Environmental Microbiology (2010)
doi:10.1111/j.1462-2920.2010.02345.x
Minireview
In quest of the nitrogen oxidizing prokaryotes of
the early Earth
emi_2345 1..13
Siegfried E. Vlaeminck,1* Anthony G. Hay,1,2
Loïs Maignien1 and Willy Verstraete1
1
Laboratory of Microbial Ecology and Technology
(LabMET), Ghent University, Coupure Links 653, 9000
Gent, Belgium.
2
Department of Microbiology, Cornell University, Ithaca,
NY 14853, USA.
Summary
The introduction of nitrite and nitrate to the relatively
reduced environment of the early Earth provided
impetus for a tremendous diversification of microbial
pathways. However, little is known about the first
organisms to produce these valuable resources. In
this review, the latest microbial discoveries are integrated in the evolution of the nitrogen cycle according to the great ‘NO-ON’ time debate, as we call it. This
debate hypothesizes the first oxidation of nitrogen
as abiotic and anoxic (‘NO’) versus biological and
aerobic (‘ON’). Confronting ancient biogeochemical
niches with extant prokaryotic phylogenetics,
physiology and morphology, pointed out that the welldescribed ammonia and nitrite oxidizing Proteobacteria likely did not play a pioneering role in microbial
nitrogen oxidation. Instead, we hypothesize ancestral
and primordial roles of methanotrophic NC10 bacteria
and ammonia oxidizing archaea, respectively, for
early nitrite production, and of anammox performing
Planctomycetes followed by Nitrospira for early
nitrate production. Additional genomic and structural
information on the prokaryotic protagonists but also
on their phages, together with the continued search
for novel key players and processes, should further
elucidate nitrogen cycle evolution. Through the ramifications between the biogeochemical cycles, this
will improve our understanding on the evolution of
terrestrial and perhaps extraterrestrial life.
Received 18 April, 2010; accepted 9 August, 2010. *For correspondence. E-mail [email protected]; Tel. (+32) 9 264 59 76;
Fax (+32) 9 264 62 48.
© 2010 Society for Applied Microbiology and Blackwell Publishing Ltd
Introduction
Although debates on the niche and nature of the origin of
life are ongoing, it is commonly accepted that life originated in the Hadean ocean (around 4.5–4 Gyr) and that a
last universal common ancestor (LUCA) preceded the
diversification of prokaryotic life in the Archaean eon
(around 4–2.5 Gyr) (Nisbet and Sleep, 2001). Most extant
microbial conversions are considered to have emerged
and been globally distributed by 3.5 Gyr ago. During the
evolution and proliferation of life, it is clear that the introduction of the oxidized compounds nitrite (NO2-) and
nitrate (NO3-) in a relatively reduced environment had
tremendous impacts. Nitrite and nitrate are highly bioavailable due to their good solubility and negative charge
over a range of pH values. These attributes contributed to
nitrate’s potential to serve as a possible nitrogen form for
growth, a capacity that became widespread among bacteria, and later among eukaryotes (Richardson et al.,
2001; Stolz and Basu, 2002). Furthermore, thanks to the
high oxidation numbers of nitrogen in nitrite (+3) and
nitrate (+5), a variety of new redox couples came into
existence. This provided impetus for the development of
various heterotrophic and autotrophic dissimilatory pathways that allow oxidized nitrogen species to serve as
electron acceptors in the absence of free oxygen (Zumft,
1997; Stolz and Basu, 2002). However, little is known
about the first organisms to produce these valuable oxidized nitrogen forms. Here we briefly review oxidative
nitrite and nitrate producing processes, prior to discussing
the emergence of these processes from an evolutionary
perspective according to the two ruling schools of thought.
In doing so, for every process, we endeavoured to identify
likely ancestors, pioneers and followers, revealing key
roles for some recently discovered prokaryotes.
Microbial nitrogen cycling
Figure 1 reviews the different steps and enzymes
involved in the extant microbial nitrogen cycle, showing
mainly a pivotal role for nitrite, involving 10 different
enzyme systems, which have already been described for
its direct production and consumption. Ammonification
2
S. E. Vlaeminck, A. G. Hay, L. Maignien and W. Verstraete
versions (putative) nitrogen oxidation capacities are not
widespread among prokaryotes (Fig. 2), indicating that
these mostly autotrophic conversions are rather specialized and have not been subject to broad horizontal gene
transfer.
