APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Sept. 2005, p. 5371–5382
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Vol. 71, No. 9
Structure and Sequence Conservation of hao Cluster Genes
of Autotrophic Ammonia-Oxidizing Bacteria:
Evidence for Their Evolutionary History
David J. Bergmann,1Alan B. Hooper,2and Martin G. Klotz3*
Black Hills State University, Spearfish, South Dakota 577831; University of Minnesota, St. Paul, Minnesota 551082;
and Department of Biology, University of Louisville, Louisville, Kentucky 402923
Received 27 December 2004/Accepted 21 April 2005
Comparison of the organization and sequence of the hao (hydroxylamine oxidoreductase) gene clusters from
the gammaproteobacterial autotrophic ammonia-oxidizing bacterium (aAOB) Nitrosococcus oceani and the
betaproteobacterial aAOB Nitrosospira multiformis and Nitrosomonas europaea revealed a highly conserved gene
cluster encoding the following proteins: hao, hydroxylamine oxidoreductase; orf2, a putative protein; cycA,
cytochrome c554; and cycB, cytochrome cm552. The deduced protein sequences of HAO, c554, and cm552were
highly similar in all aAOB despite their differences in species evolution and codon usage. Phylogenetic
inference revealed a broad family of multi-c-heme proteins, including HAO, the pentaheme nitrite reductase,
and tetrathionate reductase. The c-hemes of this group also have a nearly identical geometry of heme
orientation, which has remained conserved during divergent evolution of function. High sequence similarity is
also seen within a protein family, including cytochromes cm552, NrfH/B, and NapC/NirT. It is proposed that the
hydroxylamine oxidation pathway evolved from a nitrite reduction pathway involved in anaerobic respiration
(denitrification) during the radiation of the Proteobacteria. Conservation of the hydroxylamine oxidation
module was maintained by functional pressure, and the module expanded into two separate narrow taxa after
a lateral gene transfer event between gamma- and betaproteobacterial ancestors of extant aAOB. HAO-
encoding genes were also found in six non-aAOB, either singly or tandemly arranged with an orf2 gene, whereas
a c554gene was lacking. The conservation of the hao gene cluster in general and the uniqueness of the c554gene
in particular make it a suitable target for the design of primers and probes useful for molecular ecology
approaches to detect aAOB.
Autotrophic ammonia-oxidizing bacteria (aAOB) obtain all
energy for growth from the oxidation of ammonia to nitrite.
Ammonia is first oxidized to hydroxylamine by the membrane-
bound hetero-trimeric copper enzyme ammonia monooxygen-
ase (AMO) (4, 22, 40). Hydroxylamine is oxidized to nitrite by
the periplasmic enzyme hydroxylamine oxidoreductase (HAO)
(61). HAO binds seven c-type hemes and an active-site heme,
known as heme P460 (3, 57). A small amount of hydroxylamine
is also oxidized by the periplasmic, monoheme cytochrome
P460 protein, proposed to scavenge hydroxylamine not oxi-
dized by HAO (9, 15, 42). aAOB occur in two phylogenetic
lineages of the Proteobacteria: the closely related genera Nitro-
somonas and Nitrosospira within the Betaproteobacteria and
several strains in the gammaproteobacterial genus Nitrosococ-
cus, including Nitrosococcus oceani (18, 43, 54). An unrelated
autotrophic nitrifying bacterium, canidatus Brocadia anam-
moxidans, phylum Planctomycetes (29), employs a hydrazine-
oxidizing HAO for part of the anaerobic conversion of ammo-
nia and nitrite to dinitrogen.
Based on studies of Nitrosomonas europaea, hydroxylamine
oxidation in extant aAOB is assumed to proceed through ca-
talysis by the octaheme HAO followed by electron transfer to
membrane ubiquinone mediated by the tetraheme cytochrome
c554 and the membrane-associated tetraheme cytochrome
cm552(22, 24). The latter passes two electrons to AMO and
channels two electrons into the electron transport chain to
cytochrome oxidase. The close functional connection of these
cytochromes is supported by the clustering of their encoding
genes in the genome of N. europaea ATCC 19718 (Schmidt):
HAO (hao)- and cytochrome c554(cycA and hcy)-encoding
genes are part of a cluster that exists in three nearly identical
copies (8, 11, 20, 21, 36, 47). In two of the three hao gene
clusters, the cycA gene is followed by cycB, which encodes the
membrane-anchored tetraheme cytochrome cm552(8). The hao
and cycA genes are separated by a hypothetical open reading
frame (orf2) in all three copies of the N. europaea hao gene
cluster (11). This conservation of Orf2 in the N. europaea
genome was peculiar, because earlier primer extension and
Northern blot studies identified a terminator for the hao tran-
script and a functional promoter of the cycA gene between the
translational controls of Orf2 (8, 21, 47).
Biochemically, HAOs have been characterized from three
aAOB: the aerobic ammonia oxidizers Nitrosomonas europaea
and Nitrosococcus oceani (23) and the anaerobic ammonia
oxidizer B. anammoxidans (52). Amino acid sequence and X-
ray crystal structure information are available only for HAO
from N. europaea (26, 47). All three enzymes share similar
spectral properties, including the unique P460 chromophore.
However, partial protein sequences from proteolytic digests of
HAO from B. anammoxidans indicate that there may be only
limited sequence similarity between HAO from B. anammoxi-
* Corresponding author. Mailing address: University of Louisville,
Department of Biology, 139 Life Science Building, Louisville, KY
40292. Phone: (502) 852-7779. Fax: (502) 852-0725. E-mail: aem
dans and N. europaea (48). A partial protein sequence for the
Hao protein from Nitrosospira multiformis C-71 was deduced
from a PCR amplicon obtained from a fragment of the hao
gene of this organism (50).
Here we present the sequence of the entire hao gene cluster
that exists as a single copy in the genome of N. oceani and
compare it to the hao gene clusters of N. europaea and N.
multiformis. Our analyses of gene cluster organization and
phylogenetic analyses of the protein sequences encoded by
genes in the hao gene cluster support the hypothesis that the
hao gene cluster evolved in the gammaproteobacterial ances-
tors of extant aAOB from genes involved in denitrification and
resides in extant betaproteobacterial aAOB as a result of hor-
izontal gene transfer events.
