplasm, and cytoplasm, respectively, are encoded in the genome of P. aeruginosa (Berks et al., 1995; Stover et al., 2000). They all contain a molybdopterin guanine dinucleotide cofactor. The membrane-bound Nar is the enzyme responsible for anaerobic nitrate respiration of P. aeruginosa in CF sputum (Palmer et al., 2007). Nar is encoded in the narK1K2GHJI gene cluster (PA3872–3877). NarG, NarH, and NarI are the structural subunits of the enzyme and NarJ is required for the assembly of the functional enzyme (Philippot and Højberg, 1999). The narK1 and narK2 genes encode putative nitrate/nitrite transporters homologous to each other, but only narK2 has been reported to be required for denitrifying growth (Sharma et al., 2006). The narXL genes (PA3878–3879) encoding a two-component transcriptional regulator, NarXL, which is required for the nitrate-responsive expression of the narK1K2GHJI operon, is located upstream from the narK1 gene (Schreiber et al., 2007). Reduction of nitrate by Nar is coupled to quinol oxidation and consumes two protons from the cytoplasm, thereby contributing to creation of a proton gradient across the membrane (Zumft, 1997).

The periplasmic enzyme Nap and related proteins are encoded in the napEFDABC gene cluster (PA1172–1177). Nap is a quinol oxidase, but nitrate reduction in the periplasm by Nap does not contribute to generation of the proton gradient. The physiological function of Nap in P. aeruginosa is not certain at present. In R. sphaeroides, Nap is regulated by both nitrate and electron supply and predicted to be involved in redox balancing by using nitrate as an ancillary oxidant to dissipate excess reductant (Gavira et al., 2002). The cytoplasmic enzyme Nas is exclusively involved in nitrate assimilation. The nasC gene (PA1779) for Nas is clustered with the nirBD genes (PA1780–1781) encoding assimilatory nitrite reductase, which catalyzes the reduction of nitrite to ammonium (Berks et al., 1995).

Nitrite reductase catalyzes the second step of denitrification, reduction of nitrite to NO. Two types of dissimilatory nitrite reductases, the copper-containing type and the cytochrome cd₁-type, have been reported so far and P. aeruginosa has the latter type (Zumft, 1997). Both types are located in the periplasm. The cytochrome cd₁ nitrite reductase consists of two identical subunits, each containing a covalently attached heme c and a non-covalently bound heme d₁, which is a prosthetic group unique to this type of nitrite reductase (Silvestrini et al., 1994). In P. aeruginosa, nitrite reductase is encoded in the nirSMCFDLGHJEN gene cluster (PA0509–0519). nirS is the structural gene for the enzyme (Silvestrini et al., 1989). nirM encodes cytochrome c-551 and nirC encodes another monoheme cytochrome c, and both these cytochromes mediate electron transfer from the cytochrome bc₁ complex to nitrite reductase (Arai et al., 1990; Nordling et al., 1990; Hasegawa et al., 2001, 2003). The nirFDLGHJE genes are necessary for the biosynthesis of heme d₁ (Kawasaki et al., 1995, 1997). nirN encodes a c-type cytochrome, which is similar to nirS, but its function is not certain (Hasegawa et al., 2001).

Reduction of NO to N₂O is catalyzed by NO reductase (Hendriks et al., 2000; Zumft, 2005). Two types of bacterial NO reductases, designated cNOR and qNOR, have been characterized. cNOR is a membrane-bound cytochrome bc complex and receives electrons from soluble cytochrome c. qNOR lacks the cytochrome c component and receives electrons from quinol. P. aeruginosa and many denitrification bacteria have cNOR. qNOR has been found in bacteria and cyanobacteria such as Ralstonia eutropha, Neisseria gonorrhoeae, and Synechocystis sp. PCC6803 (Cramm et al., 1999; Householder et al., 2000; Büsch et al., 2002). Bacterial NO reductase shows similarity to the main subunit of cytochrome c oxidases of the heme–copper superfamily and is predicted to be the ancestor of oxygen-based respiratory enzymes (Saraste, 1994; Saraste and Castresana, 1994; Zumft, 2005). NO reductases have no proton-pumping activity, but cNOR contributes to generation of the proton gradient across the membrane because the electrons for its reaction are supplied through the cytochrome bc₁ complex via soluble cytochrome c. The NO reductase of P. aeruginosa has been purified and characterized and its crystal structure has been reported recently (Kumita et al., 2004; Hino et al., 2010). NO reductase is encoded in the norCBD operon (PA0523–0525), which is clustered with the nir genes for nitrite reductase in the genome of P. aeruginosa (Arai et al., 1995a). norC and norB encode cytochrome c and cytochrome b subunits of the enzyme, respectively. norD encodes a soluble protein predicted to be required for production of the active enzyme. NorB carries the binuclear catalytic center consisting of heme b₃ and non-heme Fe_B. NorC mediates electron transfer from soluble cytochrome c to NorB (Zumft, 2005). NO reductase functions not only for anaerobic energy conservation as a respiratory enzyme, but also for detoxification of exogenous NO. The machinery for detoxification of NO and its derivative reactive nitrogen species is necessary because the infected P. aeruginosa cells are subjected to nitrosative stress by the attack of the host immune system. The NO reductase-deficient mutant of P. aeruginosa shows a reduced survival rate in NO-producing macrophages (Kakishima et al., 2007). P. aeruginosa has another NO-detoxification enzyme, flavohemoglobin, which is encoded by the fhp gene (PA2664; Arai et al., 2005). Flavohemoglobin is known to exhibit NO dioxygenase activity under aerobic conditions and NO reductase activity under anaerobic conditions (Poole and Hughes, 2000). However, flavohemoglobin of P. aeruginosa is necessary for detoxification of NO under aerobic conditions but does not support denitrifying growth by complementing the function of NO reductase under anaerobic conditions (Arai et al., 2005). The fhp gene is regulated by the NO-responsive transcriptional regulator FhpR and transcribed as an operon with the following two genes, ppyR (PA2663) and nnrS (PA2662), which encode putative membrane proteins. Disruption of these ppyR and nnrS genes has no effect on the aerobic tolerance to reactive nitrogen species, but the nnrS mutant shows very poor growth under anaerobic denitrification conditions (unpublished data). The ppyR gene has been reported to have a role in the formation of biofilm (Attila et al., 2008).