Reductive pathways
Fig. 1. The extant microbial nitrogen cycle with pathways related to
catabolism (dashed arrows) and anabolism (full arrows). Oxidation
states of the nitrogen compounds are indicated in pink, and
intermediates are shown between brackets. In alphabetical order of
the abbreviations, the key enzymes are ammonia monooxygenase
(AMO), hydroxylamine oxidoreductase (HAO), hydrazine hydrolase
(HH), hydrazine oxidoreductase (HZO), periplasmic nitrate
reductase (NAP), membrane-bound nitrate reductase (NAR),
cytoplasmic nitrate reductase (NAS), nitrogenase (NIF), siroheme
nitrite reductases (NIR and NirB), Cu-containing nitrite reductase
(NirK), cytochrome cd1 nitrite reductase (NirS), nitric oxide
reductase (NOR), nitrous oxide reductase (NOS), pentaheme
cytochrome c nitrite reductase (NrfA), nitrite oxidoreductase (NXR),
particulate methane monooxygenase (pMMO) (Stolz and Basu,
2002; Bergmann et al., 2005; Strous et al., 2006; Francis et al.,
2007; Semrau et al., 2008; Jetten et al., 2009; Ettwig et al., 2010;
Walker et al., 2010). *For archaeal nitritation, this enzyme is
unknown and proposed intermediates are NH2OH or HNO.
**Ammonia oxidation remains to be demonstrated for methane
oxidizing NC10 bacteria and Verrucomicrobia. +Enzyme unknown.
++
NAP functions in tandem with NrfA, and NAR with NirB.
and assimilation represent two redox neutral steps,
whereas nitrogen fixation, denitratation, denitritation,
nitrite reduction to ammonium and nitrite reduction by
anammox are reductive steps, and nitritation, nitratation
and oxidation of ammonium and nitrite by anammox are
oxidizing steps. To clearly distinguish between the consecutive steps consuming and producing nitrite and
nitrate, the umbrella terms ‘nitrification’, i.e. nitritation followed by nitratation, and ‘denitrification’, i.e. dissimilatory
denitratation and subsequent canonical denitritation, have
been avoided.
In the following paragraphs, the phylogeny and significance of extant nitrogen-oxidizing microorganisms is
summarized. In contrast to most reductive nitrogen con-
Biological nitrogen fixation is a crucial step in nitrogen
cycling, since it is the only biological process that makes
nitrogen bioavailable from the abundantly present nitrogen gas in the atmosphere. Note that for extant marine
ecosystems, biological nitrogen fixation (121 Tg N year-1)
exceeds the abiotic fixation from electrical discharges
(1.1 Tg N year-1) by two orders of magnitude (Galloway
et al., 2004). The genes encoding nitrogenase, the key
enzyme for nitrogen fixation, are very widespread among
the bacterial and even archaeal domains (MartinezRomero, 2006). According to phylogenetic analysis of
these genes, LUCA might already have been able to fix
nitrogen, but it is also possible that methanogenic
archaea were the first to fix nitrogen and that this capacity
was then horizontally transferred to bacteria (Raymond
et al., 2004).
The ability to use nitrate as a nitrogen source (assimilatory denitratation) is not only common for marine
heterotrophs (Allen et al., 2001), but also relatively widespread among many other bacteria (Richardson et al.,
2001; Stolz and Basu, 2002). Dissimilatory nitrite reduction to ammonium is still understudied, but the available
information indicates that this process is also relatively
widespread among bacteria (Mohan et al., 2004; Smith
et al., 2007). While nitrite can also be reduced by
anammox, this catabolic process is discussed below with
the oxidative pathways, since it also involves the oxidation
of ammonium and nitrite.
Canonical denitritation reduces nitrite via nitric oxide
and nitrous oxide to nitrogen gas (Fig. 1), a trait that
occurs mostly in combination with dissimilatory denitratation and which is broadly distributed throughout the bacterial and archaeal domain (Zumft, 1997; Stolz and Basu,
2002). In contrast, a novel, methanotrophic denitritation
route was recently described in bacteria from the NC10
candidate division, in which nitrite is reduced to nitric
oxide which is subsequently acted upon by an unknown
enzyme to nitrogen gas and molecular oxygen (Fig. 1),
the latter being used intracellularly for aerobic methane
oxidation (Ettwig et al., 2010).