MATERIALS AND METHODS
Bacterial strains, culture maintenance, and DNA isolation. The nucleotide
sequence and organization of the hao gene cluster of two strains of Nitrosococcus
oceani was determined using different methods. In the case of N. oceani strain
C-107WH, a reverse genetics approach (isolation and purification of the HAO
protein, amino acid sequencing, and PCR amplification of the hao gene using
degenerate primers) was used to obtain a partial sequence for the hao gene. The
sequences of the entire hao gene cluster from N. oceani strain ATCC 19707 and
N. multiformis C-71 have been obtained by using whole-genome shotgun se-
N. oceani strain C-107WH was obtained as a gift from the collection of Bess B.
Ward at Princeton University. Media were prepared using a mixture of 90%
artificial seawater (Tropic Marin, Wartenburg, Germany) and 10% natural sea-
water (pH 8.0) with the following additions before autoclaving: (NH4)2SO4(10
mM), MgSO4(1.45 mM), and CaCl2(0.14 mM). After autoclaving, K2HPO4was
added (final concentration, 0.65 mM), along with 0.5 ml each of two filter-
sterilized trace element solutions, A and B (solution A, FeSO4[4.66 mM], EDTA
[4.66 mM], and CuSO4[0.16 mM]; solution B, Na2MoO4[8.3 ?M], MgCl2[20
?M], CoCl2[17 ?M], and ZnSO4[700 ?M]). Cultures were grown in 1-liter
batches of medium in Fernbach flasks and titrated to pH 8.0 daily with K2CO3or
in a 10-liter fermentor culture titrated continuously with K2CO3.
Genomic DNA of N. oceani strain C-107WH was prepared from 3.2 g (wet
weight) of cells (5). Restriction digests, agarose gel electrophoresis, and transfer
of denatured restriction fragments to nylon membranes by capillary action were
performed using standard methods (45). Previous experiments (2, 32, 40) sug-
gested that the gene clusters and operons involved in ammonia catabolism exist
in the genome of N. oceani as single copies; therefore, the procedure to isolate
copy-specific template as developed for other AOB (41) was not necessary for N.
oceani. Designed oligonucleotide primers were obtained commercially (Sigma-
Genosys, The Woodlands, TX, or Life Technologies, Gaithersburg, MD). PCR
amplification was performed using a Perkin-Elmer GeneAmp 2400 thermal
cycler and Taq DNA polymerase (Platinum Taq; Life Technologies) as described
by the manufacturer with 4.0 mM MgCl2. DNA probes were labeled with32P by
the random hexamer priming method (16) using the Prime-A-Gene kit (Promega
Corp.). A probe for the cycA gene encoding cytochrome c554of N. europaea was
prepared by digestion of plasmid pE273 (8) with BamHI and EcoRI and isolation
of the 458-bp fragment containing cycA by preparative agarose gel electrophore-
sis. Southern blot assays were performed at 42°C in 6? SSPE (1? SSPE is 0.18
M NaCl, 10 mM NaH2PO4, and 1 mM EDTA [pH 7.7]), 0.5% sodium dodecyl
sulfate (SDS), 5? Denhardt’s solution overnight (45). Southern blot assays using
the longer probes were performed overnight at 50°C or 60°C in 6? SSPE, 0.5%
SDS, 0.5% BLOTTO (45) followed by washing for 30 min in buffer (0.1? SSPE,
0.5% SDS) at 30°C to 40°C before detection by autoradiography or phosphor-
Nitrosococcus oceani type strain ATCC 19707 (C-107) was grown as described
previously (2). Genomic DNA was isolated from late-exponential-phase cultures
as described by McTavish et al. (35) modified following the recommendations of
the Joint Genome Institute (DOE-JGI; Walnut Creek, CA).
In 2003, the U.S. Department of Energy decided to fund the sequencing of the
complete genomes of five nitrifying bacteria by the JGI, and among them were
Nitrosococcus oceani strain ATCC 19707 and Nitrosospira multiformis strain C-71
(Surinam). High-quality genomic DNA was prepared from Nitrosococcus oceani
strain C-107 and was provided by the Klotz Laboratory at the University of
Louisville as the sequencing template. Whereas the genome sequence data
available at this stage of the project have been provided freely by the U.S.
DOE-JGI for use in this publication/correspondence only, the five nitrifier ge-
nome sequences currently in progress will be made available in their entirety
through the JGI website following established federal data release policies
Purification of HAO from N. oceani strain C-107WH and PCR amplification of
an hao gene fragment. After harvesting 4.8 g of N. oceani cells by centrifugation,
cells were resuspended in 20 ml of 50 mM potassium phosphate buffer (pH 7.5).
Cells were broken using three passes through a French press and sonicated for
2 min, and the suspension was adjusted with EDTA to a final concentration of 5
mM. This cell extract was centrifuged at 150,000 ? g for 1 h at 5°C. The
supernatant was decanted, and the pellet was resuspended in 10 ml of 1 M KCl
in phosphate buffer and incubated at 5°C for 1 h. After a repeated centrifugation
of the suspension followed by decanting of the supernatant (KCl membrane
wash), phosphate buffer was added to the KCl membrane wash to adjust the KCl
concentration to 0.2 M and the liquid was concentrated to 1 ml on an Amicon
stirred cell with a YM 10 membrane and Centricon 10 spin filters (Amicon-
Grace, Beverly, MA). The KCl membrane wash was applied to a 1- by 105-cm
column of Sephadex G100 (Sigma Co., St. Louis, MO) equilibrated with 0.2 M
KCl in phosphate buffer. The cytochrome (HAO) eluted in the void volume. This
was concentrated with an Amicon stirred cell and a Centricon 10 spin filter. This
HAO preparation could be partially reduced by hydroxylamine and exhibited a
characteristic absorbance at 460 nm when reduced by dithionite (23).
