Review Correspondence

In the bacterial periplasm, the reduction of nitrate to nitrite is catalysed by a periplasmic nitrate reductase (NAP) system, which is a species-dependent assembly of protein subunits encoded by the nap operon. The reduction of nitrate catalysed by NAP takes place in the 90 kDa NapA subunit, which contains a Mo-bis-molybdopterin guanine dinucleotide cofactor and one [4Fe-4S] iron-sulfur cluster. A review of the nap operons in the genomes of 19 strains of Shewanella shows that most genomes contain two nap operons. This is an unusual feature of this genus. The two NAP isoforms each comprise three isoform-specific subunits - NapA, a di-haem cytochrome NapB, and a maturation chaperone NapD - but have different membrane-intrinsic subunits, and have been named NAP-alpha (NapEDABC) and NAP-beta (NapDAGHB). Sixteen Shewanella genomes encode both NAP-alpha and NAP-beta. The genome of the vigorous denitrifier Shewanella denitrificans OS217 encodes only NAP-alpha and the genome of the respiratory nitrate ammonifier Shewanella oneidensis MR-1 encodes only NAP-beta. This raises the possibility that NAP-alpha and NAP-beta are associated with physiologically distinct processes in the environmentally adaptable genus Shewanella.

In addition to being able to respire with TMAO, many species of Shewanella can respire using the alternative marine osmoregulator dimethyl sulfoxide (DMSO) (Gralnick et al., 2006).The reduction of TMAO or DMSO in Shewanella is catalysed by TMAO reductase (Czjzek et al., 1998) or DMSO reductase, respectively.TMAO reductase and DMSO reductase are both members of the DMSO reductase class of the mononuclear molybdenum-containing enzyme superfamily (Gonza ´lez et al., 2006;Hille, 1996;McEwan et al., 2002;Moura et al., 2004;Stolz et al., 2006), which also includes periplasmic nitrate reductase (NAP).The reduction of nitrate to nitrite by NAP is the first step in the reductive respiratory N-cycle in bacteria, which terminates with the production of either ammonium (respiratory ammonification) or dinitrogen (denitrification).Soluble NAP or membrane-bound nitrate reductase (NAR) systems manage catabolic respiratory nitrate reduction and the assimilatory nitrate reductase (NAS) system governs ammonium production in the cytoplasm for anabolic purposes (Gonza ´lez et al., 2006;Richardson et al., 2001;Stolz & Basu, 2002;Tavares et al., 2006;Zumft, 1997).As currently understood, the two respiratory pathways are distinguished downstream of the reduction of nitrate to nitrite by the enzymes that catalyse either the six-electron reduction of nitrite to ammonium (cytochrome c nitrite reductase, NrfA) in respiratory ammonification, or the step-wise reduction of nitrite to nitric oxide (copper nitrite reductase, NirK; or cytochrome cd 1 nitrite reductase, NirS), nitric oxide to nitrous oxide (nitric oxide reductase, NOR) and nitrous oxide to dinitrogen (nitrous oxide reductase, NOS) in denitrification (Gonza ´lez et al., 2006;Richardson et al., 2001;Stolz & Basu, 2002;Tavares et al., 2006;Zumft, 1997).
At least 11 different types of nap gene clusters have been described from bacteria (Gonza ´lez et al., 2006;Marietou et al., 2005;Richardson et al., 2001).The napA gene encodes the catalytic NapA subunit where nitrate reduction takes place and which contains a Mo-bis-molybdopterin guanine dinucleotide cofactor (Mo-bis-MGD) and one [4Fe24S] iron-sulfur cluster.The napB gene encodes the di-c-haem subunit (NapB) that transfers electrons to NapA, with the napAB genes being accompanied by different combinations and arrangements of napCDEFGHKLM genes.The napDABC genes are found in a-, b-and c-proteobacteria (Berks et al., 1995;Delgado et al., 2003;Hettmann et al., 2004;Reyes et al., 1998) and encode what were once defined as the 'essential' NAP proteins (Potter & Cole, 1999), with NapC being a membrane-anchored tetra-haem c-type cytochrome of the NapC/NirT family that mediates electron transport from the quinol pool to NapB (Cartron et al., 2002;Roldan et al., 1998) and NapD being a maturation chaperone for NapA.The napDABC gene cluster is interrupted by genes encoding an iron-sulfurcluster-based NapGH complex in the napFDAGHBC operon of the c-proteobacteria Escherichia coli (Grove et al., 1996), Haemophilus influenzae and Salmonella typhimurium (Marietou et al., 2005) and the a-proteobacterium Magnetospirillum magnetotacticum (Taoka et al., 2003).The nap operons of the e-proteobacteria Wolinella succinogenes (napAGHBFLD) (Kern & Simon, 2008;Simon et al., 2003) and Campylobacter jejuni (napAGHBLD) do not include the napC gene.The function of NapC in NAP in these species and in S. oneidensis MR-1, which also lacks NapC, may be met by the periplasmic c-type cytochrome homologue CymA, which is also from the NapC/NirT family (Gao et al., 2009;Murphy & Saltikov, 2007;Myers & Myers, 1997, 2000;Schwalb et al., 2003).The napCMADGH operon of the d-proteobacterium Desulfovibrio desulfuricans lacks the napB gene (Marietou et al., 2005).Without the napB gene, NAP from D. desulfuricans is expressed as a catalytic monomer, NapA (Dias et al., 1999;Najmudin et al., 2008).
The first X-ray crystal structure of the NapA subunit from D. desulfuricans ATCC 27774 was reported 10 years ago (Dias et al., 1999) and has recently been revised to include a sulphur atom in the Mo coordination sphere (Najmudin et al., 2008).The structures of monomeric NapA have been described from E. coli (Jepson et al., 2007) and of the NapAB heterodimer from Rhodobacter sphaeroides (Arnoux et al., 2003).In addition to the Mo-bis-MGD chelates and combinations of oxo (O 22 , OH 2 /H 2 O)-and/or sulfido (S 22 , SH 2 )-based ligands, the coordination sphere of each mononuclear Mo enzyme of the DMSO reductase family is completed by donor atoms derived from serine [TMAO reductase and DMSO reductase (Czjzek et al., 1998;Li et al., 2000;McAlpine et al., 1998)] or cysteine [NAP (Arnoux et al., 2003;Dias et al., 1999;Jepson et al., 2007;Najmudin et al., 2008)].
This review focuses upon the enzymes involved in the reductive respiratory reactions of the N-cycle in Shewanella, with a specific focus upon the periplasmic nitrate reductase (NAP).The metabolism of DMSO by DMSO reductase appears to be quite variable in Shewanella, as reflected in the multiple types of DMSO reductase gene clusters present across this genus (Fredrickson et al., 2008;Gralnick et al., 2006).The variability in DMSO reductase gene clusters in Shewanella prompted us to review NAP in species of this genus, which has revealed that most, but not all, genomes encode two NAP isoforms.The presence of two nap operons in most genomes of Shewanella is an unusual feature of this genus.

