MINIREVIEW
Nitri¢er genomics and evolution of the nitrogen cycle
Martin G. Klotz1,2,3 & Lisa Y. Stein4
1
Evolutionary and Genomic Microbiology Laboratory, Department of Biology, University of Louisville, Louisville, KY, USA; 2Department of Microbiology
and Immunology, University of Louisville, Louisville, KY, USA; 3Center for Genetics and Molecular Medicine, University of Louisville, Louisville, KY, USA;
and 4Department of Environmental Sciences, University of California, Riverside, CA, USA
Correspondence: Martin G. Klotz,
Department of Biology, University of
Louisville, 139 Life Science Building, Louisville,
KY 40292, USA. Tel.: 11 502.852.7779;
fax: 11 502.852.0725; e-mail:
[email protected]
Received 20 August 2007; accepted
25 September 2007.
First published online December 2007.
DOI:10.1111/j.1574-6968.2007.00970.x
Editor: Rustam Aminov
Keywords
nitrogen cycle; nitrification; denitrification;
anammox; catabolic modules; evolution.
Abstract
Advances in technology have tremendously increased high throughput whole
genome-sequencing efforts, many of which have included prokaryotes that
facilitate processes in the extant nitrogen cycle. Molecular genetic and evolutionary
analyses of these genomes paired with advances in postgenomics, biochemical and
physiological experimentation have enabled scientists to reevaluate existing
geochemical and oceanographic data for improved characterization of the extant
nitrogen cycle as well as its evolution since the primordial era of planet Earth.
Based on the literature and extensive new data relevant to aerobic and anaerobic
ammonia oxidation (ANAMMOX), the natural history of the nitrogen-cycle has
been redrawn with emphasis on the early roles of incomplete denitrification and
ammonification as driving forces for emergence of ANAMMOX as the foundation
for a complete nitrogen cycle, and concluding with emergence of nitrification in
the oxic era.
Introduction
The flux of nitrogen through the global biogeochemical
nitrogen cycle has undergone dramatic alterations in the
past few decades (Galloway & Cowling, 2002). Over half of
the fixed nitrogen that annually enters terrestrial ecosystems
now has its origins in anthropogenic processes including
production of ammonia-based fertilizers via the Haber–
Bosch process, cultivation of nitrogen-fixing crops, and the
combustion of fossil fuels leading to the release of nitrogen
oxides (Nevison & Holland, 1997; Galloway & Cowling,
2002). The majority of processes in the extant global
biogeochemical nitrogen cycle are facilitated by bacteria
including: (1) N2 fixation, (2) nitrification and (3) denitrification (Fig. 1). A fourth process, anaerobic ammonia
oxidation (ANAMMOX), is a more recently described
bacterial contribution to the nitrogen cycle (Dalsgaard
et al., 2005; Jetten et al., 2005).
The process of N2 fixation is carried out by a variety of
bacteria that use nitrogenase, in concert with other enzymes
and cofactors, to facilitate the highly endergonic breakage of
the triple bond in dinitrogen to yield ammonia (Postgate,
1970). Bacterial nitrification proceeds by the sequential
oxidation of ammonia to nitrite predominantly by ammonia-oxidizing bacteria (AOB) and of nitrite to nitrate by
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nitrite-oxidizing bacteria (NOB) (Prosser, 1989). However,
there is recent evidence that a number of Crenarchaea,
abundant in soils, estuarine and marine environments, are
also capable of nitrification (Könneke et al., 2005; Francis
et al., 2007) by a different mechanism than that of AOB
(Arp et al., 2007). Nitrification, whether facilitated by
bacteria or archaea can proceed only in oxic environments
or in anoxic environments by select species given an external
supply of NO2 (N2O4) (Schmidt et al., 2001). Nitrate and
nitrite are substrates for denitrification, the process that
recycles fixed nitrogen back to gaseous dinitrogen. Denitrification is facilitated by either anaerobic respiration of
bacteria in anoxic environments, i.e. ‘complete’ or ‘canonical’ denitrification (Zumft, 1997; Brandes et al., 2007), or by
reductive detoxification of nitrite to nitrous oxide in aerobic
environments, i.e. ‘incomplete’ or ‘nitrifier’ denitrification
(Lipschultz et al., 1981). ANAMMOX, performed by anaerobic ammonia-oxidizing bacteria (ANAOB), couples the
oxidation of ammonia to the reduction of nitrite to produce
dinitrogen in anoxic ecosystems. The release of ammonia
during degradation of organic matter, as well as assimilatory
and respiratory reduction of nitrate (or nitrite) to ammonia,
i.e. ammonification, are also biotic contributions to the nitrogen cycle performed by bacteria, fungi and plants (Fig. 1c).