Sequence comparison and analysis. Sequence similarities were investigated
initially using the National Center for Biotechnology Information BLAST pro-
gram (1). Protein sequences deduced from experimentally determined nucleo-
tide sequences were analyzed with the PSORT (39) or Top-Pred (12) programs
to identify hydrophobic domains that could serve as signal peptides for export
into the periplasm or constitute membrane-spanning domains. Multiple se-
quence alignments of the identified ORFs in the hao gene clusters from N.
oceani, N. multiformis, and N. europaea were performed using the ClustalW
program (56) accessible online through “Biology Workbench” provided by The
University of California San Diego Supercomputing Center (http://workbench
.sdsc.edu/). For the purpose of phylogenetic analysis of individual hao cluster
genes, highly similar sequences were obtained from the GenBank/EMBL data-
base using the BLAST program (1). After elimination of repetitive sequences,
full-length protein sequences were aligned using ClustalX version 1.83 (Gonnet
250 protein weight matrix with gap opening and gap extension penalties of 35/15
and 0.75/0.35, respectively, for the pairwise/multiple sequence alignments) (55).
To elucidate the evolutionary history of nitrification, we searched for and
analyzed proteins with significant sequence similarities to the products encoded
by the genes in the hao gene cluster of aAOB. In particular, this paper reports
on the successful inference of homology between the hydroxylamine oxidoreduc-
tase, tetrathionate reductase (ttr), and formate-dependent nitrite reductase
(Nrf), as well as between cytochrome cm552(orf4 in the hao gene cluster) and
proteins in the NapC-TorC-NirT-NrfB/H protein family. To this end, a total of
37 (Hao) and 56 (cytochrome cm552) identified protein sequences were included
in alignments, and distance neighbor-joining trees were constructed by the BioNJ
function in PAUP* v. 4.10b (53) and used as guide trees for manual refinement
of the respective ClustalX alignments. Sources and protein accession numbers of
most sequences are provided below in Fig. 5 and 6. Sources for the NapC-TorC-
NirT protein sequences used in alignments but not identified by accession num-
bers in the phylogenetic tree (see Fig. 6) were as follows: for the alphaproteobac-
teria, Agrobacterium tumefaciens strain C58 (Atu4410), Azospirillum brasiliense
(AAL36492), Bradyrhizobium japonicum (AAM47036), B. japonicum USDA 110
(BAC52305), Paracoccus denitrificans (Q56352), Rhizobium sp. strain G-179
(AAD46691), Rhodobacter sphaeroides (BAA31963), and Sinorhizobium meliloti
1021 plasmid pSymA (SMa1232); for the betaproteobacteria, Bordetella bronchi-
septica RB50 (BB2802), B. parapertussis 12822 (BPP2700), and Ralstonia eutro-
pha megaplasmid pHG1 (PHG213); for the gammaproteobacteria, Escherichia
coli O157:H7(ECs3091 andECs1151),
(HD0079), Pasteurella multicoda Pm70 (PM1598), Photobacterium profundum
SS9 (PBPRA1468, PBPRA2028, and PBPRA2364), Pseudomonas aeruginosa
PA01 (PA1172), Salmonella enterica serovar Typhi CT18 (STY2481), S. enterica
(CAA06850), S. oneidensis (SO1233), Shigella flexneri 2a strain 2457T (SF2416),
Vibrio cholerae O1 biovar el Torro strain N16961 (VC1693), Vibrio parahaemo-
lyticus (VP1161), Vibrio vulnificus YJ016 (VV1374), and Yersinia pestis KIM
The refined alignments of 37 and 56 full-length Hao-Ttr-NrfA and Cm552-
NapC-TorC-NirT-NrfB/H proteins, respectively, were used for the inference of
phylogeny. Phylogenetic relationships were investigated by using character-based
tree-searching methods with maximum parsimony or maximum likelihood object
5372 BERGMANN ET AL.APPL. ENVIRON. MICROBIOL.
functions. A maximum parsimony tree was built from the ClustalX alignment by
performing a full heuristic search with the PAUP* program and the following in
effect: 50% majority consensus, random order taxon-addition replicates with
tree-bisection-reconnection branch-swapping, mulpars, and steepest decent
functions in effect. The quality of the branching patterns was assessed by boot-
strap resampling of the data sets using 100 replications. Because inclusion or
exclusion of a few characters can highly affect bootstrap proportions of maximum
parsimony trees derived from limited data sets, we also conducted a maximum
likelihood inference by subjecting the alignment to a Bayesian inference of
phylogeny by using the program MrBayes (v. 3.0b4; written by Huelsenbeck and
Ronquist; http://morphbank.ebc.uu.se/mrbayes). In the latter, the protein se-
quence alignment was subjected to Metropolis-coupled Monte Carlo Markov
chain sampling over 100,000 generations. Four equally heated Markov chains
were used to build a sufficient number of reliable trees after the likelihoods of the
trees had converged on a stable value and to allow successful swapping between
chains. Three independent runs led to convergence on stable likelihood values
after 30,000 generations (data not shown). The searches were conducted assum-
ing an equal or a gamma distribution of rates across sites and using the WAG
empirical amino acid substitution model (60). In a postrun analysis, MrBayes
summarized the results concerning tree topology and branch lengths. By ignoring
the trees generated before the search converged on stable likelihood values
(removed as “burn-in”), a 50% majority rule consensus phylogram was con-
structed that displayed the mean branch lengths and posterior probability values
of the observed clades. Multiplication by 100 made these probability values
comparable to the bootstrap proportions calculated for the clade pattern in the
maximum parsimony consensus tree.
Nucleotide sequence accession number. The sequence of the 1,410-bp PCR-
amplified fragment of hao from N. oceani strain C-107WH determined using the
degenerate reverse primer and other custom-synthesized primers was deposited
in GenBank under accession number AY858555.
Sequence of the hao gene cluster. The hao gene cluster of N.
oceani strain ATCC 19707, obtained by whole-genome shotgun
sequencing (Fig. 1), contained four genes, hao, orf2 (encoding
a putative membrane protein), cycA (encoding cytochrome
c554), and cycB (encoding cytochrome cm552), and closely re-
sembled two of the three copies of the hao gene cluster in the
genome of N. europaea (8, 11, 47). A putative promoter with a
consensus for ?70RNA polymerase was identified upstream of
a Shine-Dalgarno sequence that precedes the hao gene. A
putative ?-independent transcriptional termination sequence
was identified downstream of cycB.