Most Shewanella genomes encode two isoforms of NAP
A review of the genomes of 19 strains of Shewanella returned 35 proteins annotated as NapA and showed that there are two napA genes in the genomes of most of these strains (Table 1).The five cysteine residues in NapA that coordinate the two metal prosthetic groups (one to the Mo-bis-MGD group and four to the [4Fe24S] cluster) (Arnoux et al., 2003;Dias et al., 1999;Jepson et al., 2007;Najmudin et al., 2008) are conserved across all NapAs in Shewanella (Fig. 1).The nap genes in Shewanella are arranged in two different types of operons.The first type is a napEDABC gene arrangement, designated the nap-a operon; and the second type is a napDAGHB gene arrangement, designated the nap-b operon.The genomes of 16 of the Shewanella strains reviewed contain both the nap-a operon and the nap-b operon.The genome of Shewanella denitrificans OS217, which from experiment has been established as a vigorous denitrifier (Brettar et al., 2002), contains only the nap-a operon.The genomes of S. oneidensis MR-1, which has been established as a respiratory nitrate ammonifier (Cruz-Garcia et al., 2007;Myers & Myers, 1997;Schwalb et al., 2002), and S. halifaxensis HAW-EB4 (Zhao et al., 2006(Zhao et al., , 2008) ) contain only the nap-b operon.
There are 12 residues that are completely conserved and 6 residues that are conservatively substituted (same charge: K or R, E or D; or similar size: I or V, S or A) within NapA-a or within NapA-b in Shewanella and that are different between NapA-a and NapA-b (Table 2).These residues will be diagnostic for NapA-a or NapA-b in ongoing Shewanella sequencing projects, which include Shewanella benthica KT99, S. putrefaciens ML-S2, S. putrefaciens W3-6-1 and S. putrefaciens 200.
In addition to the a form of NapA-a, NapB-a and NapD-a, the nap-a operon encodes NapE and NapC, but not NapG or NapH.In Shewanella piezotolerans WP3 and S. sediminis HAW-EB3, the napE gene is absent from the nap-a operon.In addition to the b form of NapA-b, NapB-b and NapD-b, the nap-b operon encodes NapG and NapH, but not NapE or NapC.In S. baltica OS155, S. baltica OS185, S. baltica OS195, S. baltica OS223, S. frigidimarina, S. piezotolerans WP3 and S. woodyi, the napGH genes are absent from the nap-b operon.In S. frigidimarina, the napD-b gene is absent from the nap-b operon (Fig. 2).