Oxidative pathways
Aerobic oxidation of ammonium is the only known way to
oxidatively produce nitrite (Fig. 1), which can be the substrate for many further oxidation or reduction reactions.
© 2010 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology
Early microbial nitrogen oxidation 3
Fig. 2. Phylogenetic 16S rRNA tree of life, displaying the taxonomic groups relevant to the evolution of oxidative nitrite and nitrate production.
‘The all-species living tree’ project release LTP 100 (September 2009) was used as a backbone for the most up-to-date topology for the
bacterial and archaeal domain, as calculated with the RAxML algorithm from about 10950 carefully selected and corrected high-quality
sequences (Yarza et al., 2008). The scale bar represents 10% divergence. From the SILVA database (Pruesse et al., 2007), aligned rRNA
gene sequences were selected for 1961 Eukaryota (sequence quality, alignment and pintail at least 94/100) and for nitrogen cycle
prokaryotes, using the selections of Hatzenpichler and colleagues (2008) and de la Torre and colleagues (2008) for ammonia oxidizing
archaea (AOA), Hoffmann and colleagues (2009) and Mohamed and colleagues (2010) for anoxic ammonia-oxidizing bacteria (AnAOB),
Hanson and Hanson (1996), Semrau and colleagues (2008) and Ettwig and colleagues (2010) for methane oxidizing bacteria (MOB), and
Bock and Wagner (2006) for aerobic ammonia-oxidizing bacteria (AerAOB) and nitrite oxidizing bacteria (NOB). In ARB software (Ludwig
et al., 2004), the selected species were added to the tree with the parsimony tool using domain-specific positional variability filter sets from the
102 SILVA release (February 2010). For clarity, taxonomic groups of less interest were not pruned but hidden instead, and the angles on
branches display their branching off, visualizing how deeply groups of interest branch. The position of the last universal common ancestor
(LUCA) is commonly thought to be between the deepest branching bacterial phylum (Thermotogae) and the node branching towards Archaea
and Eukaryota (Baldauf et al., 1996; Woese, 2000).
Extant nitritation is performed by aerobic ammoniaoxidizing bacteria (AerAOB) belonging to the b- and
g-Proteobacteria (Koops and Pommerening-Roser, 2001),
and by ammonia-oxidizing archaea (AOA) affiliated to
the marine groups 1.1a and 1.1b of the Crenarchaeota
(Francis et al., 2007) and recently proposed to represent
a new phylum, i.e. the Thaumarchaeota (BrochierArmanet et al., 2008a; Spang et al., 2010). Apart from
their oxidative capacities, AerAOB have also been
reported to encode for two of the three steps of canonical
denitritation (Fig. 1), i.e. Cu-containing nitrite reductase
(NirK) and nitric oxide reductase (NOR). As such, some
AerAOB can combine oxidative and reductive capacities
to convert ammonium to nitrogen gas under autotrophic
oxygen-limited conditions (Kuai and Verstraete, 1998). In
comparison, in the two annotated AOA genomes NirK was
also encoded, whereas matches for NOR genes were
either weak or absent (Hallam et al., 2006; Walker et al.,
2010).
The ammonia monooxygenase (AMO) of AerAOB is
closely related to the particulate methane monooxygenase (pMMO) of aerobic methane-oxidizing bacteria
(MOB) belonging to the a- and g-Proteobacteria, enabling
MOB to oxidize ammonia as well, albeit at much lower
rates (Hanson and Hanson, 1996; Nyerges and Stein,
2009). Furthermore, genes encoding hydroxylamine oxidoreductase (HAO), the second enzyme involved in nitritation, have also been retrieved in proteobacterial MOB
(Bergmann et al., 2005), showing high similarity between
the proteobacterial AerAOB and MOB nitritation path-
© 2010 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology
4 S. E. Vlaeminck, A. G. Hay, L. Maignien and W. Verstraete
ways. It should be noted, however, that one g-MOB
species was found incapable of ammonia and hydroxylamine oxidation (Nyerges and Stein, 2009). Recently, two
novel types of MOB were described. Members of the
Verrucomicrobia can oxidize methane aerobically at low
pH values (Dunfield et al., 2007; Pol et al., 2007; Semrau
et al., 2008) and members of the NC10 candidate division
can perform methanotrophy under anoxic conditions,
coupled to denitritation (Ettwig et al., 2010). So far,
however, it is not known if the pMMOs in these bacteria
have a low enough enzyme specificity to attack ammonia
as is the case for their proteobacterial counterparts. In
contrast to the MOB, other heterotrophs performing nitritation were not included in this review since their biochemical reactions are not well known (Verstraete and
Alexander, 1973; Castignetti and Hollocher, 1984; Robertson and Kuenen, 1990; Hooper et al., 1997). Overall,
heterotrophs presumably do not gain energy from nitritation (Robertson and Kuenen, 1990; Klotz and Stein,
2008), and AerAOB and AOA are considered to be the
protagonists in extant global nitrite production (Kowalchuk
and Stephen, 2001; Francis et al., 2007).