PCR amplification of an hao gene fragment. The N-terminal
sequence of the Hao protein from N. oceani strain C-107WH
was determined, by Edman degradation at the UMN Micro-
chemical Facility by using a HP241 gas-phase sequenator, to be
DIPDELYEALGVDXYXAXPXELYEAATERY. Based on
this sequence, the following degenerate forward primer for
PCR amplification was synthesized (I denotes inosine): 5?-GA
reverse primer, 5?-GTTCATIGGICCCCAICCTICAGTGTA-
3?, was based on residues 440 to 449 of Hao from N. europaea
(47). PCR was performed using a standard program (denatur-
ation for 5 min at 94°C and 25 cycles consisting of 30-s dena-
turation at 94°C, 30-s annealing at 45°C, and 60-s extension at
68°C, followed by a final 7-min extension step at 72°C).
The partial protein sequence of Hao from N. oceani strain
C-107WH (not shown) was identical to that of the first 471
residues of Hao from N. oceani strain ATCC 19707 (Fig. 2),
except at positions 22 (Leu in ATCC 19707, Phe in C-107WH),
68 (Ala in ATCC 19707, Asp in C-107WH), 168 (Val in ATCC
19707, Ala in C-107WH), 171 (Thr in ATCC 19707, Ile in
C-107WH), and 298 (Trp in ATCC 19707, Leu in C-107WH).
The PCR product containing a fragment of the N. oceani
hao gene was labeled with32P and used to probe a blot of
restriction digests of genomic DNA from N. oceani strain
C-107WH. Only one restriction fragment hybridized to the
probe in four of the five restriction digests, indicating that only
one copy of hao was present in the genome of N. oceani strain
C-107WH (Fig. 3). A probe based on a 458-bp fragment of the
cycA gene from N. europaea hybridized weakly to genomic
DNA from N. oceani strain C-107WH (data not shown), indi-
cating that the sequence identity between the cycA genes from
N. europaea and N. oceani was higher than for genes in the amo
operon, for which cross-hybridization did not occur (2). In the
cycA gene Southern blot assay, the two restriction digests re-
vealing more than one hybridizing restriction fragment were
accounted for by the presence of the respective restriction sites
seen in the nucleotide sequence within the fragment. The ge-
nome sequence of N. oceani strain ATCC 19707 contained,
indeed, only one copy of the cycA gene.
Comparison of the hao gene clusters in aerobic ammonia-
oxidizing bacteria. The sequences of the Hao proteins from N.
europaea (Schmidt), N. multiformis (Surinam), and N. oceani
(ATCC 19707) were highly similar with a high degree of con-
servation in the eight heme-binding regions (Fig. 2). It is thus
likely that the functional HAOs have similar secondary, ter-
tiary, and quaternary structures. The tyrosine residue Tyr467,
which is cross-linked to heme P460 in N. europaea (3, 22, 26),
FIG. 1. Cartoon showing ORFs of the hao gene clusters in Nitroso-
coccus oceani (Nc_oce), Nitrosomonas europaea (Nm_eur), and Ni-
trosospira multiformis (Ns_mul).
VOL. 71, 2005hao GENE CLUSTER CONSERVATION IN NITRIFYING BACTERIA5373
FIG. 2. ClustalW alignment of Hao protein sequences from Nitrosococcus oceani (NO0160), Nitrosospira multiformis (NMU1996), Nitrosomo-
nas europaea (NE0962), Methylococcus capsulatus (MCA0956), and Silicibacter pomeroyi (SPOA0200). Also included are the deduced protein
sequences of putative Hao proteins from Geobacter metallireducens (Gmet02001372), Desulfovibrio desulfuricans (Ddes02001487), Methanococ-
coides burtonii (Mbur03000734), and the likely nonfunctional Hao-like sequence from Magnetococcus sp. strain Mc-1 (Mmc10221065). Amino acid
residues conserved in the majority of sequences are shaded. The secondary structure of HAO from N. europaea (26) is shown beneath the
alignments, with the letters H designating ?-helical regions and B designating ?-turns. Heme-binding regions are underlined.
5374 BERGMANN ET AL.APPL. ENVIRON. MICROBIOL.
as well as Asp267 and His268 (but not Tyr334), which are
hypothesized to deprotonate or H-bond with substrate (22),
were also conserved in all Hao protein sequences, as were the
residues nearby. It is interesting that the sequence of amino
acid residues of ?-helix 1 appears to be repeated in ?-helix 2 in
all three Hao proteins, perhaps the result of an intragenic
nucleotide sequence duplication event within hao. The least-
conserved region of Hao from N. oceani, N. multiformis, and N.
europaea was the region between hemes 2 and 3, which was
similar in length but dissimilar in sequence, as well as the loop
between ?-helices 10 and 11, which was 11 amino acids longer
in N. oceani than N. europaea. A relatively hydrophobic C-
terminal region of Hao from N. europaea, which was not visu-
alized in the X-ray crystal structure of this protein (26), was
conserved in the Hao proteins from N. multiformis and N.
oceani and has been identified by P-SORT and TopPred anal-
yses as a membrane-spanning domain. The N. multiformis Hao
had a longer C terminus by 11 residues. In the modeled HAO
trimer (26), the three copies of this domain appear to be close
and can be imagined to interact together in the membrane.
Despite the overall sequence similarity of the Hao proteins
from the three aAOB, the two betaproteobacterial Hao pro-
teins lacked a nine-amino-acid-long fragment found between
?-helices 10 and 11 in N. oceani.
The orf2 gene was found in all hao gene clusters of the three
aAOB; however, the predicted protein sequence of Orf2 was
the least conserved among the gene cluster products (Fig. 4A;
Table 1). Orf2 was predicted by PSORT to be located in the
The protein sequences of cytochrome c554from all three
aAOB contained several conserved regions evenly distributed
throughout the sequence (Fig. 4B), indicating that the overall
secondary and tertiary structures of the cytochrome c554from
N. oceani are likely similar to those from N. europaea (28).
Heme-binding regions and the region between ?-helix 1 and
?-strand 2 were most conserved, whereas the loops joining
?-helices 4 and 5 and ?-helices 7 and 8 were extended in
cytochrome c554from N. oceani compared to those from N.
multiformis and N. europaea (Fig. 4B).
