Whole-genome analysis of the ammonia-oxidizingbacterium, Nitrosomonas eutropha C91:implications for niche adaptation

Lisa Y. Stein,1* Daniel J. Arp,2 Paul M. Berube,3

Patrick S. G. Chain,4,5,6 Loren Hauser,7

Mike S. M. Jetten,8 Martin G. Klotz,9

Frank W. Larimer,7 Jeanette M. Norton,10

Huub J. M. Op den Camp,8 Maria Shin4,5 andXueming Wei21Department of Environmental Sciences, University ofCalifornia, Riverside, CA 92521, USA.2Department of Botany and Plant Pathology, OregonState University, Corvallis, OR 97331, USA.3Department of Microbiology, University of Washington,Seattle, WA 98195, USA.4Chemistry, Materials, and Life Sciences Directorate,Lawrence Livermore National Laboratory, Livermore,CA 94550, USA.5Microbial Program, Joint Genome Institute, WalnutCreek, CA 94598, USA.6Department of Microbiology and Molecular Genetics,Michigan State University, East Lansing, MI 48824,USA.7Oak Ridge National Laboratory, Oak Ridge, TN 37831,USA.8Department of Microbiology, Institute for Water andWetland Research (IWWR), Radboud UniversityNijmegen, Toernooiveld 1, 6225 ED Nijmegen, theNetherlands.9Departments of Biology and Microbiology andImmunology, University of Louisville, Louisville,KY 40292, USA.10Department of Plants, Soils, and Climate, Utah StateUniversity, Logan, UT 84322, USA.

Summary

Analysis of the structure and inventory of the genomeof Nitrosomonas eutropha C91 revealed distinctivefeatures that may explain the adaptation ofN. eutropha-like bacteria to N-saturated ecosystems.Multiple gene-shuffling events are apparent, includ-ing mobilized and replicated transposition, as well as

plasmid or phage integration events into the 2.66 Mbpchromosome and two plasmids (65 and 56 kbp) ofN. eutropha C91. A 117 kbp genomic island encodesmultiple genes for heavy metal resistance, includingclusters for copper and mercury transport, which areabsent from the genomes of other ammonia-oxidizingbacteria (AOB). Whereas the sequences of the twoammonia monooxygenase and three hydroxylamineoxidoreductase gene clusters in N. eutropha C91 arehighly similar to those of Nitrosomonas europaeaATCC 19718, a break of synteny in the regions flank-ing these clusters in each genome is evident.Nitrosomonas eutropha C91 encodes four gene clus-ters for distinct classes of haem-copper oxidases,two of which are not found in other aerobic AOB. Thisdiversity of terminal oxidases may explain the adap-tation of N. eutropha to environments with variable O2

concentrations and/or high concentrations of nitro-gen oxides. As with N. europaea, the N. eutrophagenome lacks genes for urease metabolism, likelydisadvantaging nitrosomonads in low-nitrogen oracidic ecosystems. Taken together, this analysisrevealed significant genomic variation betweenN. eutropha C91 and other AOB, even the closelyrelated N. europaea, and several distinctive proper-ties of the N. eutropha genome that are supportive ofniche specialization.

Introduction

Nitrification, the oxidation of ammonia to nitrate, is anessential process as the activities of nitrifying bacteriaprovide oxidized inorganic nitrogen compounds – nitrite,nitrate, nitric oxide and nitrous oxide – to the environment(Wood, 1986; Arp and Stein, 2003). The aerobic oxidationof ammonia to nitrite by ammonia-oxidizing bacteria(AOB), and of nitrite to nitrate by nitrite-oxidizing bacteria(NOB), are the two steps of nitrification that support chemo-lithotrophic growth of a limited number of bacterial genera(Purkhold et al., 2000). In addition, the isolation of amarine Crenarchaeon (Candidatus ‘Nitrosopumilus mar-itimus’) that grows chemolithotrophically on ammonia(Könneke et al., 2005), and the detection of crenarchaealammonia monooxygenase genes and transcripts in soil

Received 26 April, 2007; accepted 2 July, 2007. *Forcorrespondence. E-mail lisa.stein@ucr.edu; Tel. (+1) 951 827 2704;Fax (+1) 951 827 3993.

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(Treusch et al., 2005; Leininger et al., 2006), estuarine(Beman and Francis, 2006) and marine (Wuchter et al.,2006) environments, has potentially expanded the diver-sity of microorganisms that aerobically oxidize ammoniafor the generation of energy and reductant. So far, allcharacterized aerobic AOB are phylogenetically affiliatedwith the Nitrosomonas (including Nitrosococcus mobilis)and Nitrosospira genera of the Betaproteobacteria as wellas the Nitrosococcus genus of the Gammaproteobacteria(Purkhold et al., 2000). Anaerobic ammonia oxidation(anammox) is also an ecologically significant process,particularly in anoxic marine and freshwater ecosystems(Dalsgaard et al., 2005; Penton et al., 2006). In contrast tothe aerobic AOB, anammox bacteria such as Kueneniastuttgartiensis belong to the Planctomycetales order andhave unique enzymes and cellular structures that allowthem to couple the anaerobic oxidation of ammonia to thereduction of nitrite to produce N2 (Strous and Jetten, 2004;Strous et al., 2006). Anammox bacteria are also able tomediate dissimilatory reduction of nitrate to ammonia,which may account for significant depletion of nitrate inanoxic environments (Kartal et al., 2007).

Several Nitrosomonas isolates, most notablyN. eutropha N904, can also oxidize ammonia anaerobi-cally using NO2 (which is in equilibrium with N2O4) as anoxidant while simultaneously reducing nitrite to N2O andN2 (Schmidt and Bock, 1997). The demonstrated useof N2O4 as an alternative oxidant to O2 established the‘NOx cycle’ model for aerobic and anaerobic ammonia-oxidizing metabolism of Nitrosomonas spp. (Schmidtet al., 2001). Schmidt and colleagues (2001) hypoth-esized that ammonia monooxygenase (AMO) reducesN2O4 to an NO intermediate that is either re-oxidized toNO2 (and N2O4) under aerobic conditions or released fromthe cell under anaerobic conditions. In addition to anaero-bic ammonia oxidation, N. eutropha N904, N. europaeastrain ‘Freitag’ and N. europaea ATCC 19718 can alsogrow anaerobically by so-called ‘nitrifier denitrification’ incontinuous cultures on pyruvate, H2 and nitrite; the onlyammonia-free, growth-supporting metabolism demon-strated thus far for aerobic AOB (Bock, 1995; Schmidtet al., 2004). Intriguingly, no orthologues to hydrogenasegenes were identified in the N. europaea ATCC 19718genome (Chain et al., 2003); hence, the enzymology ofthis pathway has yet to be ascertained. In addition toanaerobic metabolism of AOB, some NOB of the Nitro-bacter genus can grow by reducing nitrate to nitrite at theexpense of organic carbon (Belser, 1977; Starkenburget al., 2006). Thus, the nitrifying bacteria as a grouppossess some metabolic versatility.