Ammonification and nitrification can be regarded as short
FEMS Microbiol Lett 278 (2008) 146–156
147
N-cycle evolution
(a)
(b)
(c)
Fig. 1. Key steps in the evolution of the nitrogen cycle. (a) Very early anaerobic nitrogen metabolism, in which NO
2 and NO3 are produced by
combustion and NH3 is mainly generated from N2 at hydrothermal vent sites: Incomplete respiratory denitrification (MGD-NarGH/NapAB; cd1-NirS) and
respiratory nitrite ammonification [pentaheme-NrfAH (NrfABCD)]. See text for references. (b) Early anaerobic nitrogen metabolism: To avoid NH2OH
poisoning, invention of hydroxylamine dehydrogenation (HAO/HZO) and reduction (prismane protein), which replenished nitrite and ammonia pools.
Resulting emergence of functional HURM and hydrazine hydrolase constitute ANNAMOX process and thus complete recycling of fixed nitrogen. (c) Late
anaerobic and early aerobic nitrogen metabolism: Emergence of assimilatory ammonification (siroheme cytochrome c NIR) and N2 fixation to satisfy
increased ammonia demand. Emergence of heme-copper and copper redox centers and subsequent diversification of anaerobic and aerobic respiration
(Cu-NirK, HCOs, NOR), complete denitrification (Cu–NOS) as well as aerobic methane- and ammonia oxidation (pMMO/AMO) and thus closure of the
biotic nitrogen cycle. Elevated ammonia input from anthropogenic sources leads to elevated nitrifier denitrification in today’s nitrogen cycle.
circuits that bypass the vast dinitrogen reservoir connected
in the present cycle by denitrification and nitrogen fixation
(Fig. 1c). In addition to these biotic processes, the nitrogen
cycle is amended by abiotic processes including ammonia
production from N2 (Brandes et al., 1998; Wachtershauser,
2007 and references therein) at hydrothermal vents, the
oxidation of N2 to nitrite and nitrate by combustion (Yung
& McElroy, 1979; Mancinelli & McKay, 1988; Kasting, 1993;
Navarro-González et al., 2001) and mineralization (McLain
& Martens, 2005).
Enzymes of ammonia oxidation
Knowledge of the nitrification process and its best-studied
facilitators, the AOB and NOB, goes back more than 100
FEMS Microbiol Lett 278 (2008) 146–156
years to the work of Winogradsky (1892). In AOB catabolism, ammonia is first aerobically oxidized to hydroxylamine
by ammonia monooxygenase (AMO) followed by the dehydrogenation of hydroxylamine to nitrite by hydroxylamine
oxidoreductase (HAO), which is proposed to relay the four
extracted electrons to the ubiquinone pool via two interacting cytochromes, c554 and cM552 (Fig. 2; Hooper et al.,
2005). Owing to its soluble nature, HAO is the best-studied
functional component in the nitrification process followed
by cytochrome c554 (Hooper et al., 2005 and references
therein). Both proteins have been crystallized and their
structures resolved (Igarashi et al., 1997; Iverson et al.,
2001). In contrast, AMO, a multimeric transmembrane
copper-enzyme, has yet to be functionally isolated, crystallized and its structure solved. The process of AMO reduction
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148
M.G. Klotz & L.Y. Stein
Fig. 2. Flow of energy and reductant in the nitrification process through pertinent catabolic modules in ammonia-oxidizing (AOB) and nitrite-oxidizing
(NOB) bacteria. Solid lines show experimentally verified reactions, dotted lines with question marks indicate lack of experimental verification of reactions
assumed to occur (Hooper et al., 2005; Arp et al., 2007). REDOX reactions via dashed lines indicate that multiple nonidentical copies of c552 are
required to interact with a diverse set of reaction partners. The circuit for reverse electron flow was omitted. Colored backgrounds were used to
highlight the cyclic electron flow module (see also Fig. 3) as well as the hydroxylamine/hydrazine-ubiquinone-redox-module, HURM. Blue-colored boxes
indicate copper-dependent enzyme complexes. (c)aa3, cytochrome (c)aa3; bc1, cytochrome bc1 (complex III); NirK, Cu-dependent nitrate reductase;
c 0 -b, cytochrome c 0 -b; c550, cytochrome c550; c552, cytochrome c552; cM552, cytochrome cM552; c554, cytochrome c554; NXR, nitrite
oxidoreductase; P460, cytochrome P460; PMF, proton-motive force; Q/QH2, ubiquinone-ubiquinol pool; sNOR, cNOR, ccNOR, nitric oxide reductase
with differing electron acceptor mechanisms. See text for further details.
by electrons obtained from ubiquinone, which it needs to
oxidize ammonia, remains elusive and the study of AMO is
by far less progressed compared with the other functional
players involved in ammonia oxidation by AOB (Arp et al.,
2007).