The cycB gene, which encodes tetraheme c cytochrome
cm552, was present as Orf4 in the hao gene cluster of all three
aAOB (Fig. 4C). As with the other genes involved in ammonia
catabolism in aAOB, the genome of N. oceani contained only
one copy, whereas the betaproteobacterial aAOB contained
multiple copies. The N termini of the cm552proteins differed in
sequence in all three aAOB; however, all three were predicted
by PSORT to contain a membrane anchor region. As expected,
the central portion of the cm552proteins, containing the heme-
binding regions, was highly conserved. The C-terminal do-
mains of cm552proteins from beta-aAOB were rich in Glu and
Asp and hence negatively charged, a sequence feature absent
from the cm552protein in N. oceani.
Comparison of the hao gene cluster proteins from aAOB
with similar proteins from non-aAOB. To our surprise, genes
encoding Hao-like proteins were identified in the genomes of
six bacteria that do not utilize ammonia oxidation to gain
energy and reductant for growth. Due to significant sequence
similarity (Fig. 2; Table 1), the Hao protein sequences from
Nitrosomonas, Nitrosospira, and Nitrosococcus aligned well with
proteins annotated as Hao in the genomes of Methylococcus
capsulatus Bath (on the chromosome) and Silicibacter pomeroyi
DSS-3 (on a megaplasmid). Although the S. pomeroyi Hao
protein lacked the hydrophobic C-terminal domain that is
present in the Hao proteins in aAOB and M. capsulatus (which
may anchor functional HAO in the plasma membrane), the
presence of the critical heme and ligand-coordinating residues
(Tyr467, Asp267, and His268) suggests that these proteins can
assemble to bona fide trimeric HAOs, while genes encoding
proteins with significant sequence similarities to other genes in
the hao gene cluster were either missing (cytochrome c554) or
not near the hao gene (cytochrome cm552homologues). The
hao genes in M. capsulatus and S. pomeroyi were tandem ar-
ranged with orf2 genes (Fig. 4A). Hao stop and Orf2 start
codons overlapped by 1 nucleotide in the M. capsulatus ge-
nome and were preceded by a purine-rich region. It is thus
likely that Hao and Orf2 are coexpressed in M. capsulatus. Hao
stop and Orf2 start codons were separated by intergenic se-
quence on the S. pomeroyi megaplasmid. The conservation of
orf2 genes and the predicted association of its expression prod-
uct with the plasma membrane suggest that the Orf2 protein
may play a role in the biogenesis, arrangement, or stabilization
We also identified genes in the genomes of Magnetococcus
sp. strain Mc-1, Desulfovibrio desulfuricans G20, Geobacter
metallireducens GS15, and Methanococcoides burtoni whose
Hao protein-like expression products had lesser but significant
sequence similarities to Hao (Fig. 2). These putative Hao pro-
teins were different from the other Hao proteins in that they
lacked helices 1, 2, 3, 6, and 7 and had different sixth axial
ligand histidines for hemes 1, 6, and 7. In addition, they con-
tained long insertions between hemes P460 and 5 and between
helix 19 and heme 8. The genes encoding the putative Hao
proteins in G. metallireducens and M. burtoni were located
adjacent to genes similar in sequence to one another; however,
these genes had no significant sequence identity with either
cycA, cycB, or orf2 genes. The putative hao gene in D. desul-
furicans was found next to a gene encoding a putative tetra-
heme c cytochrome in the cm552-NrfB/H family. The Hao-like
protein-encoding gene found in the genome of Magnetococcus
FIG. 3. Southern blot of restriction digests of genomic DNA from
N. oceani, probed with the fragment of N. oceani hao amplified by
PCR. Lanes 1 to 5, restriction digests of N. oceani DNA (lane 1,
BamHI; lane 2, EcoRI; lane 3, KpnI; lane 4, PstI; lane 5, SacI); lane 6,
E. coli LE392 genomic DNA digested with EcoRI; lane 7, N. europaea
genomic DNA digested with EcoRI.
VOL. 71, 2005 hao GENE CLUSTER CONSERVATION IN NITRIFYING BACTERIA5375
can very likely not assemble into a functional HAO, since its
protein sequence lacks critical residues, including Tyr467,
Asp267, and the histidine ligated to heme 3, all of which were
present in the bona fide and putative Hao proteins.
Discussion. The high degree of conserved gene organization
(hao, orf2, cycA, and cycB) and sequence in the hao gene
cluster from both gamma- and beta-aAOB reported here sug-
gests that all four genes are essential for hydroxylamine oxida-
tion and electron transfer in aerobic aAOB. Whereas beta-
aAOB, such as N. europaea and N. multiformis, contain
multiple copies of the hao gene cluster, N. oceani has a single
copy. Correspondingly, N. oceani has a single copy of operons
encoding AMO (2) and urease (32), whereas beta-aAOB con-
tain two copies (N. europaea) (36) or three copies (Nitrosospira
spp.) (30, 40, 41) of their amo operons. Preliminary results
indicate that other gamma-aAOB have single copies of their
catabolic genes (2; M. G. Klotz, unpublished data). It has been
argued that the presence of a single copy rather than multiple
copies of ammonia catabolic genes can be an adaptation to a
low but constant ammonia concentration in the environment in
contrast to the highly varied concentrations of ammonia en-
countered by other nitrifying bacteria (40). Whereas the near
identity in sequence of multiple copies of amo and hao gene
clusters in a given organism may be explained by the operation
of a rectification mechanism (31), the synteny of these gene
clusters between organisms that do not belong to closely re-
lated taxa (i.e., the Gamma- and Betaproteobacteria) suggests
that all their member genes encode proteins that interact phys-
ically (25, 33).
AMO of aAOB exhibits considerable similarity in known
FIG. 4. ClustalW alignments of the predicted amino acid sequences of ORFs 2, 3, and 4 in the hao gene clusters from N. oceani (NO), N.
multiformis (NMU), and N. europaea (NE). Because the sequences of the multiple copies in NMU and NE are identical, only one sequence is
shown. (A) Alignment of the predicted sequences of Orf2 proteins listed by their accession numbers. To highlight the putative involvement of this
protein in catabolic electron flow during ammonia oxidation, the sequences of Orf2 proteins found in tandem arrangement with HAO as single
copies in the nucleoid of Methylococcus capsulatus (MCA0955) and on the megaplasmid of Silicibacter pomeroyi (SPOA0200) are shown.