For the generation of nitrate in the environment, nitratation is the best studied process and requires the presence of oxygen. Extant nitratation is catalysed by nitrite
oxidoreductase (NXR) and is exclusively a bacterial trait
performed by autotrophic nitrite-oxidizing bacteria (NOB)
belonging to the a-, g- and d-Proteobacteria as well as
to the Nitrospirae phylum (Koops and PommereningRoser, 2001). Until recently, the genomes of only three
a-proteobacterial Nitrobacter species (Starkenburg et al.,
2008) and one g-proteobacterial Nitrococcus species
(GenBank Accession No. NZ_AAOF00000000) were
available. However, the lately published genome of a
‘Candidatus Nitrospira’ species significantly increased
our understanding of nitratation diversity (Lücker et al.,
2010). Surpisingly, the Nitrobacter and Nitrospira
genomes also encode the denitritation enzyme NirK,
suggesting that these NOB can perform uncomplete
denitritation.
An often overlooked source of nitrate is anammox, a
unique bacterial trait performed by autotrophic anoxic
ammonia-oxidizing bacteria (AnAOB) belonging to the
Planctomycetes phylum (Jetten et al., 2009). During this
anoxic process ammonium oxidation is coupled to nitrite
reduction, although 20% of the latter is also oxidized to
nitrate anoxically (Strous et al., 1998), by a putative
membrane-bound nitrate reductase (NAR) (Strous et al.,
2006), with the released electrons being used for the
reduction of carbon dioxide:
NH4 + + 1.32 NO2 − + 0.066 HCO3 − + 0.13 H+
→ 1.02 N2 + 0.26 NO3 − + 0.066 CH2O0.5N0.15 + 2.03 H2O
(1)
Despite the lack of pure AnAOB cultures, it is very unlikely
that anammox nitrate production is an artefact, since
the nitrate production stoichiometry was confirmed from
highly enriched cultures and from physically purified cells,
consisting for 97.6 and 99.6% out of AnAOB respectively
(Strous et al., 1999; van der Star et al., 2008).
Since extant nitrogen gas production on a global scale
is estimated to be 175–450 Tg N year-1 (Codispoti et al.,
2001), for which anammox is likely 30–50% responsible
(Devol, 2003; Dalsgaard et al., 2005), anammox nitrate
production amounts to 7–29 Tg NO3--N yr-1 (Eq. 1). Since
oxygen minimum zones occupy 8% of the extant oceans
(Paulmier and Ruiz-Pino, 2009), anammox nitrate production can, especially on a local scale, represent a considerable nitrate input for the environment. In the Black Sea
for instance, aerobic and anoxic ammonium oxidation
rates are of the same order of magnitude, and AerAOB/
AOA and AnAOB abundance shows the same spatial
profile (Coolen et al., 2007; Lam et al., 2007). This suggests that a significant part of the nitrite produced during
nitritation is consumed by anammox, rather than by nitratation. The inclusion of the anammox process and its role
in nitrate production in global nitrogen models should
therefore be considered.
The great ‘NO-ON’ time debate
A key event in the Archaean eon was the appearance of
oxygenic photosynthesis, which evolved from anoxygenic
photosynthesis and became the first source of free
oxygen in a relatively reduced environment (Kasting,
1993). Although Cyanobacteria first produced oxygen
possibly as early as 3.8 Gyr ago, local consumption by
abiotic and biotic processes prevented the escape of
oxygen from the ocean to the atmosphere until the start of
the Proterozoic eon (around 2.5–0.5 Gyr), the so-called
‘Great Oxidation Event’ (Buick, 2008). Hence, over a significant time frame in the Archaean eon, the ocean water
column had a large range of redox gradients, with oxidizing conditions in parts of the top layer, and reducing
conditions elsewhere (Kasting, 1993). Similar conditions
in extant oxygen-minimum zones like the Black Sea
suggest that these may be a useful model for this early
ocean, despite differences in the levels of atmospheric
oxygen.