Environmentally derived 16S rRNA gene sequencesrelated to N. eutropha and N. europaea cluster withinthe taxonomic group ‘Nitrosomonas cluster 7’ (Stephenet al., 1996), also called the ‘Nitrosomonas europaea/

Nitrosococcus mobilis lineage’ (Purkhold et al., 2000).16S rRNA gene sequences with similarity to Nitrosomo-nas cluster 7 are frequently identified in sewage treatmentplants and in soils with high ammonia content, but havealso been found in estuarine sediments and hypersalinelakes (Stehr et al., 1995; Kowalchuk et al., 2000; Wardet al., 2000; Oved et al., 2001; Carnol et al., 2002;Webster et al., 2002; Mortimer et al., 2004; Koops et al.,2007). Close inspection of gene sequences from all thesestudies revealed that most AOB within Nitrosomonascluster 7 isolated from moderate to low N-load environ-ments tend to group closer with N. europaea-like speciesthan with N. eutropha-like species, suggesting thatN. eutropha-like species tend to be better adapted to loca-tions with very high N-loads. Indeed, N. eutropha spp.tolerate a maximum of 600 mM ammonium, whereasN. europaea spp. tolerate only up to 400 mM ammonium(Koops et al., 2007). Both N. europaea and N. eutrophaare halotolerant, surviving in up to 400 mM NaCl, andboth lack urease activity (Koops et al., 2007). UnlikeN. europaea, N. eutropha contains carboxysomes (Koopset al., 2007). On the other hand, N. europaea ATCC19718 can be grown chemolithoheterotrophically usingammonia as an energy source and fructose as a carbonsource (Hommes et al., 2003); this physiology has not yetbeen demonstrated for N. eutropha.

The type strain N. eutropha C91, also known as Nm57and formerly as ATCC 25984, was isolated by S.W.Watson from a sewage disposal plant in Chicago, IL, USA(Watson and Mandel, 1971; Koops et al., 1991). Thecurrent study describes the structure and contents of theN. eutropha C91 genome sequence and its comparisonwith completed genome sequences of the aerobic AOBN. europaea ATCC 19718 (Chain et al., 2003), Nitroso-coccus oceani ATCC 19707 (Klotz et al., 2006) and Nitro-sospira multiformis ATCC 25196 (J.M. Norton, D.J. Arp,P.J. Bottomley, P.S.G. Chain, L.J. Hauser, M.G. Klotz,M.L. Land, F.W. Larimer, M.W. Shin, S.R. Starkenburgand L.Y. Stein, unpublished); the anammox bacterium,K. stuttgartiensis (Strous et al., 2006); and, where rel-evant, the NOB Nitrobacter winogradskyi Nb-255(Starkenburg et al., 2006) and Nitrobacter hamburgensisX14 (S.R. Starkenburg, P.S.G. Chain, L.J. Hauser, M.G.Klotz, F.W. Larimer, L. Sayavedra-Soto, M.W. Shin, L.Y.Stein and B.B. Ward, unpublished).

Results and discussion

Genome structure, general characteristics andidentification of regions with distinctive properties

The N. eutropha C91 genome is composed of a singlecircular chromosome (2 661 057 bp, 48.5% G + Ccontent) and two large plasmids, pNeutP1 (65 132 bp,49.8% G + C) and pNeutP2 (55 635 bp, 49.7% G + C)

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(Table 1). The two plasmids each contain an identical11 414 bp (56.1% G + C) transposon (Tn_Neut) flankedby short inverted repeats (Table 2). This element appearsto have integrated into, and likely inactivated, the TrbG(VirB9) gene (Neut_2548/41) in pNeutP1. PlasmidpNeutP1 also contains a putative aldehyde dehydroge-nase gene (Neut_2587) and an additional transposon

(Tn_mer) of 7 951 bp (61.24% G + C and flanked by a39 bp inverted repeat), which is predicted to have dis-rupted a type III restriction helicase (Neut_2572/61). Thissecond large transposon carries a conserved Gram-negative mercury-resistance operon (Barkay et al., 2003),encoded by merTPCADE with the regulatory merR geneencoded divergently upstream of the operon, while three

Table 1. Summary of N. eutropha C91 genomecontents.Total bases in genome 2 781 824

Bases in chromosome: total/coding 2 661 057/2 279 595Bases in plasmid pNeutP1: total/coding 65 132/57 768Bases in plasmid pNeutP2: total/coding 55 635/45 285Bases in mobile elementsa 63 453GC content (%): chromosome/pNeutP1/pNeutP2 48.49/49.8/49.68Coding density (%): chromosome/pNeutP1/pNeutP2 85.6/88.6/81.3

Total number of CDSs 2 687Protein coding: chromosome/pNeutP1/pNeutP2 2 443/55/52With predicted function: chromosome/pNeutP1/pNeutP2 2 001/44/28Without predicted functionb 442/11/245S/16S/23S 1 eachtRNA 41Miscellaneous RNA 3Pseudogenes: chromosome/pNeutP1/pNeutP2 90/1/1

a. Transposon, phage and plasmid components.b. Proteins of unknown function, conserved hypothetical and hypothetical proteins.

Table 2. Summary of complex repeat sequences in N. eutropha C91 genome sequence.

Name # CopiesSize(bp) CDSs % ID Details

amo 2 4 899 AMO > 99 amoCAB with ORF4,5 (amo1: Neut_2078-74; amo2:Neut_2319-15)

hao 3 4 452 HAO > 98 two copies with both c-type cytochromes; one lacks the lastCDS and is shorter at 3610 bp (hao1: Neut_1672-70; hao2:2235-32; hao3: Neut_1793-90)

ISNeut1 22 (1) 984 1 transposase/integrase gene > 99 (1) truncated

ISNeut2 11 (2) 850 2 transposase genes � 99 (2) at > 96%

ISNeut3 11 (1) 1 310 2 transposase genes � 99 (1) truncated fragment flanked by ISNeut2 and ISNeut6

ISNeut4 8 (6) 999 2 transposase genes > 96 (6) truncated degrading fragments (3 each of 5′ and3′ fragments), 1 interrupted by a truncated ISNeut7,3 others interrupted by another ISNeut4 or undergoinginternal deletions

ISNeut5 3 (1) 1 222 1 transposase, mutator type > 99 (1) interrupted by another IS element (not repetitive)

ISNeut6 3 (1) 1 130 2 transposase genes > 99 (1) degrading (> 80%) truncated fragment flanked by ISNeut3and another IS element (not repetitive)

ISNeut7 1 (1)+ 1 450 1 or 2 transposase genes(degradation observed)

> 98 (1) interrupted by two other IS elements (1 ISNeut1, othernot repetitive); an additional (> 98%) short degradingfragment is flanked by ISNeut1 and by a truncated anddegrading ISNeut4

REPNeut1 2 950 3 genes of unknown functions 100 flank a region (Neut_2352-57) not found in N. europaea andof low G + C

REPNeut2 2 1 332 1 phage integrase gene > 99 both adjacent to a Ser-tRNA

REPNeut3 2 12 167 plasmid functions andphage integrase

100 Both copies on chromosome; separated by 1.69 Mbp(Neut 0665-0679; 2266-2252)

Tn_mer 2 7 951 mercury-resistance operonand transposase

100 1 chromosomal copy (Neut_1305-14), 1 copy on pNeutP1(Neut_2562-71); (Fig. 1A)

Tn_Neut 2 11 414 recombinase, integrase, typeII sec-pathway ATPase,UvrD-helicase, adeninemethylase hypothetical protein

100 plasmids only (Neut_2603-08; 2542-47)

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genes downstream of the mer operon encode the trans-position functions (a TnpM transposition modulator/regulator, a resolvase and a TnpA-like transposase)(Fig. 1A; Table 2). The nucleotide sequence of the entiretransposon is 100% identical to a chromosomal copy.Interestingly, a second highly similar chromosomal copyof the mer operon (e.g. a 1210 bp spanning the merRTPregion is 90.3% identical to the other two copies) is notadjacent to any transposase-related genes (Fig. 1A).