Sequence identity of the subunits of NAP-a and NAP-b in Shewanella
The sequence identity of NapA-a among the NAP-aencoding Shewanella species (all 19 strains excluding S. oneidensis MR-1 and S. halifaxensis HAW-EB4) is 61.5 %.The sequence identity of NapA-b among the NAP-bencoding Shewanella species (all strains excluding S. denitrificans OS217) is 74.6 %.The sequence identity values for the other subunits of the NAP-a and NAP-b isoforms are given in Table 3.Based upon experimental work with S. denitrificans (NAP-a-based system) (Brettar et al., 2002) and S. oneidensis MR-1 (NAP-b-based-system) (Cruz-Garcia et al., 2007;Myers & Myers, 1997;Schwalb et al., 2002), both NAP isoforms must be catalytically active.It may be that in species of Shewanella that contain both NAP-a and NAP-b, the NAP isoforms are differentially regulated under different environmental conditions (pH, oxygen availability, quinol pools).There is no significant difference in the predicted pI values of any of the isoformspecific subunits (NapA, NapB, NapD) (Fig. 2).

Other species that encode more than one NAP isoform
A review of 165 nap-encoding prokaryotic genomes returned 184 proteins annotated as NapA.These 184 NapAs were present in 165 species across 53 genera (see Supplementary Table S1, available with the online version of this paper).Since there are more NapAs than species, the genomes of some bacterial species have more than one nap operon.The nap operons were mapped in the genomes of 136 species across 51 genera for the 155 cases in which the nap genes were contiguous.Apart from members of the genus Shewanella, which has been noted as having two nap operons (Stewart et al., 2009), only three other species were found with genomes with two nap operons: Azoarcus sp.
The reduction of nitrite to ammonium in the respiratory ammonification pathway is catalysed by a pentahaem nitrite reductase, NrfA.Of all the 19 Shewanella species, the vigorous denitrifier S. denitrificans is the single case in which the genome does not encode NrfA.This accords with the denitrifying phenotype of this strain and establishes the NAP-a system as functional in denitrification.In ten of the Shewanella species, there is more than one form of NrfA; these forms cluster into three groups: NrfA1, NrfA2 and NrfA3 (minor group: S. sediminis and S. *x5a where the protein pool is NAP-a and x5b where the protein pool is NAP-b (i.e.identity within each subunit isoform: intra-isoform subunit identity).The entries in the bottom row represent the identities among the entire set of NapA-a and NapA-b, NapB-a and NapB-b or NapD-a and NapD-b (inter-isoform subunit identity).
DThe N-terminal extensions of 8 or 18 residues in length from S. amazonensis or S. denitrificans, respectively, have been omitted.piezotolerans).All of the c-type haem binding sites in all of the NrfA proteins are conserved.The presence of multiple copies of genes for NrfA in a single species is an unusual feature.In addition to the catalytic NrfA subunit, NRF consists of a pentahaem cytochrome c (NrfB), an ironsulfur cluster protein on the periplasmic face of the membrane (NrfC) and a transmembrane domain (NrfD), which as the NrfABCD cluster is commonly found in c- proteobacteria (Clarke et al., 2007;Hussain et al., 1994;Simon et al., 2000).The NapC homologue, CymA, has been found to be essential in S. oneidensis MR-1 for the anaerobic reduction of nitrate, nitrite, Fe(III), Mn(IV), DMSO or V(V) (Carpentier et al., 2005;Gao et al., 2009;Myers & Myers, 1997, 2000;Schwalb et al., 2003).Putative CymA proteins with 68.1 % identity are encoded in the genomes of all Shewanella species that encode NAP-b.The genome of S. denitrificans does not encode CymA.
Shewanella halifaxensis HAW-EB4 produces nitrous oxide but not N 2 during anaerobic growth on nitrate-augmented media (Zhao et al., 2006(Zhao et al., , 2008)).The lack of N 2 production from N 2 O by S. halifaxensis HAW-EB4 is consistent with the absence of nosZ in the genome.The absence of nirK and nirS and the presence of norZ on the S. halifaxensis HAW-EB4 genome suggests that, similar to the majority of Shewanella species that are respiratory nitrate ammonifiers (Krause & Nealson, 1997), S. halifaxensis HAW-EB4 is likely to produce nitrous oxide as a product of NorZcatalysed NO detoxification.