There has been much discussion over the last 35 years
(Egami, 1973; Broda, 1975), about the timing of nitrogen
oxidation relative to the appearance of free oxygen. To
date, however, this great ‘NO-ON’ time debate as we call
it, remains unresolved (Ducluzeau et al., 2009; Godfrey
and Falkowski, 2009). Uncertainty about the composition
of the primordial atmosphere lies at the base of this
debate. The amount of solar radiation received by the
early Earth from its younger Sun was much less than it is
© 2010 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology
Early microbial nitrogen oxidation 5
today (Kasting, 1993). Hence, the primordial ocean would
have completely frozen over unless there was an insulating layer containing a sufficient concentration of greenhouse gases. Specifically, the amount of carbon dioxide
present in the atmosphere is an item of special contention
since, as discussed below, it would have been a major
factor governing chemical nitrogen oxidation.
Overall, the basic evolutionary concept of the ‘NO-ON’
debate is that the unprecedented accumulation of a nitrogen compound would, by virtue of its mere presence,
have provided an impetus to evolve enzymes, yielding
products or reactions being beneficial to the host. In the
nitrite/nitrate then oxygen (‘NO’) view, significant abiotic
sources of nitric oxide (NO) led to the existence of nitrite
and nitrate before the advent of oxygen, providing the first
incentive for the development of anoxic processes that
consume these oxidized nitrogen species (Egami, 1973).
As shown in detail below, high atmospheric levels of
carbon dioxide were expected in the ‘NO’ school of
thought. In contrast, according to the oxygen then nitrite/
nitrate (‘ON’) view, the first significant introduction of nitrite
and nitrate in the environment occurred aerobically and
biologically, and hence, nitrite and nitrate consuming processes could only have evolved after oxygenic photosynthesis (Broda, 1975). In this school, the atmosphere
consisted of lower levels of carbon dioxide in the presence of methane.
The ‘NO’ school
Egami (1973) can be considered as the founder of the ‘NO’
school, advocating the presence of nitrite and nitrate on
Earth before oxygen. Later models and lab-scale simulations confirmed the plausibility of this school in an atmosphere rich in carbon dioxide. Carbon dioxide was a likely
candidate gas to insulate the early Earth, requiring an
atmospheric partial pressure of at least 0.1 bar in the
period from 4.5–3.5 Gyr (Kasting, 1993). If the carbon
dioxide level exceeded 50%, electrical discharges would
have led to the abiotic production of nitric oxide from
carbon dioxide and nitrogen gas (Navarro-Gonzalez et al.,
2001). High temperature sources for such discharges
would have included volcanic gases, volcanically induced
lightning, thunderstorm lightning and meteorite impacts.
These combined events could have rendered an abiotic
nitric oxide production of 3 Tg N year-1 in the most optimistic scenario (Ducluzeau et al., 2009). In addition to
small amounts of nitrous oxide, further reaction of nitric
oxide with water vapour in the presence of UV light
would have yielded mainly nitrate and nitrite, in a ratio
of approximately 4/1 (Mancinelli and McKay, 1988;
Summers and Khare, 2007). In the ‘NO’ school of thought,
the anoxic bioprocesses consuming nitrite or nitrate would
therefore have emerged before the oxygen requiring pro-
cesses nitritation and nitratation (Fig. 3; Egami, 1973;
Klotz and Stein, 2008; Ducluzeau et al., 2009). It should be
noted that nitrite, and to a minor extent nitrate, could also
have been reduced abiotically with Fe2+ (Summers and
Chang, 1993; Ottley et al., 1997), if the early Archaean
ocean had a temperature above 25°C and a pH above 7.3.
The high carbon dioxide partial pressure required to
achieve abiotic nitric oxide formation would likely have
rendered the early ocean relatively acidic however, rendering Fe2+ dependent reductions less likely. Furthermore,
at high carbon dioxide levels, Fe2+ precipitates as a carbonate, requiring high temperatures (80°C) to obtain
detectable nitrate reduction (Summers and Chang, 1993).