Similar to the chromosome of N. europaea, N. eutrophaalso harbours several (at least seven) families of insertionsequence (IS) elements, repeated up to 22 times withinthe chromosome (Table 2). While two of these families arehighly similar to IS elements found in N. europaea, four ofthe families are not present at all in N. europaea and thusconstitute recent integration followed by replicative trans-position, or numerous independent acquisitions. Two

additional 950 bp duplicated elements (REPNeut1) flanka 5732 bp, six-gene region (Table 2). The G + C content ofthe entire region flanked by REPNeut1 elements is37.6%, far lower than the chromosomal average, andnone of the genes have homologues in N. europaea, sug-gesting that this region may have been acquired recentlyby N. eutropha. Two nearly identical copies of a phageintegrase (Neut_1125/1755) and its promoter region(REPNeut2) lie directly downstream of two Ser-tRNAs.Incidentally, each also lies upstream of another tRNA witha copy of ISNeut4 and a number of conserved hypotheti-cal coding sequences (CDSs) in between.

In addition to these repeated elements, the N. eutrophachromosome also contains two identical copies of a large12 167 bp DNA fragment (REPNeut3; 57.4% G + C;Table S1) separated from one another by 1.69 Mbp.Unlike the transposons, this repeat encodes several

Fig. 1. Comparison of conserved geneclusters for (A) mercury resistance and (B)copper tolerance in the N. europaea ATCC19718 and N. eutropha C91 genomesequences. Black arrows represent CDSsfound only in the N. eutropha genomesequence. Percentages of shared amino-acidsequence identities to similar sequences inN. europaea are indicated in bold;percentages of shared amino-acid sequencesidentities to similar sequences within thechromosomal/plasmid mer gene cluster ofN. eutropha are also indicated (plain text) forcopy 2 of the chromosomal mer gene cluster.

N. eutropha – chromosomal copy 2

Neut0034 0033 0032 0031 0030 0029 0028

86% 82% 85% NP 81% 54% 85%89% 88% 95% merF 82% 63% 92%

A. Conserved mer gene clusters

B. Conserved copper tolerance gene clusters

N. eutrophacopA copB copC

Neut0021 0022 0023

N. eutropha – chromosomal copy 1 and plasmid copy

Neut1305NeutP1_2571

1306 1307 1308 1309 1310 1311 1312 1313 13142570 2569 2568 2567 2566 2565 2564 2563 2562

91% 93% 85% 78% 90% 85% 89% 72% 84% 87%

N. europaea

NE0843 0842 0841 0840 0839 0838 2575 0837 0836 0835

merR merT merP merC merA merD merE TnpM TnpR TnpA

N. europaeapcoA pcoB

NE0279 0280

N. eutropha83% 83%

Neut0311 0312

NE2064 2063 2062 2061 2060 2059 2058 2057

amoC amoA amoB ORF4 ORF5 copC copDN. europaea

97% 97% 91% 77% 75% 63% 71% 54%

Neut2319 2318 2317 2316 2315 2314 2313 2312

N. eutropha

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plasmid-related (replication proteins A and C, stabilizationprotein StbC, several conjugal transfer proteins) andphage-related (integrase, AlpA-like phage transcriptionalregulator) proteins and may thus have originated from aplasmid or a phage. In addition, nine of the genes in thisrepeat share no significant similarity with genes in thegenomes of other AOB.

The beginning of the N. eutropha chromosome containsa ca. 117 kbp region (Neut_0006-0126) with features of agenomic island (Dobrindt et al., 2004): its G + C contentdiffers significantly (56.1%) from the rest of the genome(Fig. 2A), it is flanked by tRNA genes (tRNA-Gly at bp6613, tRNA-Cys at bp 123 441), it begins with a 12 bpdirect repeat sequence (two copies) preceding a phage-related integrase (Neut_0006), and the contents are richin CDSs conferring resistance to heavy metals (Table S2),especially copper and mercury (25/126 CDSs including

the divergent copy of the mer operon shown in Fig. 1A),as well as an ISNeut2 element, and several conservedhypotheticals. Of the 126 CDSs within this region, 64(51%) have no homologues within the other AOBgenomes. Notably, this region also contains a secondcomplete cluster of ccm genes, the products of whichtransport the haem prosthetic group into the periplasmand attach it to apocytochrome c (Table S2).

Taken together, these genomic features suggest thatmultiple insertion, duplication and rearrangement events,propagated at least in part by bacteriophage and trans-position (IS and transposons) throughout the life history ofNitrosomonas spp., have helped shape the currentgenome structure of both N. eutropha and N. europaea(Chain et al., 2003). Indeed, a comparison of genesynteny and G + C content between the genomesequences of N. eutropha and N. europaea shows the

X

A

B

N. europaea

N. eutropha

amo1 hao2 hao1 amo2 hao3

hao1 hao3 amo1 amo2 hao2

NE0940 0946-58 0963-70 // 2040/41 2045-52 // 2058/59 // 2318-35 2340comEA pheST (0953/54) trmU (0963) rhlE/orfM rpoBC (2045/46) copCD lepAB (2334/35) rpsT

rhlE/orfM rpsT lepAB (1789/90) rpoBC (1794/95) TSehp DCpocAEmoc (2326/27) trmU (2336)Neut_1668/69 1673 // 1772-90 1794-1802 // 2073 // 2313/14 2320 -31 2336-43

X

hao3amo2hao2amo1 hao1

hao2amo2hao3 amo1hao1 X X

XXX

X

XXXX

XXXX X

X

Fig. 2. A. Comparison between genome sequences of N. europaea ATCC19718 (top) and N. eutropha C91 (bottom) using the ArtemisComparison Tool (Carver et al., 2005). Red and blue lines (blocks) define regions of similarity; red for co-linear, blue for inverted with respectto one another. G + C graphs with a window size of 10 000 bp are shown outside the alignment window. Gene clusters for amo and hao areindicated for both genomes.B. Organization of the amo and hao gene clusters and their environments. Conserved neighbouring genes in syntenous regions are indicatedby arrowheads, with ‘X’ indicating the break point in synteny between genomes. Colours indicate conserved cluster neighbourhoods.

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significant extent of structural rearrangement, not neces-sarily associated with the many repetitive sequences, thathas occurred since the divergence of these closely relatedspecies, leading to a non-syntenous arrangement of theirhighly similar genome inventories (Fig. 2).

Catabolic gene inventory

Nitrosomonas eutropha C91, like N. europaea ATCC19718, contains two and three copies of the gene clustersthat encode functional AMO (amoCAB) and hydroxy-lamine oxidoreductase (HAO; hao) and associated cyto-chromes respectively (Norton et al., 2002). Both amoCABclusters are succeeded by a tandem of conserved genes,ORF4 and ORF5, which has only been found in thegenome sequences of betaproteobacterial AOB (Beta-AOB). A homologue of ORF5, but not ORF4, is alsopresent in the genomes of N. oceani and the methan-otroph, Methylococcus capsulatus Bath (Ward et al.,2004; Klotz et al., 2006).