Functional implications of two NAP isoforms in Shewanella
The bioinformatics review of the enzymes involved in the reductive respiratory N-cycle (NAP, NRF, NIR, NOR, NOS) in Shewanella shows that the S. oneidensis MR-1 genome encodes only NAP-b and NrfA1.Therefore, in S. oneidensis MR-1, NAP-b is functional in the catalysis of the first step of the respiratory nitrate ammonification pathway of the N-cycle; and, of the two forms of NRF (NrfA1 and NrfA2), the NrfA1 form is functional.This is supported by experiment, where it has been shown that: (i) the sole nitrate reductase of S. oneidensis MR-1 was NapA; (ii) S. oneidensis MR-1 did not catalyse any of the further steps of denitrification; and (iii) anaerobic cultures of S. oneidensis MR-1 performed respiratory nitrate ammonification (Cruz-Garcia et al., 2007).The genome of the respiratory nitrate ammonifier Wolinella succinogenes (Kern & Simon, 2008) has the nap-b operon, which supports the posited association between NAP-b and respiratory ammonification.The only species of Shewanella that does not encode either NAP-b or NRF is S. denitrificans, which is a vigorous denitrifier (Brettar et al., 2002).Therefore, in S. denitrificans, the NAP-a form of NAP is self-evidently functional in denitrification.
It is apparent, however, that not all Shewanella species that have a gene for NapA-a are capable of denitrification (defined here as the conversion of soluble nitrate or nitrite  to gaseous NO) owing to the absence of the NO-genic nitrite reductase NirK (Table 4).In some denitrifying bacteria such as Bradyrhizobium japonicum, NapA-a is also the sole respiratory nitrate reductase and so must again be driving denitrification (Delgado et al., 2003).However, in the paradigm denitrifying bacterium Paracoccus denitrificans, the reduction of nitrate for denitrification is driven by the membrane-bound nitrate reductase NarG.In P. denitrificans, the nap-a operon is also present, but this NAP-a system is synthesized under aerobic conditions.The nap-based electron-transfer system in P. denitrificans, which involves ubiquinol (UQH 2 ) and NapC, plays a role in cellular redox balancing, dissipating the free energy available in the UQH 2 -nitrate redox couple rather than conserving it as proton-motive force (Ellington et al., 2006;Richardson et al., 2001).Thus in different genera of bacteria NapA-a has two distinct physiological roles (denitrification or redox balancing) and it seems probable that it may play both of these roles in the genus Shewanella (Fig. 3).Genes that encode the NAR protein subunits, NarG, NarH and NarI, are present in the genomes of S.
Most Shewanella species are facultative anaerobes, which suggests that oxygen levels could play a role in the differential regulation of metabolic pathways, including nitrate reduction.Studies of the oxygen-dependent regulation of quinone biosynthesis in Shewanella show that most, but not all, species produce ubiquinones and menaquinones.The available quinone pool may in turn direct the regulation of the electron transfer from quinol to the NAP isoforms (Brondijk et al., 2002(Brondijk et al., , 2004;;Potter et al., 2001;Potter & Cole, 1999).The transcriptional regulators that mediate global gene expression between anaerobic and aerobic growth might also play a role in the regulation of the expression of the NAP isoforms in Shewanella.