Note that significant abiotic nitrate reduction to ammonium
is also possible in the presence of green rust (Hansen
et al., 1996), but the presence of such iron hydroxides
prior to the presence of free oxygen is unclear.
As time passed, atmospheric carbon dioxide levels
would have gradually decreased due to lower production
from decreasing meteorite impact rates and higher consumption from the weathering of the growing continents
(Kasting and Siefert, 2001). With insufficient carbon
dioxide levels, abiotic nitric oxide production would have
eventually ceased, possibly limiting bioavailable nitrogen.
This may have acted as a stimulus for the emergence
of biological nitrogen fixation (Navarro-Gonzalez et al.,
2001) in the event that LUCA was not capable of biological nitrogen fixation. However, given the large range of
possible Archaean carbon dioxide levels (Kasting, 1993),
it is not possible to infer whether abiotic nitric oxide
production ceased before or after the onset of oxygenic
photosynthesis.
The ‘ON’ school
Broda (1975) was the first to hypothesize the basics of
the ‘ON’ school: nitrite and nitrate could only be formed
biologically after the onset of biogenic oxygen production. In the ‘ON’ school of thought, carbon dioxide levels
were not high enough to obtain significant nitric oxide
formation. Models confirmed that at lower carbon dioxide
levels, methane could have been an important greenhouse gas in the early atmosphere (Pavlov et al., 2000).
Significant methane sources could have included hydrothermal vents and, after the emergence of LUCA,
methanogenic archaea, which are generally considered
to be among the earliest prokaryotes (Nisbet and Sleep,
2001).
Only after the emergence of Cyanobacteria and their
introduction of free oxygen in the Archaean ocean
(! 3.8 Gyr), could aerobic ammonia oxidation to nitrite
emerge. This created the impetus for the development
of processes oxidizing nitrite to nitrate (nitratation and
anammox) and reducing nitrite, and later nitrate, to
© 2010 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology
6
S. E. Vlaeminck, A. G. Hay, L. Maignien and W. Verstraete
Fig. 3. Representation of the ‘NO-ON’ time debate on a geological timeline showing a different appearance order of the nitrogen cycle
processes consuming or producing nitrite or nitrate for both schools of thought. For simplicity, only the substrates and products for the nitrite
and nitrate generating processes are depicted. Note that the cessation of abiotic nitric oxide (NO) formation in the ‘NO’ school could also have
occurred after the onset of oxygenic photosynthesis, as discussed in the text (see The ‘NO’ school). LUCA, last universal common ancestor.
ammonium or nitrogen gas (denitritation, anammox and
later denitratation) (Fig. 3; Broda, 1975; Falkowski,
1997; Godfrey and Falkowski, 2009). In contrast to the
‘NO’ school, the order in which these processes
emerged would have had major implications for
Archaean biological nitrogen availability. Indeed, in the
‘ON’ school dissolved nitrogen compounds could only be
converted to gaseous compounds after the onset of biogenic oxygen production (Fig. 3). As a result, the first
closure of the nitrogen cycle occurred relatively late,
thereby limiting the nitrogen availability in the Archaean
ocean. Note that the formation of nitrogen gas was not
necessarily a complete loss to all microbes, as some
use the gas to alter their buoyant density, thereby allowing aggregated cells to travel through the water column
(Vlaeminck et al., 2007), and profit from the access to
additional resources thanks to the induced advective
flow (De Schryver et al., 2008).
© 2010 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology
Early microbial nitrogen oxidation
Evolution of biological nitrogen oxidation
Emergence of oxidative biological nitrite production
Candidate processes for early microbial ammonia oxidation to nitrite are proteobacterial nitritation (AerAOB),
archaeal nitritation (AOA) and aspecific methane oxidation under anoxic (NC10 MOB) or aerobic (other MOB)
conditions. Except for the NC10 MOB, the organisms
performing nitritation or methanotrophy need free oxygen
from their environment, and so they could only emerge
after the biogenic production of oxygen. Consistent with
this, the proteobacterial AerAOB and MOB as well as the
verrucomicrobial MOB branch off later from the tree of life
than the Cyanobacteria (Fig. 2). Furthermore, free oxygen
was also required to liberate a key element for nitritation.