(B) Alignment of the predicted sequences of cytochrome c554proteins listed by their accession numbers. These cytochromes have been reported,
so far, only from aAOB. Signal peptides, predicted by PSORT, have been removed from the sequences. The secondary structure of cytochrome
c554from NE (28) is shown below the alignment, with letters H designating ?-helical regions and B designating ?-turns. Heme-binding regions are
underlined. (C) Alignment of the predicted sequences of cytochrome cm552proteins from aAOB listed by their accession numbers. The result of
a phylogenetic analysis of these and 53 related sequences is provided below in Fig. 6.
5376BERGMANN ET AL.APPL. ENVIRON. MICROBIOL.
aspects of structure, amino acid sequence, and enzymatic prop-
erties to the membrane-bound particulate methane monooxy-
genase (pMMO) of methanotrophs (7, 19, 40), which are found
in the ?- and ?-subdivisions of the Proteobacteria (17). It is
notable that the amino acid sequences of the Amo proteins
from N. oceani are considerably more similar to the sequences
of pMmo proteins of gammaproteobacterial methanotrophs
than to Amo protein sequences from betaproteobacterial
aAOB (40, 43). Unlike AMO from N. europaea, which has a
much lower affinity for methane than for ammonia, AMO from
N. oceani has nearly equal affinities for methane and ammonia
(58). Because N. oceani is a member of the oldest gammapro-
teobacterial lineage, the Chromatiales (purple sulfur bacteria),
extant genes encoding AMO and pMMO may have evolved
from an ancestral gene cluster that was present in the pro-
teobacterial ancestor before the divergence of the Alphapro-
teobacteria from the Gamma- and Betaproteobacteria. Such an
ancestral monooxygenase may have been able to oxidize both
methane and ammonia at low rates, thereby producing low
levels of the diffusible intermediate, methanol, and the poten-
TABLE 1. Hao and Orf2 protein sequence identities and similarities
% Identity/% similarity to Orf2 or Haoaof:
N. oceaniN. europaea N. multiformis S. pomeroyiM. capsulatus
aIdentities and similarities for the Hao proteins are given in bold.
VOL. 71, 2005hao GENE CLUSTER CONSERVATION IN NITRIFYING BACTERIA 5377
tial toxin and mutagen hydroxylamine (4). This monooxygen-
ase required oxygen and reductant to incorporate oxygen into
methane or ammonia. Therefore, if this ancestral monooxy-
genase evolved as a copper enzyme during the radiation of
Proteobacteria, it was useful only in locally oxic microhabitats
of the globally anoxic environment. Production of hydroxyl-
amine would have been wasteful of reductant and may have
driven evolution towards a more complex and energy-conserv-
ing hydroxylamine-scavenging system than the mono-heme cy-
tochrome P460, a hydroxylamine dehydrogenase (9, 10) that
may have been active during this preoxic evolutionary stage.
With the exception of cytochrome cm552/NirT (8, 61), no
homologues for the four proteins (HAO, Orf2, cytochrome
c554, and cytochrome cm552) have been reported in bacteria
other than chemolithoautotrophic nitrifiers. In addition to re-
porting the sequence and structural conservation of the hao
gene cluster in aAOB, we report here the presence of hao-like
genes in the genomes of six prokaryotes that do not use cata-
bolic ammonia oxidation. It has been noted that the operonic
order of orthologous genes (synteny) in different genomes is
less preserved than their presence and that synteny is low when
protein sequence identity between orthologous proteins in dif-
ferent genomes is lower than 50% (25, 33). It was thus signif-
icant to find that the sequences of Hao proteins from N. eu-
ropaea and N. multiformis were 68% identical, a value similar
to the identity of homologous Amo proteins (40). As also
reported for Amo proteins (40), identity of the Hao proteins
from N. oceani and beta-aAOB was lower and ranged between
50 and 60% (Table 1). Protein sequence similarity between
Hao proteins from beta-aAOB was near 80%, and similarities
among Hao proteins from N. oceani, M. capsulatus, and S.
pomeroyi were in the lower-70 percentile (Table 1). Compari-
sons of all other bona fide Hao protein sequences yielded
values in the upper-60 percentile (Table 1). As seen by analysis
of the region stretching from heme-binding sites 3 to 8, the N.
oceani Hao protein (NO0160) had low similarity to putative
Hao proteins from three other non-aAOB (35%, 38%, and
41% for G. metallireducens [Gmet02001372], D. sulfuricans
[Ddes02001487], and M. burtoni [Mbur03000734], respec-
tively). This was largely due to numerous and large gaps. The
low level of sequence identity/similarity correlated with the loss
of synteny in that the hao genes in these organisms were not
arranged with any that are found in the four-gene cluster of
In two of the non-aAOB that contain Hao, M. capsulatus and
S. pomeroyi, the hao genes were tandem arranged in an operon
with an orf2 gene. These sequences are remarkable finds, as
they contribute to our understanding of several aspects of the
evolutionary history of aerobic ammonia oxidation:
First, an hao-orf2 gene tandem was identified on a megaplas-
mid in the marine sulfur-oxidizing alphaproteobacterium S.
pomeroyi that appears to lack further inventory suited for am-
monia or methane oxidation (37). Existence of a vector-borne
hao gene strengthens the hypothesis of horizontal exchange of
the hao gene among the Proteobacteria. Potential donors in-
clude the ancestors of extant marine ammonia-oxidizing purple
sulfur bacteria, such as Nitrosococcus.