Whereas the sequence availability of individual genes
encoding AMO and HAO led to a surge in information
about AOB distribution and abundance through design and
use of molecular probes (Purkhold et al., 2000; Kowalchuk &
Stephen, 2001 and references therein), aspects of the molecular biology and biochemistry of these organisms aside
from carbon assimilation and use of ammonia as an energy
source have received little attention. In particular, very little
to nothing is known about the regulation of gene expression
required for nitrifier denitrification by AOB (Fig. 2). This is
surprising given that nitrifier denitrification produces reactive nitrogen species (RNS), competes for electrons with
primary bioenergetic processes such as reverse electron flow
and the electron transport chain that produces proton
motive force (PMF), and may reduce the availability of
nitrite, the catabolic substrate to NOB (Fig. 2).
ANAOB, on the other hand, were discovered less than two
decades ago (Dalsgaard et al., 2005; Jetten et al., 2005) such
that the molecular mechanisms and genetic inventory
involved in ANAMMOX (Fig. 3) are just beginning to take
shape (Strous et al., 2006). Although AOB and ANAOB both
oxidize ammonia as their primary source for energy and
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reductant, they interconnect the pools of fixed and gaseous
nitrogen in different metabolic contexts. Whereas aerobic
AOB and NOB oxidize ammonia to nitrate collaboratively
and independently of denitrification (Fig. 2), ANAOB
combine nitrification (ammonia oxidation) and denitrification (nitrite reduction, dinitrogen production) activities in a
single process (Fig. 3; Kartal et al., 2007).
Evolutionary relatedness of ammoniaand methane oxidation
Nitrification, denitrification and ANAMMOX have always
been understood per se as different processes performed by
different organisms with different evolutionary histories.
Because the mechanism of ANAMMOX has been worked
out only recently (Strous et al., 2006; Kartal et al., 2007),
little attention has been given to its evolution. Further, the
great diversity of denitrifying organisms has prevented
formulation of a plausible model for the evolution of
denitrification. In contrast, many have speculated on the
evolution of nitrification mostly by focusing on AMO
(Teske et al., 1994; Holmes et al., 1995; Klotz & Norton,
1998; Purkhold et al., 2000; Norton et al., 2002). It was
found that AMO is homologous with particulate methane
monooxygenase (pMMO), a copper enzyme like AMO that
carries out an analogous function in methane-oxidizing
bacteria (MOB, Hanson & Hanson, 1996; Murrell &
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N-cycle evolution
et al., 2004; Bergmann et al., 2005). This was an important
finding because it cemented the importance of HAO as a
central player in the ammonia-oxidizing pathway of both
AOB and MOB. Although the processes of chemolithotrophic ammonia-catabolism and chemoorganotrophic
methanotrophy are evolutionarily linked by common descent of the nitrification module (AMO/pMMO, HAO and
electron-transferring cytochromes), they represent totally
different catabolic lifestyles, which is a conundrum to some
and a beautiful demonstration of prokaryotic diversity
created by the modular evolution of catabolism (Spirin
et al., 2006 and references therein) to others including these
authors.
Nitrogen cycle evolution on the anoxic
earth
Fig. 3. Comparison of cyclic electron flow modules, HURM (hydroxylamine/hydrazine-ubiquinone-redox-module) and the flux of nitrogen in
(a) ANAOB (Kuenenia stuttgartiensis) and (b) AOB (Nitrosococcus oceani). Dashed lines represent external source and sink for NO2 and NO,
respectively, in the proposed NOx cycle of AMO under anoxic conditions
(Schmidt et al., 2001); dotted lines indicate lack of experimental verification of reactions assumed to occur (Hooper et al., 2005; Strous et al.,
2006; Arp et al., 2007; Kartal et al., 2007). Broken lines indicate
alternative start points of linear electron flow pathways. bc1, cytochrome
bc1 (complex III); HZH, hydrazine hydrolase; HZO, hydrazine oxidoreductase; NirS, Fe-cytochrome cd1 nitrite reductase; PMF, proton-motive
force; Q/QH2, ubiquinone-ubiquinol pool. See text for further details.
Holmes, 1996). While the first published hypothesis on the
evolutionary relatedness of AMO and pMMO relied on an
AmoA sequence from the g-AOB Nitrosococcus oceani that,
in hindsight, turned out to be a PmoA of a contaminating
MOB in the culture, later comparisons of available AmoA
and PmoA sequences and enzyme properties confirmed that
AMO and pMMO are, indeed, homologous enzymes (Lontoh
et al., 2000; Purkhold et al., 2000; Norton et al., 2002).