Indeed, the catabolism of AerAOB, AOA and MOB
requires copper (Hanson and Hanson, 1996; Arp and
Stein, 2003; Walker et al., 2010), whose release into the
early ocean is only thought to have occurred once oxygen
was present (Lewis and Landing, 1992; Klotz and Stein,
2008).
For proteobacterial nitritation, the relatedness of
AerAOB with b- and g-proteobacterial anoxygenic phototrophs at the 16S rRNA level and the similarity of the
cytoplasmatic membrane arrangements indicate that
AerAOB might have inherited their cell plan from photosynthesizing Proteobacteria (Teske et al., 1994). More
recent bioinformatic analyses of proteobacterial AerAOB
and MOB has suggested that the genes encoding
nitritation might have been transferred from the
g-proteobacterial MOB to the AerAOB of the same
subphylum, and later to the b-proteobacterial AerAOB
(Klotz et al., 2008). This implies that the well-studied
b-proteobacterial AerAOB are likely relatively young, consistent with their 16S position on the tree of life (Fig. 2).
The recently discovered MOB in the Verrucomicrobia
and the NC10 candidate division might bring clarity on the
origin of pMMO. These two phylogenetic groups possibly
harbour the oldest MOB, since they branch off early from
the 16S tree of life (Fig. 2). Furthermore, the Verrucomicrobia contain purportedly ancient structural features, as
discussed more in detail below (see Emergence of biological nitrate production). The ability of NC10 MOB to
produce their own oxygen from nitric oxide under anoxic
conditions potentially has important evolutionary consequences. In the ‘ON’ school, this oxygenic and methanotrophic denitritation did not play a key role, since it could
have only evolved after oxygenic photosynthesis and the
subsequent first biological nitrite production. In strong
contrast, however, the discovery of Ettwig and colleagues
(2010) changes the traditional view of the ‘NO’ school,
rendering the aerobic oxidation of methane, and possibly
of ammonium and other reduced compounds, possible
before the onset of oxygenic photosynthesis. It is hoped
7
that future experiments with these newly discovered MOB
confirm the pMMO aspecificity for ammonia oxidation and
lend credence to an ancestral role of the NC10 pMMO in
microbial nitrite production.
Concerning archaeal nitritation, phylogenetic/genomic,
enzymatic and physiological arguments are consistent
with a pioneering role for AOA in microbial nitrite production. First, the 16S rRNA tree of life indicates that the AOA
branch off relatively early, although their relative emergence with respect to the Cyanobacteria cannot be
resolved from the tree, given the range of the possible
LUCA positions (Fig. 2). Molecular clock analyses of the
sequences of 16S rRNA, 23S rRNA and proteins are
consistent with the emergence of the Crenarchaeota prior
to the advent of oxygenic photosynthesis by as little as
0.1–0.4 Gyr (Blank, 2009) or more than 1.0 Gyr (Battistuzzi et al., 2004). The distinct phylogenetic and genomic
branching of AOA early in the archaeal domain even corroborated the suggestion that AOA represent a novel
phylum, i.e. the Thaumarchaeota (Brochier-Armanet
et al., 2008a; Spang et al., 2010). Second, the ancestral
status of the AOA is further supported by the shared
presence of a DNA topoisomerase and an unsplit RNA
polymerase subunit in AOA and Eukaryota, in contrast to
other Archaea and Bacteria (Kwapisz et al., 2008;
Brochier-Armanet et al., 2008b). Also, the archaeal AMO
genes are quite distantly related from the bacterial AMO
and pMMO genes (Ettwig et al., 2010). Third, if extant
physiology is any indication of historical physiology, conditions conducive to AOA growth likely arose shortly
after the onset of oxygen production and only gradually
changed over the next millions of years to conditions
which favour AerAOB. Specifically, AOA have a hypothesized preference for lower dissolved oxygen concentrations and thus would have been better suited to a
microaerobic ocean (Francis et al., 2007; Erguder et al.,
2009). Furthermore, prior to the availability of free oxygen,
phosphate is considered to have been a limiting nutrient
(Bjerrum and Canfield, 2002). Since AOA are thought to
have low phosphate requirements (Erguder et al., 2009),
they would have been better suited to those conditions
than AerAOB which have relatively high phosphate needs
(e.g. Purchase, 1974; Hue and Adams, 1984; Nordeidet
et al., 1994; Zhang et al., 2009). The aforementioned
points clearly show that the AOA should not be overlooked
as potential early ammonia oxidizers. In contrast to proteobacterial AerAOB and MOB, no HAO was retrieved in
AOA (Hallam et al., 2006; Walker et al., 2010). The latter
study recently revealed major differences in AerAOB and
AOA catabolism, including the possible involvement of
nitroxyl (HNO) as intermediate in archaeal nitritation,
instead of hydroxylamine (NH2OH). Future elucidation of
the other involved enzyme will likely shed more light on
the evolutionary origin of the genetic module encoding
© 2010 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology
8
S. E. Vlaeminck, A. G. Hay, L. Maignien and W. Verstraete
archaeal nitritation, as will the physiological characterization of more AOA isolates.