The nirQOP operon (PA0520–0522) is located between the structural genes for nitrite reductase (nirS) and NO reductase (norCB; Arai et al., 1994, 1996, 1998). The activities of nitrite reductase and NO reductase should be coordinately regulated in order to avoid accumulation of highly cytotoxic intermediate, NO. The function of NirQ is predicted to be the fine tuning of the expression and activation of nitrite reductase and NO reductase (Jüngst and Zumft, 1992; Arai et al., 1996). The nirO and nirP genes encode transmembrane proteins. NirO has sequence similarity to subunit III of cytochrome c oxidases. The function of the nirOP genes is not certain, but it has been proposed that these genes are involved in efficient energy conservation under anaerobic conditions (Arai et al., 1996, 1998).

The final step of the denitrification pathway, reduction of N₂O to N₂, is catalyzed by N₂O reductase. This enzyme has been intensively studied in P. stutzeri and P. denitrificans (Zumft, 1997; Zumft and Kroneck, 2007) and has also been purified and characterized from P. aeruginosa (SooHoo and Hollocher, 1991). N₂O reductase is a periplasmic enzyme and predicted to receive electrons from the cytochrome bc₁ complex via soluble cytochrome c or pseudoazurin. It has two types of copper centers, the mixed-valent dinuclear Cu_A species at the electron entry site and the tetranuclear Cu_Z center at the catalytically active site (Zumft and Kroneck, 2007). The Cu_A site of N₂O reductase shows similarity to the corresponding electron entry site in subunit II of cytochrome oxidases of the heme–copper superfamily, suggesting an evolutionary relationship between the two enzymes for anaerobic and aerobic respiration (Saraste, 1994; Saraste and Castresana, 1994). In P. aeruginosa, N₂O reductase is encoded in the nosRZDFYL operon (PA3391–3396; Arai et al., 2003). The structural gene for the enzyme is nosZ. The nosDFY gene products are thought to be involved in the processing and insertion of copper into the enzyme. The nosL gene product is proposed to be an outer membrane disulfide isomerase. nosR encodes a membrane protein of unknown function. Many denitrifying bacteria can grow on N₂O as the only electron acceptor under anaerobic conditions. However, P. aeruginosa cannot grow on exogenous N₂O as the only electron acceptor, although it can utilize endogenous N₂O for the generation of energy for growth during denitrification. This is probably because the nosR promoter is regulated by NO and exogenous N₂O does not induce the nos genes (SooHoo and Hollocher, 1990; Arai et al., 2003).

Denitrification enzymes are induced under anaerobic or low oxygen conditions in the presence of nitrate or nitrite (Arai et al., 1991a,b; Figure 3). Two transcriptional regulators, ANR and DNR (dissimilatory nitrate respiration regulator), are required for full expression of all denitrification genes (Arai et al., 1994, 1995b, 1997, 1999, 2003; Ye et al., 1995; Schreiber et al., 2007). NarXL regulates some of the denitrification promoters, such as narK1, nirQ, and dnr (Schreiber et al., 2007). Denitrification is also regulated by quorum-sensing signal molecules (Yoon et al., 2002; Toyofuku et al., 2007, 2008).

The dnr gene (PA0527) encoding DNR is clustered with the nir–nor genes for nitrite reductase and NO reductase. Both ANR and DNR belong to the CRP/FNR superfamily of transcriptional regulators and activate a synthetic promoter that has a consensus FNR-binding motif (TTGAT----ATCAA; Hasegawa et al., 1998). However, the promoters of the denitrification genes, which have sequences similar to the FNR-binding motif, are activated only by DNR, but not by ANR. Expression of DNR is under the control of ANR. Thus, the ANR-mediated anaerobic induction of the denitrification genes is an indirect event that occurs by way of DNR (Arai et al., 1997). The hemN, hemF, and narK1 promoters are known to be recognized by both ANR and DNR (Rompf et al., 1998; Schreiber et al., 2007). It is not certain how ANR and DNR distinguish their target promoters. Transcriptome analysis of the ANR and DNR regulons revealed that DNR specifically regulates the denitrification genes (Trunk et al., 2010). DNR specifically senses NO and does not respond to CO (Arai et al., 2003). Heme is required for the in vivo activity of DNR (Castiglione et al., 2009). The crystal structures of the apo-forms of full-length and truncated DNR have been solved recently (Giardina et al., 2008, 2009, 2011). The structural analyses showed that DNR undergoes a very large conformation rearrangement on activation.