Although it has been known for over a decade that MOB
contribute both to nitrification (Hanson & Hanson, 1996)
and nitrifier denitrification (Sutka et al., 2003 and references
therein), genes involved in this process, aside from MMO,
were only recently identified (Ward et al., 2004; Bergmann
et al., 2005). Hydroxylamine oxidation in Methylococcus
capsulatus Bath was initially thought to be performed solely
by cytochrome P460, a monoheme cytochrome capable
of oxidizing hydroxylamine and nitric oxide in both AOB
(Fig. 2) and MOB (Bergmann et al., 1998; Sutka et al., 2003;
Hooper et al., 2005). Recent findings, however, assigned
cytochromes P460 and c 0 -b a NO-detoxification role (Fig. 2;
Elmore et al., 2007), an important function to nitrifier
denitrification. Analysis of the M. capsulatus Bath genome
sequence has now established residence of HAO in this
methanotroph in addition to cytochrome P460 (Ward
FEMS Microbiol Lett 278 (2008) 146–156
A major milestone on the path to understanding the
evolution of the nitrogen cycle was the recent availability of
genomes from ecotypically different AOB [freshwater sediment/soil, Nitrosomonas europaea (Chain et al., 2003);
marine, Nitrosococcus oceani (Klotz et al., 2006); sewage,
Nitrosomonas eutropha (Stein et al., 2007); soil, Nitrosospira
multiformis (J.M. Norton et al., unpublished data)] as well
as that of an ANAOB, Kuenenia stuttgartiensis (Strous et al.,
2006). These genomes made it possible to distill common
catabolic denominators and differences in aerobic and
ANAMMOX. The genome inventories combined with existing literature on the genetics and physiology of AOB and
ANAOB allowed identification of individual catabolic modules including the definitive catabolic core to extract and
recycle electrons from ammonia (Figs 2 and 3), a finding
that has sparked efforts to reformulate the evolutionary
history of the nitrogen cycle.
Diverse speculations on the emergence of both biotic and
abiotic components of the biogeochemical nitrogen cycle,
based on geochemical and oceanographic data (i.e. chemical
composition and isotopic signatures in sediments) over
geological time scales, have been presented (Mancinelli &
McKay, 1988; Falkowski, 1997; Brandes et al., 1998; Navarro
-González et al., 2001; Raymond et al., 2004; Canfield et al.,
2006; Wachtershauser, 2007 and references therein). Some
authors interpreted these data as evidence for a late emergence of denitrification; even after nitrification became
established in the post-Neoproterozoic era during the successive oxygenation of the world’s oceans (Falkowski, 1997).
Furthermore, some authors proposed that N2 fixation was a
very early evolutionary development urgently needed because of the rapid depletion of the primordial fixed nitrogen
pool (Navarro-González et al., 2001). While iron and
molybdenum, key trace metals for nitrogenase function,
were likely abundant in the Archaean era (Anbar & Knoll,
2002; Canfield et al., 2006 and references therein), the
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150
encoding gene complexity and high cost of the N2 fixation
process were likely prohibitive to early evolution and selection of this function (Mancinelli & McKay, 1988). Ongoing
N2 fixation, even with the less efficient Fe-nitrogenase
(rather than Fe–Mo nitrogenase) and at a slower pace than
in modern times, would have led to a remarkable decrease of
N2 in the atmosphere in the absence of sufficient recycling
by denitrification or ANAMMOX. Although the wide taxonomic distribution of nif genes in extant bacteria and
archaea has been a frequent argument for early emergence
of N2 fixation (Falkowski, 1997; Navarro-González et al.,
2001), nif genes are clustered and hence prone to lateral
transfer (Raymond et al., 2004). Finally, the lack of N2-fixing
organelles in eukaryotes provides an additional argument
against an early evolution of the N2 fixation inventory in
prokaryotes (McKay & Navarro-González, 2001). Therefore,
the following postulates are in congruence with the hypothesis of a late emergence of N2 fixation and early emergence
of denitrification (Mancinelli & McKay, 1988; Capone, 2000;
Capone et al., 2006; Canfield et al., 2006), occurring soon
after the stabilization of the independent lineages (domains)
of cellular life (Koonin & Martin, 2005; Wachtershauser,
2007 and references therein) and when nitrogen flux was still
largely controlled by abiotic activities (Yung & McElroy,
1979; Mancinelli & McKay, 1988).
In the modern nitrogen cycle (Fig. 1c), only denitrification in a wider sense is suited to return fixed nitrogen and
sustain the atmospheric N2 pool (Capone et al., 2006);
however, an often overlooked point is that not all enzymes
that function in the extant nitrogen cycle were likely around
during the anoxic Archaean and reducing Proterozoic eras
because their metal cofactors were not widely available.
Based on pertinent isotope signatures, enzymes with nickel
(e.g. hydrogenase), iron (e.g. sulfur–iron and cytochrome c
proteins) and molybdenum (e.g. formate dehydrogenase
and nitrate reductase) cofactors were likely functional in
the Archaean, while enzymes with Class B transition metals
such as copper, zinc and cadmium were not. Class B
transition metals likely became even scarcer in the Proterozoic era, in which the sulfidic nature of the oceans locked
metals like copper into biounavailable sulfidic minerals
(Lewis & Landing, 1992; Canfield, 1998; Anbar & Knoll,
2002; Poulton et al., 2004).