The amo1 gene cluster in N. europaea and the amo2

gene cluster of N. eutropha are flanked at their 5′ endswith conserved sets of 12 genes, which is interrupted by amerR homologue in N. europaea (Fig. 2B). These sets ofconserved genes are followed in both genomes by thehao2 gene clusters (hao-orf2-cycAB), with further extend-ing synteny (Fig. 2B). In contrast, the upstream flankingregions of the amo2 gene cluster of N. europaea and theamo1 gene cluster of N. eutropha are not conserved(Fig. 2B). However, the 3′ flanks of each amo gene clusterbetween N. europaea and N. eutropha are conserved: theamo2 clusters of both genomes are succeeded by atandem that encodes copper binding/resistance proteinsCopCD (Figs 1 and 2B), whereas the DNA sequencedownstream of the amo1 clusters are non-identical asidefrom a single comEA-like gene whose product is impli-cated in DNA processing (Fig. 2B). Hence, these cataboli-cally crucial regions in both nitrosomonad genomes haveonly partial synteny. Because the break of synteny occursbetween the up- and downstream regions of the amogene clusters and the genes within the clusters are nearlyidentical (Norton et al., 2002), this break in synteny couldbe the consequence of the rectification process respon-sible for maintaining sequence identity of the multiplegene clusters (Klotz and Norton, 1998).

Whereas the hao2 gene clusters in both nitrosomonadgenomes are in close proximity to amo gene clusters, asdescribed above, the hao1 and the hao3 gene clustershave conserved 3′ but variable 5′ flanking sequences(Fig. 2B). These 5′ flanking regions have likely beenswapped in one of the two nitrosomonad genomesequences. As with the amo gene clusters, the break insynteny for the hao gene clusters occurs between the up-and downstream regions of the nearly identical cluster

copies, which is likely also the result of the rectificationprocess. It is still unclear how the rectification mechanismoccurs as an ongoing process in the nitrosomonads,including which of the multiple gene cluster copies servesas the template for sequence correction. Thus, compara-tive analysis of these regions in additional genomes fromclosely related Beta-AOB will be necessary to resolve thisquestion. In addition to the break in synteny, the hao1

gene clusters in genomes of both N. europaea andN. eutropha lack the cycB gene. It is most likely that theabsence of cycB is due to a deletion that occurred in thecommon ancestor of the N. europaea/N. eutropha lineage(Arp et al., 2007), as all three hao gene clusters arecomplete in the genome sequence of N. multiformis(J.M. Norton et al., unpublished), a representative of theother genus within the Beta-AOB.

Based on the analysis of other AOB genome sequences(Chain et al., 2003; Klotz et al., 2006) and the comparisonof individual AOB gene inventories (Norton et al., 2002;Bergmann et al., 2005; Elmore et al., 2007), the expectedinventory for extraction of energy and reductant fromammonia (amoCAB, hao, cycAB, ubiquinone), primaryelectron flow (cytochrome c552, cytochrome bc1, cyto-chrome c and quinol oxidases, and NADH-ubiquinoneoxidoreductase) and secondary electron flow (nirK, norB,cytochrome c peroxidase, cytochrome P460 and cyto-chrome c ′-beta), as well as ATP synthesis (F0F1-typeATPase), has been identified in the N. eutropha genome.Despite its reported sodium tolerance, genes encodingNa+-NDH and Na+-dependent V-type ATPases were notidentified in the N. eutropha genome but were found in thegenome of the Gamma-AOB, N. oceani, as an example ofniche adaptation to the marine environment (Klotz et al.,2006). The N. eutropha genome also did not reveal genesencoding candidate proteins that may facilitate recyclingof electrons from ubiquinone to AMO. However, the pres-ence of a homologue for nitrosocyanin (red copperprotein; Neut_2173) in the N. eutropha genome, and itsabsence in genomes of non-nitrifying bacteria, supportsthe earlier hypothesis that nitrosocyanin is AOB-specificand likely essential to the obligate ammonia lithotrophy ofAOB (Klotz et al., 2006).

Haem-copper oxidase gene clusters

Bacteria that experience fluctuating O2 levels tend tomaintain flexible respiratory chains by encoding multipleclasses of proton-pumping haem-copper oxidases(HCOs) in addition to different terminal reductases(Saraste and Castresana, 1994). Two types, the(ubi)quinol and cytochrome c oxidases, form the HCOsuperfamily of respiratory oxidases. Three classes ofcytochrome c oxidases have been described in bacteria:cytochrome aa3 (ba3) and cytochrome caa3 (cao3)-type

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oxidases generally function under high O2 tension,whereas cytochrome cbb3-type oxidase functions atreduced O2 tension (Garcia-Horsman et al., 1994; Pereiraet al., 2001; Pitcher and Watmough, 2004). In a compari-son of seven AOB and NOB genome sequences, onlyN. multiformis contained a complete gene cluster for cyto-chrome caa3; all the other genomes contain one to threecomplete gene clusters for cytochrome aa3 (Table 3).Interestingly, only N. eutropha contained a complete genecluster for cytochrome bo3-type quinol oxidase, a function-ally equivalent enzyme to cytochrome aa3 and cyto-chrome caa3 oxidases except for its respective electrondonor (Garcia-Horsman et al., 1994). It is not uncommonfor a single bacterium to encode both cytochrome coxidase and quinol oxidase gene clusters (Garcia-Horsman et al., 1994).

Perhaps more interestingly, of the seven comparedgenome sequences, only N. eutropha and K. stutt-gartiensis contained a gene cluster encoding at leastthree of the four subunits for cytochrome cbb3 oxidase(ccoNOP or fixNOP) (Table 3). The N. eutropha genomelacks a ccoQ/fixQ gene, similarly to that reported for other

bacteria (Pitcher and Watmough, 2004). Instead, thefixNOP gene cluster of N. eutropha is adjacent to a senCgene. In Rhodobacter capsulatus, the SenC protein isinvolved in maximizing expression and activity of cyto-chrome cbb3 oxidase during photosynthesis (Buggy andBauer, 1995; Swem et al., 2005). In-frame deletionmutants of fixNOQP genes showed that FixQ was notessential for cytochrome cbb3 oxidase assembly or activ-ity in Bradyrhizobium japonicum (Zufferey et al., 1996),suggesting that the gene cluster of N. eutropha may befunctional. On the other hand, the fixGHIS gene cluster,required to assemble a functional cytochrome cbb3

oxidase in most bacteria (Pitcher and Watmough, 2004),was not found in the N. eutropha genome. TheN. eutropha genome also contains the genes needed toexpress a functional cNOR-type nitric oxide reductase(norCBQD), of which the catalytic subunit, NorB, shares21% protein sequence identity with the FixN catalyticsubunit of the cytochrome cbb3 oxidase. Taken together,the cytochrome cbb3 oxidase gene cluster may be oneof the critical genome inventories that distinguishesN. eutropha and K. stutgartiensis from the other AOB and

Table 3. Classification of haem-copper respiratory oxidases (EC:1.9.3.1) in the genome sequences of ammonia- and nitrite-oxidizing bacteria.