Quinones and transcriptional activators in Shewanella
The NAP-a and NAP-b isoforms differ in the composition of transmembrane subunits, with NAP-a (NapEDABC) possessing the tetrahaem cytochrome-c NapC and NAP-b (NapDAGBH) possessing the iron-sulfur cluster ferredoxins, NapGH.The functional differences between the cytochrome c-and iron-sulfur-based transmembrane subunits, and the interactions of these subunits with different membrane quinol pools (ubiquinol and menaquinol), may be important to the putative functional difference between NAP-a and NAP-b in Shewanella.In aerobic cultures of Shewanella, ubiquinols (ubiquinol 7 and 8) are present in higher concentrations than menaquinols (menaquinol 7 and methylmenaquinol 7) (Akagawa-Matsushita et al., 1992;Nishijima et al., 1997;Venkateswaran et al., 1999), which accords with the classification of ubiquinol as the quinol (Soballe & Poole, 1999).Anaerobic cultures of S. oneidensis MR-1 produce four to fivefold more menaquinols than ubiquinols (Myers & Myers, 2000).In E. coli, it has been established that irrespective of the quinol pool (ubiquinol or menaquinol), NapC is essential for NAP activity (Brondijk et al., 2002;Potter et al., 2001;Potter & Cole, 1999).The role of the NapGH complex was found to be dependent upon the quinol source (Brondijk et al., 2002(Brondijk et al., , 2004)).Where ubiquinol was the sole quinol source, NapGH was essential as an ubiquinol dehydrogenase that mediated electron transfer between NapC and NapAB; this NapGH-based mediation was not essential in E. coli constructs where menaquinol and ubiquinol were available (Brondijk et al., 2002(Brondijk et al., , 2004)).Therefore, it may be that the activity of the NapC-based complex (NAP-a) or the NapGH-based complex (NAP-b) in Shewanella is directed by the composition of the available quinol pool.Biochemical studies using Shewanella species that encode NAP-b without NapGH (Table 1, Fig. 2) will be useful to better understand the electron-transfer role played by the NapGH complex in NAP-b.
The genes for menaquinone biosynthesis proteins, which include MenD (2-succinyl-5-enolpyruvyl-6-hydroxy-3cyclohexene-  (Meganathan, 2001).Therefore, at least for these denitrifying bacteria, there appears to be a positive relationship between the presence of the nap-a operon and ubiquinone biosynthetic gene clusters.Apart from S. denitrificans, the genomes of all other species of Shewanella examined contain both menaquinone and ubiquinone biosynthetic gene clusters, which suggests that for species which encode both NAP-a and NAP-b the activity of these enzymes may be differentially regulated by the ubiquinone or menaquinone pool, respectively.These notions accord with the renowned environmentally adaptable phenotype of Shewanella (Fredrickson et al., 2008;Hau & Gralnick, 2007).
The transcriptional activators oxygen-responsive fumarate nitrate regulator (Fnr) protein and cAMP receptor protein (Crp) may play a role in the regulation of the expression of NAP-a and NAP-b in Shewanella.In E. coli, transcription of the periplasmic napF operon is regulated under anaerobic and nitrate-limited conditions by Fnr (Stewart et al., 2009).Under energy-limited growth conditions, Crp has been found to stimulate napF operon expression in E. coli (Stewart et al., 2009), with Crp also implicated in catabolite repression and the regulation of anaerobic respiration in S. oneidensis MR-1 (Saffarini et al., 2003).Although the electron-transport regulator A (EtrA) identified in S. oneidensis MR-1 has 73.6 % sequence identity with Fnr (Saffarini & Nealson, 1993), the retained ability of a S. oneidensis MR-1 etrA knockout strain to grow on a wide variety of electron acceptors, including nitrate, showed that EtrA does not regulate anaerobic respiratory gene expression (Maier & Myers, 2001).To date, all of the studies on the transcriptional regulators EtrA and Crp in Shewanella have focused only upon S. oneidensis MR-1 (Beliaev et al., 2002;Maier & Myers, 2001;Saffarini & Nealson, 1993;Saffarini et al., 2003;Stewart et al., 2009), which encodes only NAP-b.Future studies of the roles of the transcriptional activators in NAP regulation in strains of Shewanella that encode both NAP-a and NAP-b will be useful.