Because copper was not bioavailable to serve as a redox
cofactor in catalysis, present day nitrous oxide reductase
(NOS), a copper enzyme found in all extant canonical
denitrifiers that produce dinitrogen (Zumft, 1997; Brandes
et al., 2007), either was (1) preceded by a functional
noncopper NOS lost from or not yet recognized in current
genome inventories, (2) evolved from a noncopper NOS, or
(3) was a de novo invention of the oxic era (Klotz, NSF
Microbial Genome Sequencing Program workshop 2007,
San Diego, CA). The authors favor the last hypothesis since
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M.G. Klotz & L.Y. Stein
early denitrifiers would have had a sufficient NO
3 pool to
support anaerobic respiration (Fig. 1a) and incomplete
denitrification lacking NOS activity argues against an efficient early N2 fixation process. Early emergence of both N2
fixation and incomplete denitrification would have caused
marked depletion of the atmospheric N2 pool for which
there are no supporting data (Capone & Knapp, 2007).
In addition, the authors support the hypothesis of
Mancinelli & McKay (1988) that ammonification was a key
process providing fixed-nitrogen to early microbiota in the
early nitrogen cycle rather than N2 fixation (Fig. 1a).
Respiratory nitrite ammonification, the electrogenic reduction of nitrite to ammonia via formate or H2, is facilitated by
pentaheme cytochrome c nitrite reductases (NrfABCD/
NrfAH) found in a wide range of obligate and facultative
chemolithotrophic bacteria (Simon, 2002). With abundant
nitrate, NrfAH could receive nitrite from the respiratory,
membrane-associated, Mo-containing, nitrate reductase
(NarGHJI) or with scarce nitrate from the Mo-containing
periplasmic nitrate reductase (NapFDAGHBC) (Lin & Stewart,
1998; Gunsalus & Wang, 2000; Simon, 2002; McLain &
Martens, 2005). The NrfAH complex, whose structure has
been solved from several Proteobacteria (Einsle et al., 2002;
Rodrigues et al., 2006), functions independently of oxygen
and is dependent on the availability of heme-iron; requirements likely met in the Archaean era.
In addition to nitrite reduction, extant NrfA (EC 1.7.2.2)
also reduces nitric oxide and hydroxylamine to ammonia
without the release of RNS intermediates (Simon, 2002 and
references therein). This may not have applied to early NrfA,
however, which may have leaked catalytic intermediates
including hydroxylamine (Fig. 1b). Because hydroxylamine
is a potent mutagen, its production would have provided
functional pressure for the evolution of two unrelated,
oxygen-independent, catalytic complexes capable of
scavenging hydroxylamine, and hence, the evolution of the
ANAMMOX process (Fig. 1b). The first hydroxylaminescavenging complex is the ammonia-forming hydroxylamine reductase (EC 1.7.99.1), a soluble iron–sulfur protein
known as prismane or hybrid-cluster protein as it contains a
combination of a 4Fe–4S cluster and a novel 4Fe–2S–2O
cluster (Pino et al., 2006). Genes encoding prismane protein
are found in the genomes of many extant strict and
facultative anaerobic bacteria and archaea. The second
complex is the hydroxylamine/hydrazine oxidoreductase,
an octaheme cytochrome c dehydrogenase (Hooper et al.,
2005), found in both AOB and ANAOB.
The hydroxylamine/hydrazine oxidoreductase is called
HAO in AOB where it oxidizes hydroxylamine to nitrite
(Hooper et al., 2005), and HZO in ANAOB where it oxidizes
hydrazine to N2 (Schalk et al., 2000). Biochemically, HAO
is currently misclassified as both an oxygen-dependent
enzyme (EC 1.7.3.4) and a ‘hydroxylamine (acceptor)
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N-cycle evolution
oxidoreductase (EC 1.7.99.8).’ Because HAO/HZO interact
with cytochromes c as acceptors and both are hydroxylamine/hydrazine (donor) oxidoreductases (Fig. 3), a reclassification as ‘EC 1.7.2./’ is in order. Electron flow from
hydroxylamine to ubiquinol in AOB involves both a cyclic
path, to provide reductant to AMO, and a linear path for the
generation of PMF (Fig. 3; Arp et al., 2007). Comparison of
the cyclic electron flow in AOB and ANAOB (Strous et al.,
2006) revealed a striking similarity and a central positioning
of HAO/HZO (Fig. 3). HAO and HZO are functional
analogs as both dehydrogenate hydroxylamine and hydrazine and deliver the extracted electrons to ubiquinol via
cognizant cytochromes c. Thus, HAO and HZO are interchangeable, and define the hydroxylamine/hydrazine-ubiquinol redox module (HURM) of AOB and ANAOB (Figs 2
and 3; Klotz, NSF Microbial Genome Sequencing Program
workshop 2007, San Diego, CA).