Second, the genomes of Alphaproteobacteria (Magnetococ-
cus), iron- and sulfate-reducing Deltaproteobacteria (Geobacter
and Desulfovibrio), and the cold-adapted and methanogenic
euryarchaeon Methanococcoides burtoni contained a gene that
encodes a potentially functional Hao protein, reinforcing the
hypothesis that horizontal transfer of the hao gene has likely
occurred more than once between obligate or facultative litho-
Third, an hao-orf2 gene tandem was identified in the Methy-
lococcus capsulatus genome. Thus, HAO may be the agent that
ameliorates the potential toxicity of hydroxylamine produced
by a substrate-promiscuous (ammonia-oxidizing) pMMO of
this and related type X and type I methanotrophs. We note
that the Hao proteins of the non-aAOB lack N- and C-terminal
regions, which are thought to be involved in docking for elec-
tron transfer to cytochrome c554(27) or membrane association
in the vicinity of cm552and ubiquinone. The putative absence of
the electron transfer to ubiquinone would deprive the system
of reductant for pMMO and force electrons (from hydroxyl-
amine) to cytochrome oxidase, bypassing the proton motive
force-generating cytochrome bc1complex. These factors may
account for the inability of M. capsulatus to grow on ammonia
and CO2despite having a functional Calvin cycle (6).
Fourth, the inability of M. capsulatus to function as an
aAOB, the presence of a diverse catabolic genomic inventory
in the M. capsulatus genome (59), and because pMMO from M.
capsulatus is more closely related to AMO from gamma-aAOB
than to pMMO from alphaproteobacterial type II meth-
anotrophs (40) suggest that hao-orf2 and other genes otherwise
found only in aAOB (e.g., the operon containing a multi-
copper oxidase-encoding gene and orf5 that flanks the amo
operon in aAOB [Klotz, unpublished]) likely arrived in the
ancestor of the Methylococcaceae by transfer from donors such
Cytochrome cm552, the product of the fourth gene in the hao
gene cluster, has been grouped within a large family of mem-
brane-associated tetraheme and pentaheme cytochromes that
includes NirT of Pseudomonas stutzeri and NapC of Paracoccus
denitrificans, several of which have been shown to carry elec-
trons from a pool of quinols in the plasma membrane to nitrite
reductase, nitrate reductase, or other periplasmic reductases
during anaerobic respiration (22). It is thus not surprising that
the NapC-TorC-NirT proteins have considerable overall se-
quence similarity except for their C-terminal regions, perhaps
reflecting differences in their periplasmic electron acceptors
(44), which include oxidoreductases containing heme, flavin, or
molybdopterin (22). In contrast to the NapC-TorC-NirT pro-
teins, cm552cytochromes apparently relay electrons from HAO
via cytochrome c554to the membrane quinone/quinol pool.
Our initial sequence analysis showed that cytochromes cm552
from aAOB have much greater sequence similarity to one
another than to any member of the NapC-TorC-NirT protein
family. The octaheme HAO of N. europaea and the formate-
dependent pentaheme c-cytochrome nitrite reductase (NrfA)
have been shown to have congruent heme stacking arrange-
ments and secondary and tertiary structures (13, 14, 22) as well
as common N-oxide intermediates. Although hydroxylamine is
a bound intermediate during the reduction of nitrite to ammo-
nia by extant NrfA proteins (13), free hydroxylamine also re-
acts with the enzyme. The Nrf systems were of interest to us
because they operate in numerous bacteria, including the Pro-
teobacteria and Bacteroidetes (51). Of particular interest was
the Nrf system from the Delta- and Epsilonproteobacteria, such
5378 BERGMANN ET AL.APPL. ENVIRON. MICROBIOL.
as Wollinella succinogenes and Sulfurospirillum delelyianum,
since they are likely more ancient and appear to be structurally
simpler. Delta- and epsilonproteobacterial NrfAH systems in-
clude only two interacting proteins, whereas the gammapro-
teobacterial system consists of four proteins, NrfABCD (51).
Hence, we hypothesized that the HAO-c552and NrfA-NrfH
redox chains are evolutionarily related. Noting that the con-
served heme stacking in HAO/NrfA was also seen in the oc-
taheme tetrathionate reductase (Ttr) (38), we hypothesized
that HAO evolved in parallel with tetrathionate reductase
from a common ancestor of extant delta- and epsilonpro-
teobacterial NrfA cytochromes. This hypothesis meant in gen-
eral terms that the inventory involved in nitrification evolved
partly from nitrogen and sulfur-based anaerobic respiration.
There is evidence that nitrite respiration has preceded oxygen
respiration (46), and it has been suggested that the genomic
inventory needed for denitrification was established long be-
fore molecular oxygen was available to allow nitrification (34).
Our analysis revealed homology between Hao, Ttr, and
NrfA proteins (Fig. 5). Based on the phylogenetic tree (Fig. 5),
we propose here that the octaheme Hao proteins have evolved
in parallel with octaheme tetrathionate reductases from a com-
mon ancestor octaheme reductase protein in Delta- and Epsi-
lonproteobacteria. The latter protein is thought to have
emerged by fusion/incomplete duplication processes from a
pentaheme nitrite reductase. The pentaheme nitrite reductase
systems are found in extant Delta-, Epsilon-, and Gammapro-
teobacteria (Fig. 5); however, they are absent from Alpha- and
Betaproteobacteria. It cannot be concluded from the current
data whether the genes were lost in these classes during the
radiation of the Proteobacteria or whether these enzymes ex-
panded horizontally only into the Gammaproteobacteria, as was
likely the case for the nrf operon found in the Bacteriodetes and
Actinobacteria (Fig. 5).
The quinol dehydrogenase in Delta- and Epsilonproteobac-
teria that delivers electrons directly to NrfA by forming a stable
complex with NrfA is the membrane-bound cytochrome c
NrfH (14, 51). Encouraged by the discovered homology be-
FIG. 5. Most parsimonious phylogenetic tree constructed from 36 ClustalX-aligned sequences of multi-heme cytochrome proteins in the
proposed tetrathionate reductase (Ttr), hydroxylamine oxidoreductase (Hao), and formate-dependent nitrite reductase (Nrf) protein family. The
tetrathionate reductase and hydroxylamine oxidoreductase protein subfamilies likely evolved from a common ancestral octaheme cytochrome c
reductase (?) that emerged from the formate-dependent pentaheme nitrate reductase (Nrf) systems of ancestral delta-/epsilonproteobacteria.
Bootstrap values for the clades are shown at the branch points. Protein accession numbers are indicated.