Haem-copperoxidase type

Subunit composition

ICOG 0843

IICOG 1622

IIICOG 1845

IVCOG 3245/3175

SenCCOG1999

Cytochrome NmulA_0184 NmulA_0183 NmulA_0187 NmulA_0185 NPc oxidase caa3

(low-affinityO2 reductase)

NmulA_ 1776 NmulA_ 1777 NmulA_ 1775 NP NPNmulA_0459 NmulA_0460 NmulA_0458 NP NmulA_0454Noc_ 1766 Noc_ 1767 NP NP NP

Cytochromec oxidase aa3

(low-affinityO2 reductase)

Neut_2394 Neut_2395 Neut_2392 Neut_2393 NPNE1016 NE1017 NE1013 NE1015 NPNoc_1245 Noc_1244 Noc_1246 Noc_1247 NPNoc_3046 Noc_3047 Noc_3044 Noc_3045 NPNwin_0224 Nwin_0223 Nwin_0228 Nwin_0227 NPNwin_0762 Nwin_0761 Nwin_0766 Nwin_0765 NPNham_4179 Nham_4180 Nham_4178 Nham_4177 NPNham_0256 Nham_0255 Nham_0260 Nham_0259 NPNham_3452 NP NP Nham_3454 NPNham_3462 Nham_3463 Nham_3458 Nham_3459 NP

Cytochrome coxidase aa3

(NO reductase sNOR)

Neut_ 1875 Neut_ 1874 NP NP Neut_ 1876NE0683 NE0684 NP NP NE0682NmulA_2666 NmulA_2667 NP NP NmulA_2665Noc_2970 Noc_2971 NP NP Noc_2969Nham_2711 Nham_2712 NP NP Nham_2710

Quinol oxidasebo3 (low-affinityO2 reductase)

(COG3125)Neut_0696 Neut_0697 Neut_0695 Neut_0694 NP

Cytochrome coxidase cbb3 (high-affinity O2 reductase)

FixN/CcoN FixO/CcoO FixP/CcoP FixQ/CcoQNeut_1582 (COG3278) Neut_1583 (COG2993) Neut_1584 (COG2010) NP Neut_1585Kustc0429 (N-term) Kustc0429 (C-term) Kustc0427 (COG2863) NPa NP

a. Kustc0428 is in this gene cluster and encodes a 56-amino-acid protein with no significant sequence similarity to ccoQ.NP, not present; Nmul, Nitrosospira multiformis ATCC 25196; NOC, Nitrosococcus oceani ATCC 19707; Neut, Nitrosomonas eutropha C91; NE,Nitrosomonas europaea ATCC 19718; Nwin, Nitrobacter winogradskyi Nb-255; Nham, Nitrobacter hamburgensis X14; Kustc, Kueneniastuttgartiensis.

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NOB in their ability to metabolize high concentrations ofammonia and withstand high concentrations of nitrogenoxide intermediates produced under aerobic or anaerobicconditions (Schmidt et al., 2001; 2002). Furthermore,cytochrome cbb3 oxidase may confer a selective advan-tage to N. eutropha over other aerobic AOB in eutrophicsystems where competition for O2 with heterotrophicorganisms is acute.

Each nitrifier genome, except for that of N. wino-gradskyi, also contains an additional gene cluster forsubunits II and I (coxBA) of a cytochrome aa3-typeoxidase plus a gene encoding SenC (Table 3). As senCgenes are immediately adjacent to both the fixNOP andcoxBA gene clusters of N. eutropha, this suggests a sig-nificant role of SenC in the functionality of these cyto-chrome c oxidases. Conversely, a third homologue ofsenC (Neut_1987) in the N. eutropha genome is not in thevicinity of any oxidase gene. Expression of the coxBAportion of the coxBA-senC gene cluster (NE0684-83) wasupregulated in nutrient-starved (Wei et al., 2006a) andnitrite reductase-deficient (Cho et al., 2006) N. europaea,suggesting a role of this particular gene cluster in medi-ating stress responses, particularly to nitrosative stress.Thus, we hypothesize that the coxBA-senC gene clusters,found thus far only in aerobic AOB, N. hamburgensis, anda few other Proteobacteria (i.e. Azoarcus sp. EbN1, Geo-bacillus kaustophilus HTA426, Deinococcus geothermalisDSM 11300 and Silicibacter pomeroyi DSS-3), encode anovel subfamily of cytochrome c oxidases (sNOR) thatfunction as NO reductases to detoxify NOx intermediatesproduced in the periplasm during the metabolism of AOB(J. Hemp, M.G. Klotz, L.Y. Stein and R.B. Gennis,unpublished).

H2 oxidation

Nitrosomonas eutropha N904 was reported to grow withH2 as an electron donor at higher rates than those forN. europaea (Bock, 1995). However, genes with similarityto characterized hydrogenase genes were not identified ineither nitrosomonad genome sequence. Thus, the geneticbasis for H2 oxidation by N. europaea and N. eutropharemains enigmatic.

Denitrification genes

Aside from oxidation of ammonia to nitrite, aerobic AOBalso reduce nitrite to NO and N2O in a process known asnitrifier denitrification (Wrage et al., 2001). N2 has beenmeasured as an end-product of ammonia-oxidizingmetabolism of nitrosomonads (Poth, 1986; Shresthaet al., 2002; Schmidt et al., 2004), even though genesencoding nitrous oxide reductase were not found in any ofthe AOB genomes. Instead of the haem-dependent nitritereductase NirS (Zumft, 1997), most AOB encode ortho-

logues to the copper-containing nitrite reductase, nirK(Cantera and Stein, 2007). Interestingly, analysis of thediversity of nirK gene sequences among aerobic AOBsuggests origins from multiple evolutionary lineages andthe occurrence of numerous gene-transfer events(Cantera and Stein, 2007). The nirK gene encoded byN. eutropha (Neut_1403) is most similar to, and phyloge-netically clusters with, nirK of N. europaea (Cantera andStein, 2007). Both nitrosomonad nirK genes are at the 3′end of a four-member gene cluster that includes a multi-copper oxidase (ncgA; Neut_1406) and two cytochrome cproteins (ncgBC; Neut_1404-05) (Beaumont et al., 2005).The nirK gene cluster of both N. eutropha andN. europaea is preceded by a divergently encoded nsrRregulatory gene (Neut_1407), which flanks a conservedNsrR binding element in the promoter region of the nirKgene cluster (Cantera and Stein, 2007). NsrR was shownto regulate expression of the nirK gene cluster ofN. europaea in response to nitrite, and is thought to bepart of the mechanism that protects the bacterium fromnitrite toxicity (Beaumont et al., 2004). NsrR is alsoinvolved in regulating the nitrosative stress response ofenteric bacteria, which is stimulated by exposure to NO(Bodenmiller and Spiro, 2006). Surprisingly, the completenirK gene cluster of N. eutropha and N. europaea is con-served in the three genomes of the alphaproteobacterialNOB Nitrobacter isolates, whereas the nirK genes ofN. multiformis and N. oceani do not cluster with ncgABCorthologues (Klotz et al., 2006; Cantera and Stein, 2007).Because nitrosomonads and nitrobacters form coherentnitrifying granules in wastewater (Mobarry et al., 1996),horizontal transfer of the nirK gene cluster between thesetaxonomically disparate, but functionally cooperating,bacteria has been suggested (Cantera and Stein, 2007).

The gene cluster encoding a membrane-bound cNORnitric oxide reductase, norCBQD, has been identified inthe genomes of all aerobic AOB (Table 4). The expres-sion products of the norCBQD genes of N. eutropha(Neut_0518-0521) are most similar to their orthologuesin N. europaea (sharing 88%, 90%, 89% and 70%protein sequence identity respectively). To our surprise,the NorCBQD sequence identities between N. eutrophaand the Gamma-AOB, N. oceani were much higher(78%, 78%, 77% and 57%, respectively) than theNorCBQD sequence identities between N. eutropha andN. multiformis (37%, 41%, 56% and 32%, respectively),a representative of the second genus in the Beta-AOB.This suggests that the norCBQD genes in N. multiformishave likely been acquired by horizontal transfer andrecombinational substitution after divergence of the twogenera in the Nitrosomonadaceae family. NorCBQDsequence information from other nitrosospiras is neces-sary to delineate whether this replacement occurredearly or late during evolution of the Nitrosospira genus.