Whereas the evolutionary history of hydroxylamine reductase is still elusive (Cabello et al., 2004; Pino et al., 2006),
sequence and phylogenetic analyses link the evolution of
HAO to pentaheme cytochrome c nitrite reductase, NrfA
(Bergmann et al., 2005). Recent analysis of HZO with other
putative octaheme cytochrome c oxidoreductases revealed
that HAO and HZO are homologs evolving from NrfA via
N-terminally expanded octaheme cytochrome c nitrate reductases found in a few sulfur-dependent Deltaproteobacteria
(Geobacter, Pelobacter) and the g-proteobacterial genus
Thioalkalivibrio, purple sulfur bacteria like the g-AOB
Nitrosococcus (M.G. Klotz et al., unpublished data). This
evolutionary decent of HAO/HZO with a common root in
octaheme cytochrome c oxidoreductases of sulfur-dependent
nitrite-respiratory bacteria is remarkable as it reflects the
selection of inventory capable of hydroxylamine detoxification formed as a byproduct of nitrite reduction by NrfA and
represents an evolutionary path supportive of an early
incomplete denitrification pathway depicted in Fig. 1a and b.
Although likely not suitable to sustain global primary
production on the early Earth (Canfield et al., 2006), it is
proposed that ANAMMOX evolved shortly after the incomplete denitrification pathway with the inclusion of Fedependent cytochrome cd1 nitrite reductase (NirS) and a
unique protein, hydrazine hydrolase (Strous et al., 2006).
According to this model, ANAMMOX likely provided the
first complete recycling of fixed nitrogen to the dinitrogen
pool (Dalsgaard et al., 2005) and fulfilled this role until the
emergence of copper enzymes like Cu-NOS; NirK, the
copper-dependent version of nitrite reductase (Cantera &
Stein, 2007b); and the many members of the heme-copper
oxidase (HCO) superfamily (Garcia-Horsman et al., 1994).
At the same time, early evolution of HURM by ANAMMOX-performing microorganisms also provided the
opportunity for later utilization of hydroxylamine as a
reductant and energy source in oxic environments by AOB,
FEMS Microbiol Lett 278 (2008) 146–156
leading to the evolution of nitrification and closure of the
nitrogen cycle (Fig. 1c, Klotz, NSF Microbial Genome
Sequencing Program workshop 2007, San Diego, CA).
The closed nitrogen cycle: emergence of
copper enzymes and nitrification
Aerobic ammonia oxidation likely evolved into an efficient
catabolic process only after a large enough pool of reduced
inorganic nitrogen became available to sustain it (Fig. 1c).
Aerobic ammonia oxidation also likely coincided with or
quickly succeeded the evolution of copper-containing enzymes, which necessarily required the oxygenation of the
Earth’s oceans to release bio-available copper. The most vital
copper-containing enzymes in AOB are the O2-dependent
AMO and oxygen-reducing terminal HCOs at the beginning
and the end, respectively, of the electron transport chain
(Fig. 2). Availability of copper likely also promoted the
evolution of a large amount of multicopper blue proteins
(Nakamura & Go, 2005) including Cu-NirK (Cantera &
Stein, 2007b). Likewise, the emergence of Cu-NOS and its
lateral distribution in many prokaryotes, but not AOB and
ANAOB, led to the highly efficient complete denitrification
process, as it is known today.
AMO and pMMO are homologous and the authors
support de novo emergence of their ancestor, a promiscuous
ammonia-methane monooxygenase, in a-proteobacterial
MOB and its holophyletic evolution or early lateral dispersal
to recipients in the g-proteobacterial orders Chromatiales
(Nitrosococcus) and Methylococcales (all g-MOB). Once in
place as a modular extension of existing catabolic units,
AMO (to extend HURM) and pMMO (to extend methanol
catabolism) were subject to niche-dependent adaptation
towards higher affinity for the substrate best suited to the
ammonia- or methane-catabolic lifestyle in g-proteobacterial AOB or MOB (Arp et al., 2007). The authors further
support lateral transfer of the ammonia-oxidizing inventory
to the ancestor of the b-proteobacterial family of the
Nitrosomonadaceae from g-AOB. This model is supported
by recent comparative analyses of nitrifier genomes (Arp
et al., 2007) and by the enzymic properties of AMO of
Nitrosococcus oceani, which has nearly equal affinity for
methane and ammonia (Lontoh et al., 2000).
The noted inability of AOB to catabolize alternative
natural energy sources combined with their strict dependence on O2 suggests that g-AOB and b-AOB may have
evolved by genome economization and reductive evolution
(i.e. loss of uptake and processing capacity for other energy
sources including organics) and that this process of genome
reduction likely occurred in concert with their adaptation to
specific environmental niches (Arp et al., 2007; Stein et al.,
2007). Furthermore, adaptation to specific niches appears to
have created specialized inventories for each AOB ecotype as
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152
reported from the genomes of the marine AOB, Nitrosococcus oceani (Klotz et al., 2006), and the sewage AOB,
Nitrosomonas eutropha (Stein et al., 2007). The inventory
unique to the soil AOB, Nitrosospira multiformis, is included
in a forthcoming report.