Concluding remarks
The genomes of most species of Shewanella contain two different nap operons that have been designated nap-a (napEDABC) and nap-b (napDAGHB).This is an unusual feature of this genus.The genome of the denitrifier S. denitrificans contains only the nap-a operon and the genome of the respiratory ammonifier S. oneidensis MR-1 contains only the nap-b operon.This raises the possibility that in Shewanella species that contain both the nap-a and nap-b operons the activity of NAP-a and NAP-b might be differentially regulated by oxygen levels and/or quinone pools and could be associated with physiologically distinct processes, including respiratory ammonification (species that encode NAP-b), denitrification (species that co-encode NAP-a and NirK) and redox balancing (species that encode NAP-a but not NirK).Transcriptomics and proteomics studies on S. oneidensis MR-1 and S. denitrificans will be useful, given the presence of the distinct nap operons on these respective genomes.Ultimately, a programme of Shewanella knockouts and the construction of NAP-a and NAP-b chimeras will be required to better understand the physiological roles of the NAP isoforms in this genus.
BH72 (nap-a and nap-b), Photobacterium profundum SS9 (two forms of nap-a) and Rhodobacter sphaeroides ATCC 17025 (nap-a and nap-b).The presence of two nap operons on the genome of Azoarcus sp.BH72 has been noted previously(Krause et al., 2006).Therefore: (i) it is unusual for a bacterial genome to have more than one nap operon; and (ii) the presence of two nap operons in the genomes of most Shewanella species (observed in 16 of the 19 strains examined) is an unusual feature of this genus.The 16 genomes surveyed of Escherichia species have only the nap-

Fig. 1 .
Fig. 1.Partial sequence alignment of NapA-a (e.g.Sama_a) and NapA-b (e.g.Sama_b) from selected Shewanella species.The cysteine residues bound to the [4Fe-4S] cluster or the Mo ion in NapA as characterized by X-ray crystallography(Arnoux et al., 2003;Dias et al., 1999;Jepson et al., 2007;Najmudin et al., 2008) are denoted by * or 3, respectively.The 4 symbol denotes residues that are conserved within the NapA-a isoform and which are different from residues in the Nap-b isoform.

Fig. 2 .
Fig. 2. The resolution, distribution and average predicted pI values of NAP-a and NAP-b across the genus Shewanella.

Fig. 3 .
Fig. 3. Gram-negative prokaryotic nitrate reductase enzymes (assimilatory nitrate reductase, NAS; and periplasmic nitrate reductase, NAP) and the enzymes involved in the downstream assimilation of nitrite or in the respiratory processing of nitrite to ammonium (respiratory ammonification) or nitrogen (denitrification) as adapted from the literature (solid lines) (Gonza ´lez et al., 2006; Kroneck & Abt, 2002; Richardson et al., 2001).In Shewanella, two isoforms of NAP (NapEDABC, NAP-a; and NapDAGHB, NAP-b) are encoded in the genomes of most species.The genome of the denitrifier S. denitrificans encodes only NAP-a and the genome of the respiratory ammonifier S. oneidensis MR-1 encodes only NAP-b.This raises the possibility that NAP-a and NAP-b are associated with physiologically distinct processes in Shewanella (broken lines), including respiratory ammonification (species that encode NAP-b), denitrification (species that co-encode NAP-a and NirK) and redox balancing (species that encode NAP-a but not NirK).

Table 2 .
Residues conserved within NapA-a or NapA-b that differ between NapA-a and NapA-b in Shewanella species *Coordinate numbering according to the overall alignment of NapA-a and NapA-b across Shewanella (Supplementary Fig.S1).

Table 3 .
Sequence identity values of the subunits (NapA, NapB, NapD, NapC, NapE, NapG, NapH) of the NAP-a and NAP-b isoforms in Shewanella NA, Not applicable.The nap-a operon does not encode NapG or NapH.The nap-b operon does not encode NapC or NapE.