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All AOB, including N. eutropha, also contain cytochromeproteins that have recently been suggested to serve assoluble periplasmic nitric oxide reductases: members ofthe cytochrome P460/c′-beta family (Neut_0132, 1345)and cytochrome c554 (Neut_1670, 1791, 2333)(Upadhyay et al., 2006; Elmore et al., 2007). Similar toNorCBQD proteins, the cytochrome P460 protein ofN. eutropha shares 87% and 63% protein sequenceidentity to cytochrome P460 proteins of N. europaea andN. oceani, respectively, but only 26% sequence identityto the cytochrome P460 protein of N. multiformis.

Carbon fixation

All of the aerobic AOB fix CO2 via the Calvin–Benson–Bassham cycle using ribulose bisphosphate carboxylase/

oxygenase (RuBisCO). All four aerobic AOB specieshave one copy of the RuBisCO-encoding cbb operon(Fig. 3). Both N. eutropha and N. europaea have thegreen-like Form I RuBisCO, whereas Nitrosospira spp.generally have a red-like Form I RubisCO (Utåker et al.,2002). The large and small subunits of RuBisCO,encoded by cbbL and cbbS, of N. oceani are mostsimilar to that of N. multiformis (92% and 82% amino-acid sequence identity, respectively), suggesting thatN. oceani also has a red-like Form I RuBisCO (Klotzet al., 2006). The cbbQON genes of N. eutropha aremost similar to those in N. europaea (Chain et al., 2003).Both N. multiformis and N. oceani lack the cbbQONgenes, but instead encode cbbX like many photosyn-thetic bacteria such as Rhodobacter sphaeroides andSynechococcus sp. (Shively et al., 1998) (Fig. 3). All cbb

Table 4. Comparison of transporter and other selected CDS inventories among the genome sequences of N. eutropha C91, N. europaea,N. multiformis, N. oceani and K. stuttgartiensis.

Comparison strain(s)with N. eutropha C91

% of OrthogulousCDSsa Transporters Other CDSs

N. eutropha C91 19b arsenite efflux (2), ArsR regulatory proteins (2);Co/Zn/CD efflux cation-translocating ATPaseMarC-like multiple antibiotic transporter; ABCantimicrobial peptide/lipid efflux pump (2)

carboxysome shell proteins, cbb3 cytochromec oxidase, quinol oxidase, asparaginesynthases (Neut_1735/0910)

N. europaea 12 heavy metals, copper, iron, mercury, multidrug,ABC antimicrobial peptide, ABC nitrate/sulfate/bicarbonate, Na+/H+ antiporter, potassium

Pan1 multicopper oxidase with c-typecytochromes (ncgABC) of the nirKoperon, carbon fixation (cbbQON)

N. multiformis 1 ABC antimicrobial peptide (3), potassium (1) F1F0 ATP synthase (copy 2)

N. oceani 3 ABC phosphate, potassium, TRAP mannitol/chloroaromatic, TRAP dicarboxylate, NhaPantiporter, cyanate transporter

SufI multicopper oxidase, cytochrome cperoxidase, siderophore synthetase

N. europaea &N. multiformis

14 ABC organic solvents, ABC multidrug, ABCsulfate/molybdate ABC antimicrobial peptide,ABC bacteriocin/lantibiotic, ABC spermidine/putrescine, arabinose transporter, iron, copper,Na+/H+ antiporter, cation efflux, DMT transporter

N-acyl-L-homoserine lactone synthetase,NAD(P)H-nitrite reductase (nirB), c-typecytochromes

N. europaea& N. oceani

5 arabinose transporter, iron/lead transporter,ABC multidrug, potassium

receptor for ferrienterochelin and colicins,cytochrome P460, transcriptionalregulator (NnrS), caa3 cytochromec oxidase

N. multiformis& N. oceani

1 Mg2+ and Co2

+, Co/Zn/Cd efflux,tellurite transporter

cytochrome c peroxidase; cytochrome b1

All 4 AOB species 45 ABC dipeptide/oligopeptide/nickel, ABCmultidrug, ABC Fe-S cluster biosynthesis, ABCpolysaccharide/polyol phosphate export, ABCorganic solvents, nitrite/formate (focA), sulfatetransporter, 3 components of phosphotransferasesystem for mannose/fructose, Mg/Co/Ni

ammonia monooxygenase, hydroxylamineoxidoreductase, cytochrome c-554,nitrosocyanin, F1F0 ATP synthase(copy 1), nitrite reductase (nirK), nitricoxide reductase (norCBQD), coxBA+senC,glutamate dehydrogenase, alaninedehydrogenase, glutamine synthetase

K. stuttgartiensis 22 Iron receptors, P-type Mg2+ transport ATPase,

P-type copper translocating ATPase, nitrite/formate (focA) transporter, Fe2

+/Pb2+ transporter,

multidrug efflux, silver efflux, ABC phosphatetransport, ABC Fe-S cluster biosynthesis, ABC-typenitrate/sulfonate/bicarbonate, ABC spermidine

hydroxylamine oxidoreductase, ferredoxin,sufI multicopper oxidases, NADHdehydrogenase, cytochrome b (of thebc complex), 2 cytochrome c CDSs(of the N. eutropha nirK gene cluster)

a. Percentage of orthologous CDSs was determined for each individual comparison by dividing the number of shared genes within an orthologuegroup (as described in Experimental procedures) by the total number of protein coding CDSs in the N. eutropha C91 chromosome (2443). Plasmidgenes were not included in the analysis. Tables of orthologue groupings including gene tags can be found at http://nitrificationnetwork.org/.b. Nineteen per cent of protein coding CDSs in the N. eutropha chromosome are not found in other AOB genomes.

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operons are regulated by CbbR, a LysR-type transcrip-tional regulator (Shively et al., 1998). The N. eutrophaCbbR, CbbL and CbbS proteins (encoded by Neut_0803-05, respectively) are more similar to that of the NOBN. winogradskyi Nb-255 than to orthologous genes ofAOB (data not shown).

The major difference between the cbb operon ofN. eutropha and the other aerobic AOB is the presenceof carboxysome biosynthetic genes (Fig. 3). In thisregard, the N. eutropha cbb operon is similar to thatof other autotrophs, like Thiobacillus denitrificans(Beller et al., 2006) and Hydrogenovibrio marinus, whichalso have a bacterioferritin gene (Neut_0812) encodeddownstream of the carboxysome biosynthesis genes(Shively et al., 1998; Cannon et al., 2001; Yoshizawaet al., 2004). The carboxysome biosynthetic genes ofN. eutropha include structural proteins, carbon dioxideconcentrating proteins and carboxysome shell proteins,which comprise the main structural components ofcarboxysomes in chemoautotrophic bacteria (Cannonet al., 2001). Both N. winogradskyi and T. denitrificanspossess paralogous copies of the carboxysome shellproteins like N. eutropha, and the csoS1 genes of N. eu-tropha are most similar to those of N. winogradskyi andother Nitrobacter spp., suggesting a shared ancestralorigin (Beller et al., 2006; Starkenburg et al., 2006). Theaverage G + C content of carboxysome genes of N. eu-tropha (60.1%) are more similar to the average genomicG + C content of Nitrobacter spp. (61.8%) than theN. eutropha genome (48.5%), and the N. eutrophacbbLS genes have a higher G + C content (54.5%) thancbbLS genes in N. europaea (45.8%), which further sup-ports different evolutionary lineages for N. eutropha andN. europaea carbon fixation genes with the exception ofcbbQON.