Tipping balance of the extant nitrogen
cycle: anthropogenic nitrogen and
nitrifier denitrification
Ever increasing and disproportionally high inputs of anthropogenically produced nitrogen have greatly stimulated nitrifier denitrification activities by AOB (Fig. 1c) and other
aerobic bacteria in terrestrial, freshwater and marine ecosystems, leading to concomitant increases in atmospheric
nitrous oxide (Colliver & Stephenson, 2000; Houghton
et al., 2001; Wrage et al., 2001; Beaumont et al., 2002; Stein
& Yung, 2003). Stimulation of nitrifier denitrification is
particularly acute in marine environments where fertilizer
runoff into large rivers and estuaries and aquaculture
operations have led to increased productivity, and hence,
large oxygen-depleted zones (Dore & Karl, 1996; Shailaja
et al., 2006). The globally distributed marine AOB (Ward &
O’Mullan, 2002) are considered a major source of global
N2O production as the oceans cover 71% of Earth’s surface
and regulate weather, climate, and composition of the
atmosphere (Nevison & Holland, 1997). Furthermore,
methane consumption rates, particularly in upland soils,
tend to decrease with increased N-inputs as methanotrophs
cometabolize ammonia at the expense of methane (Robertson et al., 2000) and are inhibited by nitrite produced by
AOB (King & Schnell, 1994; Bodelier & Laanbroek, 2004). In
addition, more recent studies showed that interactions
between ammonia and methane flux change methanotroph
community structure and dynamics (Mohanty et al., 2006),
and preliminary results suggest that methanotrophs lacking
HURM (Figs 2 and 3) are particularly vulnerable to hydroxylamine rather than to nitrite toxicity (L.Y. Stein and M.G.
Klotz, unpublished results). Taken together, this information suggests that mitigation of present and future imbalances in the global nitrogen cycle will be required to reduce
microbial production of greenhouse gases and slow global
warming.
The authors are only beginning to define the genetic
inventory involved in nitrifier denitrification in the AOB,
some of which may have originated in AOB as these
organisms adapted to toxic NOx intermediates produced
during ammonia-oxidizing metabolism. For example, most
AOB encode cytochromes P460 (cytL) and c 0 -b (cytS), both
of which mediate NO toxicity in other bacteria (Elmore
et al., 2007 and references therein). Nitrosocyanin (ncyA)
has thus far been found only in AOB genomes, and is hence
believed necessary for the obligate chemolithotrophy of
2007 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
c
M.G. Klotz & L.Y. Stein
these bacteria (Arp et al., 2007 and references therein).
Interestingly, NcyA synthesis was up-regulated in NO- and
NO
2 -exposed Nitrosomonas europaea biofilms (Schmidt
et al., 2004), and its structure suggests that it can bind and
reduce NO (Arciero et al., 2002). Also, the ncyA gene of
Nitrosomonas eutropha is preceded by a conserved FNR
binding motif (L.Y. Stein, unpublished results), a regulatory
protein involved in response to NO or low oxygen (Van
Spanning et al., 1995; Cruz-Ramos et al., 2002). Physiological investigations of nitrifier denitrification in Nitrosomonas
spp. have implicated involvement of the Cu-NirK nitrite
reductase and the NorB nitric oxide reductase (Beaumont
et al., 2002, 2004, 2005; Schmidt et al., 2004; Cantera &
Stein, 2007a) and cultivated AOB from all ecotypes encode
both nirK and norCBQD in their genomes (Casciotti &
Ward, 2005; Cantera & Stein, 2007a; Garbeva et al., 2007).
Both NirK and NorB were shown to be involved in nitrifier
denitrification under aerobic and anaerobic conditions in
the b-AOB, Nitrosomonas europaea (Schmidt et al., 2004).
Furthermore, the four-member nirK gene clusters of Nitrosomonas and Nitrobacter genera are preceded by the nsrR
gene, which encodes a Rrf2 family transcriptional regulator,
and are separated from nsrR by a conserved NsrR-binding
motif (Beaumont et al., 2004; Cantera & Stein, 2007b). In
Nitrosomonas europaea, nirK expression is up-regulated
under high nitrite concentrations by derepression of NsrR
(Beaumont et al., 2004). Although putative nsrR genes are
present in the genomes of Nitrosospira multiformis and
Nitrosococcus oceani, they are not near the nirK genes nor
are the nirK genes preceded by NsrR-binding motifs; therefore, NsrR-specific regulation of nirK appears to be only in
nitrifiers adapted to ammonia-rich environments (Cantera
& Stein, 2007b). Interestingly, neither nirK- nor norBknockout mutants of Nitrosomonas europaea had completely
diminished N2O production (Beaumont et al., 2002;
Schmidt et al., 2004), and the nirK mutant actually produced more N2O than wild-type Nitrosomonas europaea
(Cantera & Stein, 2007a). Thus, additional nitrite and nitric
oxide reductases apparently contribute to nitrifier denitrification. A global transcriptome study of NirK-deficient
Nitrosomonas europaea revealed selective up-regulation of
genes in a cluster encoding subunits I and II of HCO
(coxAB) and a gene encoding SenC/ScoI (Cho et al., 2006).