VOL. 71, 2005hao GENE CLUSTER CONSERVATION IN NITRIFYING BACTERIA5379
tween HAO and NrfA, we decided to include all known NrfH
sequences (and gammaproteobacterial NrfB homologues) in
our phylogenetic analysis of the cm552proteins from aAOB. By
way of verifying our hypothesis, the analysis revealed homology
between cm552and NrfH/B proteins (Fig. 6), and we propose
here that the gene cluster including hao and cm552-encoding
genes in extant aAOB evolved from ancestors of the nrfA-nrfH
operon, found in extant Delta- and Epsilonproteobacteria. The
data depicted in Fig. 6 also suggest that the ancestral cm552/
NrfH/NrfB protein may itself have a common ancestor with
other members of the NapC-TorC-NirT protein family before
being recruited into an operon with NrfA.
The present work established for the first time a molecular
evolutionary basis for the congruent heme stacking in HAO,
cytochrome c nitrite reductase, and tetrathionate reductase.
Despite conserved heme stacking, secondary and tertiary struc-
ture, HAO and cytochrome c554of Nitrosomonas (27, 28) and
the tetraheme portion of fumarate reductase (49) did not dis-
play high enough sequence similarity with Hao to justify their
inclusion into a phylogenetic analysis. In marked contrast to
the observed distribution of hao-orf2 gene tandems in aAOB
and non-aAOB, cytochrome c554(cycA) has been found only in
aAOB. The ancestry of the cycA (cytochrome c554) gene of the
hao gene cluster and the tetra-c-heme domain of the periplas-
mic fumarate reductase remain interesting unknowns.
Our data and their interpretation suggest that the hao gene
cluster has evolved as a structural and functional unit from
pentaheme nitrite reductase under pressure of increasing hy-
droxylamine concentrations associated with more efficient am-
monia catabolism. Factors including the intersubunit covalent
cross-linking of a tyrosine to the catalytic heme and the expan-
sion of the pentaheme to the octaheme configuration likely
contributed to the conversion of an ancient electron disposal
system into a mechanism capable of using hydroxylamine as an
electron donor at the ubiquinone level. We propose that ex-
pansion of the ancient hao-cycB tandem into a gene cluster
with four genes (hao-orf2-cycA-cycB) took place rather early in
purple sulfur bacterial ancestors of extant gamma-aAOB in the
family Chromatiaceae. The conserved organization and high
sequence similarity of the hao gene cluster in a single family
each of the Gamma- and Betaproteobacteria suggests that the
hao gene cluster arrived in the common ancestor of the beta-
aAOB likely by a lateral gene transfer event before extensive
speciation of these nitrifiers. Because one of three copies each
FIG. 6. Unrooted most parsimonious phylogenetic tree constructed from 56 ClustalX-aligned protein sequences of the membrane-associated
tetraheme cytochromes in the proposed Cm552/NrfB/H&NapC/TorC/NirT protein family. The clade with members of the NapC/TorC/NirT
protein subfamily was collapsed to focus on the clade with the members of the Cm552/NrfB/H protein subfamily in the phylogram. Bootstrap values
for the clades are shown at the branch points. See Materials and Methods for accession numbers not provided in the figure.
5380BERGMANN ET AL.APPL. ENVIRON. MICROBIOL.
of the hao and amo gene clusters are adjacent in the genome
of N. multiformis, it is possible that amo and hao gene clusters
were jointly transferred as a structural and functional meta-
bolic unit before operon duplication led to multiple copies in
betaproteobacterial aAOB. The observed differential regula-
tion of hao and cycAB genes in N. europaea (8, 21, 47), the
unique presence of orf3 in the hao gene cluster in N. multifor-
mis, and the low sequence identity (below 50%) between Orf2
protein homologues (Table 1) suggest that the region between
the hao and cycA genes in the hao gene cluster has experienced
change at higher rates since the hao cluster evolved indepen-
dently in the gamma- and betaproteobacterial lineages of the
In addition to improving our understanding of the molecular
evolution of nitrification, our data have significance for molec-
ular ecology. The presence of unique and highly conserved
regions within the Hao and cytochrome c554proteins of N.
europaea, N. oceani, and N. multiformis suggests that cycA
genes could be used to design new PCR primers and molecular
probes to amplify and detect this gene in order to better assess
and characterize the diversity of aerobic aAOB in environmen-
tal samples. To date, the presence of aAOB within both the
Beta- and Gammaproteobacteria precludes that a single 16S
rRNA sequence-based set of primers (or probe) can be em-
ployed for detection/identification of both beta- and gamma-
proteobacterial aAOB. Further, the rather low variability of
16S rRNA genes within the betaproteobacterial aAOB makes
it difficult to separate the sequences of PCR products amplified
from different strains of aAOB (40). A partial solution to this
problem has been the design of PCR primers to amplify that
part of the amoA gene which encodes the 27-kDa subunit of
AMO in aAOB. However, the amoA gene sequences of gam-
maproteobacterial aAOB are more similar to gammapro-
teobacterial pmoA sequences than they are to amoA sequences
of Betaproteobacteria (43); hence, different PCR primer sets
must be used to obtain amoA-based amplicons from Betapro-
teobacteria and Gammaproteobacteria. The present demonstra-
tion that cycA is unique to aAOB suggests that PCR primer
sets and probes based on cycA gene sequences may be very
useful tools for estimating aAOB diversity in the environment.
We thank Mike Morton for culturing and Vilasack Thamvongsa for
preparation of genomic DNA from N. oceani C-107SW, Briana Jack-
son for help in primer design, David Arciero for advice on the protein
isolation, and Eric Ecleston for preparing protein samples for sequenc-
ing. Our thanks also go to Amal El-Sheikh for culturing of and
genomic DNA isolation from N. oceani ATCC 19707.
This project was supported, in part, by DOE’s G2L initiative, incen-
tive funds provided to M.G.K. by the University of Louisville VPR
office, andfunds to A.B.H.
95ER20191.A004) and NSF (MCB-0093447).
ADDENDUM IN PROOF
Genes of putative Hao proteins not affiliated with orf2 genes
have been identified in three additional genomes: Rhodoferax
ferrireducens (IMG500457640), Anaeromyxobacter dehaloge-
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