Heavy metal and multidrug resistance

Searches for orthologous CDSs among the genomesequences of N. eutropha, N. europaea, N. multiformis,N. oceani and K. stuttgartiensis revealed some intriguinginsights into evolutionary history of several gene clusters,particularly those for heavy metal and multidrug transportfunctions (Table 4; Table S3). Orthologous operons formercury and copper resistance in N. eutropha were foundonly in the N. europaea genome (Fig. 1). Similar toN. eutropha, the mer operon in N. europaea is adjacent toa transposase and a site-specific recombinase (Fig. 1A).N. eutropha also encodes three copper-resistance geneclusters, one of which is absent from the other AOBgenome sequences and has similarity to the copABCoperon found commonly in plant pathogens (Cha andCooksey, 1993) (Fig. 1B). The copABC operon and thethird, divergent, mer operon are encoded within the117 kbp genomic island, as are at least 18 other genesinvolved in heavy metal resistance (Table S2). Somegenes for heavy metal resistance (e.g. arsenite) and mul-tidrug transporters were found only in the N. eutrophagenome among the aerobic AOB (Table 4; Table S3).Among these multidrug transporters are the MarC-liketransporter implicated in multidrug resistance of someenteric bacteria (Alekshun and Levy, 1997), and ABCmultidrug and antimicrobial peptide/lipid efflux pumps,both of which are adjacent to convergently encoded TetRtranscriptional regulators (Table S3). In short, the multi-tude of metal- and multidrug-resistance gene clusters,particularly those found only in the N. eutropha, but not inthe other aerobic AOB genome sequences, may be para-mount to enhanced survivability and niche specializationof N. eutropha to environments with high contaminantloads like wastewater or eutrophic sediments.

Iron uptake

Ammonia oxidizers are noted for their tremendousiron (Fe) requirement due to the many cytochromesand haem-containing enzymes essential to ammonia-oxidizing metabolism (Whittaker et al., 2000; Chain et al.,2003; Klotz et al., 2006; Wei et al., 2006b). Unlike mostbacteria, N. europaea does not produce its own sidero-phores, but rather encodes about 90 genes for iron acqui-sition, which allows for efficient scavenging and uptake ofmultiple forms of iron in iron-limited environments (Weiet al., 2006b). Unlike N. europaea, the N. eutrophagenome has a six-gene cluster that resembles the aero-bactin biosynthesis operon (Franza et al., 2005); twogenes in this cluster are orthologous to aerobactin syn-thases of N. oceani (Klotz et al., 2006) (Table S4). Sidero-phore biosynthesis by N. eutropha has yet to beinvestigated. While N. europaea maintains 22 fecI/fecR/

N. europaea cbbR cbbL cbbS cbbQ cbbO cbbN

N. eutropha Neut_0803 0804 0805 0815 0816 0821

Putative genesfor carboxysome structure and CO2 concentrating

Neut_0806 0807 0808 0809 0810 0811 0812 0814csoS2 csoS3 cso cso csoS1A csoS1B bfr par

unrelated

NE1922 NE1921

unrelated

NE1920 NE1919 NE1918 NE1917

H. marinuscbbL2 cbbS2 csoS2/S3 ORF csoS1CAB bfr

A B

cbbR cbbL cbbS cbbXN. multiformis & N. oceani

N. winogradskyi(copy 1) Nwi1988 Nwi1987/1986 Nwi1975

cbbR cbbL cbbS csoS3/S2 ccmL cbbO

Nwi1985/1984 Nwi1983

Fig. 3. Organizational comparison of the RubisCO (cbb) operonsof N. europaea, N. eutropha, Hydrogenovibrio marinus,N. winogradskyi, N. multiformis and N. oceani. bfr: bacterioferritin,par: chromosome partitioning, ccm: CO2 concentratingmechanism/carboxysome structure.

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fecA-type [s factor/anti-s factor/TonB-dependent outermembrane (OM) siderophore transporter] gene clusters(Chain et al., 2003), N. eutropha has only one suchcluster (Table S4). N. eutropha also has a single ferricuptake regulator gene. Thus, N. eutropha likely relies ondifferent mechanisms for regulating iron uptake genescompared with N. europaea.

N. eutropha has 12 predicted fecA-type genes for OMsiderophore transporters and has the potential to take upmajor types of siderophores and related compounds(Table S4). Interestingly, 8 of its 12 OM siderophore trans-porters have the highest similarity to homologues inN. europaea, reflecting the relatedness between the twospecies (data not shown). N. eutropha contains con-served genes encoding the energy-transducing TonB–protein complex (Table S4). Unlike N. europaea andN. multiformis, N. eutropha appears to lack an ABC trans-porter for Fe3+/siderophores. However, an MFS familytransporter in the aerobactin synthase gene cluster couldpotentially import loaded siderophores and/or exportunloaded ones, making it a good candidate for themissing ABC Fe3+/siderophore transporter. N. eutrophahas bacterioferritin and associated genes for intracellularFe storage (Table S4). Similarly to the other AOB,N. eutropha has a gene encoding a high-affinitysiderophore-independent ferric transporter (Ftr1-type,similar to NE0294) (Wei et al., 2006b). Finally,N. eutropha, like the other aerobic AOB, has no geneswith strong similarity to the characterized ferrous (Fe2+)transporter gene, feoB.

Transport and assimilation of nitrogen

The genomes of the four aerobic AOB include CDSs forglutamate dehydrogenase, glutamine synthetase andalanine dehydrogenase for assimilation of ammonia.Other amino-acid biosynthetic pathways are largelysimilar between N. eutropha and N. europaea (Chainet al., 2003). N. eutropha has multiple putativeglutamine-dependent asparagine synthase-encodinggenes (asnB). Neut_0901 and 1419 are over 85% iden-tical to corresponding protein sequences in N. europaeaand have similar genomic context, whereas the diver-gent Neut_1735/0910 are less than 30% identical toAsnB protein sequences in N. oceani (Noc_0777/1965respectively).

In contrast to N. europaea and N. multiformis,N. eutropha lacks a recognizable orthologue to ammoniapermease (amtB). Interestingly, K. stuttgartiensis doespossess an amtB gene whose translated product shares49% protein sequence identity with AmtB of N. europaea.In contrast, all four aerobic AOB and K. stuttgartiensisshare an orthologue to the focA-type nitrite/formate trans-porter (Table 4).

Like N. europaea, the N. eutropha genome lacks thestructural and accessory genes that encode urease (ureaamidohydrolase) that are found in many AOB (Koperet al., 2004). In contrast to N. europaea, N. eutrophalacks the urea transporter gene. While bothnitrosomonad genomes contain the gene encoding forurea carboxylase (EC: 6.3.4.6), also known as subunitone of urea amidolyase (UAL), genes coding for a bonafide second subunit of UAL, allophanate hydrolase(EC:3.5.1.54), were not found. This search was based onallophanate hydrolase sequences such as that fromalphaproteobacterium Oleomonas sagaranensis, whichcan grow on urea as a sole N-source facilitated by UALactivity (Kanamori et al., 2004; 2005). Neither N. eutro-pha nor N. europaea can grow in media with urea as asole N- or energy source, even though N. europaea hasa gene with high similarity to the predicted urea trans-porter of N. multiformis and N. oceani (82% and 52%protein sequence identity, respectively) and bothgenomes contain the inventory to express ABC-typebranched amide transporters. While both N. eutrophaand N. europaea genomes contain genes which havesimilarity to recognized allophanate hydrolase domains(Neut_2475, NE2416), there is no indication that theseare specific for the hydrolysis of carboxy-urea (allopha-nate). Overall, we predict that N. eutropha and N. euro-paea lack both UAL and urease activities, which maydisadvantage them in low-N environments relative toNitrosospira spp. that have urease activity.