It was recently proposed that this particular CoxBA complex, found only in AOB and a handful of genomes from
bacterial sulfur oxidizers, is actually a NO reductase now
termed sNOR and encoded by norYS (Stein et al., 2007;
J. Hemp et al., manuscript in preparation). In Nitrosomonas
europaea, the norYS-senC gene cluster is preceded by a
conserved binding motif for FNR, indicating its regulation
by NO or low O2. Although other AOB also encode fnr
genes, it is yet unknown whether FNR mediates NOx toxicity
and nitrifier denitrification in the AOB, as an FNR mutant
FEMS Microbiol Lett 278 (2008) 146–156
153
N-cycle evolution
of Nitrosomonas europaea did not affect NirK or NorB
expression (Beaumont et al., 2002; Schmidt et al., 2004).
Finally, Nitrosomonas eutropha, an AOB that occupies only
high N-load niches, is the only known AOB to encode a
fixNOP gene cluster, which encodes a high-affinity cbb3
cytochrome c oxidase (Stein et al., 2007). While, still
speculative in part, all this inventory is currently under
investigation for involvement in nitrifier denitrification and
mediation of NOx toxicity in AOB representative of different
ecotypes.
Conclusions
Attempts to reconstruct the evolutionary history of the
biotic nitrogen cycle revealed the important molecular
starting points of an incomplete denitrification process and
ammonification, all based on Fe–Mo-, Fe–S- and Fe-hemecontaining enzymes. The emergence of HURM, molecularly
rooted in these processes and based on Fe-heme cytochrome
c biochemistry, paved the way for both a complete recycling
of reduced fixed nitrogen to the atmospheric N2 pool
(ANAMMOX) as well as the evolution of nitrification once
the world’s oceans became oxygenated. The bioavailability
of copper was a critical determinant in both completing the
canonical denitrification pathway (NOS) and in constructing the nitrification pathway. The proposed evolutionary
scenario also supports a late emergence of N2 fixation as the
main process for making fixed nitrogen available after the
oxygen-enabled explosion in biodiversity created a demand
that exceeded its availability.
Perspectives
Comprehensive and comparative assessments of extant
inventories, including their genomic environments, need to
be combined with unequivocal mechanisms to capture
molecular echoes of the past. Specifically, the complete
extant metabolic inventory of the nitrogen cycle (i.e. Nir,
Nar, Nor, Nos, Nrf) needs to be structurally and functionally
defined and scrutinized by molecular evolutionary inference
to establish molecular lineages of decent and distribution.
These parallel approaches will help identify original pools of
donors and recipients involved in horizontal transfer of the
inventory required to support and retain niche-dependent
metabolic functions (see relevant literature on lateral gene
transfer and gene fitness, i.e. Lawrence & Hendrickson,
2005). Such analyses might clarify whether successful gene
transfers were niche-specific (e.g. creating ecotypic guilds of
recipients) or function-specific (e.g. for respiratory, assimilative, or detoxification purposes) whereby the functionspecific amelioration likely expanded existing metabolic
modules. Investigations of regulatory strategies that control
expression of the inventory in extant organisms (i.e. by
effectors like ammonia, nitrite or nitric oxide, and by DNAFEMS Microbiol Lett 278 (2008) 146–156
binding proteins such as FNR, NnrR, or NsrR) would also
clarify function(s) of the transferred genes.
A particularly urgent task at hand is the discovery of
inventory in AOA and its comparative analysis with the
respective inventory in AOB. For instance, initial analyses of
available AOA genome sequences revealed the absence of
cytochrome proteins and HURM, the essential components
of ammonia-oxidation in AOB and ANAOB (see above text
and Figs 2 and 3). Thus, alternative models for archaeal
ammonia-oxidation remain to be established. These analyses will contribute to both a more complete picture of the
evolution of the nitrogen cycle as well as to assess the
contributions of extant bacteria and archaea in global
nitrogen cycling.
Acknowledgements
The authors thank all colleagues in the nitrification network
(http://nitrificationnetwork.org) for sharing data and discussions, to Dr James Hemp (University of Illinois-Urbana)
for critical reading of the manuscript, and to three
anonymous reviewers for constructive advice. M.G.K. was
supported, in part, by incentive funds provided by the UofLEVPR office, the KY Science and Engineering Foundation
(KSEF-787-RDE-007), and the National Science Foundation
(EF-0412129). L.Y.S. was supported by funds from the
Agricultural Experiment Station of UCR.
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