Pseudomonas aeruginosa is able to grow in the absence of oxygen and nitrogen oxides, although very slowly, by using arginine as an energy source in rich medium (Shoesmith and Sherris, 1960; Vander Wauven et al., 1984). Anaerobic degradation of arginine to ornithine through the arginine deiminase (ADI) pathway is a non-redox process but produces 1 mol of ATP per arginine by a substrate-level phosphorylation. The ADI pathway enzymes are encoded in the arcDABC operon (PA5170–5173; Lüthi et al., 1990). The arcABC genes encode arginine deiminase, catabolic ornithine carbamoyltransferase, and carbamate kinase, respectively. arcD encodes an arginine-ornithine antiporter. The arc genes are induced by ANR in response to oxygen depletion (Gamper et al., 1991). An arginine-responsive regulator, ArgR, which is involved in the regulation of many arginine metabolic pathways, enhances the arcD promoter activity (Lu et al., 1999). The expression of the arc genes is partially repressed by NarXL in the presence of nitrate, indicating that the more energetically efficient denitrification is preferred over arginine fermentation (Benkert et al., 2008).

Pyruvate fermentation allows long-term anaerobic survival of P. aeruginosa under stationary conditions, although it does not sustain significant anaerobic growth (Eschbach et al., 2004). Acetate kinase and phosphotransacetylase encoded by the ackA-pta operon (PA0835–0836) and NADH-dependent lactate dehydrogenase encoded by ldhA (PA0927) are responsible for pyruvate fermentation. ATP is produced by the acetate kinase activity. Lactate dehydrogenase functions for reoxidation of NADH produced by the pyruvate dehydrogenase activity. ANR and the integration host factor (IHF) are required for the expression of the ackA-pts operon (Eschbach et al., 2004).

Pseudomonas aeruginosa produces a small redox-active phenazine compound, pyocyanin. Pyocyanin generates reactive oxygen species and acts as an antibiotic agent in the soil or a virulence factor during infection. Recently, Newman's group proposed that pyocyanin has additional roles for maintenance of redox homeostasis and control of multicellular behavior (Price-Whelan et al., 2007; Dietrich et al., 2008; Ramos et al., 2010; Wang et al., 2010; Figure 4). Pyocyanin decreases the intracellular NADH levels under energy starvation conditions, indicating that the reoxidation of NADH could be coupled to the reduction of pyocyanin. Because pyocyanin is auto-oxidized by oxygen, it could function as an extracellular electron shuttle. When the P. aeruginosa cells form a biofilm or colony, a steep oxygen gradient is present in the cell community. The cells at the bottom are limited for oxygen but pyocyanin could serve as an alternative electron acceptor. The reduced pyocyanin could be reoxidized after diffusion to the oxygen-rich surface. Thus, pyocyanin contributes to the redox homeostasis and survival of the cells in the deeper anaerobic niches. Further experimental evidence would be required to certify this hypothesis. Pyocyanin is also reported to stimulate pyruvate excretion by decreasing the carbon flux through the central metabolic pathway in P. aeruginosa PA14 at the late stationary phase (Price-Whelan et al., 2007). The pyruvate secreted by the cells at the aerobic niches, such as the surfaces of biofilms, might support the survival of the cells at anaerobic niches of the community as a substrate of anaerobic pyruvate fermentation.

Another example of the relationship between pyocyanin and biofilm lifestyle has been reported in a mixed-species biofilm (Narisawa et al., 2008; Figures 4B,C). The pyocyanin-overproducing P. aeruginosa strain P1 forms a multilayered multispecies biofilm when cocultured with a pyocyanin-resistant Raoultella strain R1 and a pyocyanin-sensitive Brevibacillus strain S1. The layer of strain P1 at the bottom was covered with the layers of resistant and sensitive strains. The sensitive strain was separated from strain P1 by the layer of the resistant strain. This multilayered structure is expected to be advantageous for strain P1 for protection from antibiotics, grazing, desiccation, and many other stresses, although the bottom of the biofilm is disadvantageous for oxygen acquisition. A pyocyanin non-producing derivative of strain P1 forms an intermingled multispecies biofilm with the pyocyanin-resistant and -sensitive strains. P. aeruginosa PAO1, which poorly produces pyocyanin under anaerobic conditions, does not construct the multilayered biofilm. These results indicate that pyocyanin has the ability to construct the organized layered structure in the multispecies biofilm. Pyocyanin might also serve as an electron acceptor for the anaerobic preservation of the P. aeruginosa cells at the oxygen-depleted bottom of the multilayered biofilm.