Two genes involved in organic-N metabolism werefound in N. eutropha that were distinct comparedwith genes annotated with similar functions in theother AOB genomes: amidohydrolase-2 (Neut_1622),which hydrolyses the C–N bond of amide groups, andN-acetylmuramoyl-L-alanine amidase (Neut_1623), whichcleaves the amide bond between N-acetylmuramoyl andL-amino acids in the bacterial cell wall. Genes for cyano-phycin (a nitrogen storage polymer) synthetase werefound in N. eutropha and also in N. europaea andN. multiformis. However, orthologues for cyanophycinase,the enzyme that degrades cyanophycin, were not found inN. eutropha or N. europaea. Orthologues for nitrilase/cyanide hydratase, which releases NH3 from cyanide,were found in all four aerobic AOB.

Orthologues in K. stuttgartiensis

Although 22% of genes in N. eutropha were orthologousto genes in K. stuttgartiensis, only a few were identifiedwith annotated transport or metabolic functions (Table 4).Based on this small inventory, it appears that ammoniaoxidation and transport pathways in the Proteobacteriaand the Planctomycetales share little, if any, evolutionaryorigin.

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Conclusions

As the 16S rRNA genes of N. eutropha C91 and N. eu-ropaea ATCC 19718 share 97% identity, they necessar-ily occupy a tightly clustered lineage in the 16S rRNAphylogenetic tree. However, comparison of genomicorganization (Fig. 2) and content (Tables 1–4; Figs 1 and3) of both nitrosomonads has shown distinct differencesthat provide hypotheses for why N. eutropha-like speciesare better adapted to eutrophic ecosystems than themore broadly distributed N. europaea-like species. Inparticular, the two additional gene clusters for respiratoryHCOs and the many heavy metal and multidrug trans-port functions distinguish the N. eutropha C91 from otheraerobic AOB and anammox genome sequences. Thesignificant amount of horizontal gene transfer to, andrearrangement within, the N. eutropha genome has,perhaps, provided unique qualities for survival andfitness of these bacteria to specialized, N-saturated,ecological niches. Certainly, confirmation of unique prop-erties in genomes of other N. eutropha isolates, andconfirmation that the genes produce functional enzymesgiven appropriate environmental signals, would providegreater evidence for their involvement in niche adapta-tion of N. eutropha, and are ideal targets for futureresearch.

Experimental procedures

Construction and sequencing of genomic library

Nitrosomonas eutropha C91 was obtained by J.M. Nortonfrom F. Valois, from the culture collection of S.W. Watson(Woods Hole Oceanographic Institute). Batch cultures ofN. eutropha were grown as described previously forN. europaea ATCC 19718 (Hyman and Arp, 1992). GenomicDNA was purified and randomly sheared. Shotgun librarieswith approximate insert sizes of 3, 7 and 40 kbp were con-structed and sequenced at the JGI Production GenomicsFacility as previously described (Klotz et al., 2006).

Sequence assembly

A total of 44 937 draft sequence reads were assembled,producing an average of 11.7-fold coverage across thegenome. The whole genome sequence was assembledusing the Phred/Phrap/Consed package (P. Green, Univer-sity of Washington) (Ewing and Green, 1998; Ewing et al.,1998; Gordon et al., 1998). The reads were assembledinto 50 high-quality draft sequence contigs, which werelinked into seven larger scaffolds by paired-end sequenceinformation. Gaps in the sequence were closed by walkingon gap-spanning clones and by sequencing polymerasechain reaction (PCR) products generated from genomicDNA. Physical (uncaptured) gaps were closed by combina-torial (multiplex) PCR. Sequence finishing and polishingadded 1015 reads, and a final genome assembly was

validated by fosmid end sequence and long-range PCRdata congruence.

Sequence analysis, annotation and nucleotide sequenceaccession numbers

Automated gene modelling was completed using multipledatabases and modelling packages as detailed elsewhere(Chain et al., 2006; Klotz et al., 2006). The chromosome andthe 65 and 56 kbp plasmids of N. eutropha (pNeutP1 andpNeutP2, respectively) can be found in the GenBank/EMBL/DDBJ database under accession numbers CP000450,CP000451 and CP000452 respectively.

Cross-genomic comparisons

Orthologous and paralogous groups were determined us-ing OrthoMCL version 1.4 (http://orthomcl.cbil.upenn.edu/ORTHOMCL/) (Li et al., 2006). A peptide database of 10 746sequences was assembled from the CDS translationsfrom N. eutropha (CP000450, CP000451, CP000452),N. europaea (AL954747), N. multiformis (CP000103) andN. oceani (CP000127). An all-versus-all analysis was per-formed using BLASTP (BLASTALL 2.2.13) using SEG, withe = 10-5. The results were processed by OrthoMCL (mcl-06-21) using an inflation factor of 1.5. The output of OrthoMCLwas parsed to separate orthologous and paralogous groups.

The genome of N. eutropha was also compared withthe genome of K. stuttgartiensis (CT030148, CT573071–CT573074) using the MaGe database (https://www.genoscope.cns.fr/agc/mage/wwwpkgdb/anammoxoscope),courtesy of Genoscope, Evry, France. The similarity thresh-old was set at 25% similarity over a minimum of 75% of thegene length.

Tables of orthologue groupings can be found on the Nitri-fication Network web pages at: http://nitrificationnetwork.org/.

Acknowledgements

Funding for this project was provided by the Department ofEnergy through the Microbial Genomes Program of the Officeof Science. The Joint Genome Institute (JGI) managed theoverall sequencing effort, which was carried out primarily atthe Lawrence Livermore National Laboratory. Computationalannotation was carried out at Oak Ridge National Laboratoryand the Production Genomics Facility. The authors gratefullyacknowledge Dr Marc Strous for comparisons of theK. stuttgartiensis genome sequence with those of the aerobicammonia oxidizers and for critical analysis. We also thankNorman Hommes for analysis and input on transporterfunctions. Support for L.Y.S. was from the Agric. Exp. Sta.and Academic Senate of UCR. M.S.M.J. was supported byGrant npc6352 from the Foundation of Applied Sciences(STW). M.G.K. was supported by NSF (EF-0412129), theKentucky Science and Engineering Foundation (KSEF-148-502-05-140) and incentive funds from the UofL EVPResearch. J.M.N. was supported by UT Agric. Exp. Sta. andBARD US-3377-02. Work at Lawrence Livermore NationalLaboratory was performed under the auspices of the U.S.Department of Energy, Contract W-7405-Eng-48.

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Supplementary material

The following supplementary material is available for thisarticle online:Table S1. Composition of 12 167 kbp REPNeut3 sequencein N. eutropha C91 genomeTable S2. Heavy metal resistance and ccm genes encodedin the 117 kpb genomic island.Table S3. Heavy metal- and multidrug-resistance genes inN. eutropha, excluding those encoded within the genomicisland (see Table S2).Table S4. Comparison of Fe acquisition genes inN. eutropha and N. europaea.

This material is available as part of the online article fromhttp://www.blackwell-synergy.com

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