Emergence of biological nitrate production
Based solely on the phylogenetic distances of their specialized hosts from LUCA, it is difficult to infer whether
anammox or nitratation evolved first (Fig. 2). Both sides of
the ‘NO-ON’ time debate agree that nitratation could only
emerge after oxygenic photosynthesis had provided the
required oxygen. From the 16S tree of life, Nitrospira
appears to be the most ancient NOB group whereas
Nitrobacter is likely the youngest group to perform nitratation (Fig. 2). Similar to the proteobacterial AerAOB,
the close 16S rRNA relationship between the a- and
g-proteobacterial NOB, Nitrobacter and Nitrococcus, and
anoxygenic phototrophic Proteobacteria, together with
the shared presence of intracytoplasmatic membrane
arrangements, is suggestive of shared inheritance with
anoxygenic photosynthesizing cells (Teske et al., 1994).
In contrast, the genera Nitrospina and Nitrospira are at the
16S level not closely related to phototrophs and, consistent with this observation, their cells do not harbour peculiar membrane arrangements (Bock and Wagner, 2006).
Nitrobacter is the only NOB genus without obligately halophilic species (Bock and Wagner, 2006), but since the
colonization of land probably preceded the emergence of
oxygenic photosynthesis (Battistuzzi et al., 2004), no conclusions can be drawn from that.
Until recently, the a-proteobacterial Nitrobacter was the
only NOB group for which the genes encoding nitratation
had been fully described and annotated. Early reports
already showed the reversibility of the Nitrobacter NXR
reaction (Sundermeyer-Klinger et al., 1984). The relatedness of Nitrobacter nitratation and denitratation was confirmed at the molecular level by a remarkable similarity
between NXR in Nitrobacter and the NAR-type enzymes
involved in dissimilatory denitratation (Kirstein and Bock,
1993; Starkenburg et al., 2008). Furthermore, the similarity between the genes encoding NirK in Nitrobacter and
the AerAOB Nitrosomonas led to the hypothesis that
these nitrogen metabolism genes were acquired through
horizontal gene transfer between species sharing the
same ecological niche (Starkenburg et al., 2008). A late
emergence of Nitrobacter is further indicated by the relatively young 16S positions of Nitrobacter and Nitrosomonas (Fig. 2), and the likely emergence of Nitrosomonas as
the youngest AerAOB group (see Emergence of oxidative
biological nitrite production). Furthermore, if extant and
historical physiology are related to some extent, Nitrobacter likely emerged after Nitrosomonas when the
ambient oxygen levels were higher, since Nitrobacter
shows a lower affinity for oxygen than Nitrosomonas
(Laanbroek and Gerards, 1993).
The recent release of genomic data on a Nitrospira
species allowed an important step forward in our understanding of NOB catabolic and anabolic diversity (Lücker
et al., 2010). NXR of Nitrospira was found to be only
distantly related to those of Nitrobacter and Nitrococcus
and to the denitratation enzyme NAR. Furthermore, the
location and electron transport chain of Nitrospira NXR
and the Nitrospira carbon fixation pathway differed fundamentally from the other NOB. Overall, these arguments
suggest that nitratation independently evolved multiple
times (Lücker et al., 2010). Interestingly and in contrast to
Nitrobacter, Nitrospira was detected as dominant NOB
under oxygen-limited conditions (Schramm et al., 1999;
2000; Gieseke et al., 2003; Vlaeminck et al., 2010), and
even prefers low oxygen levels (Park and Noguera, 2008;
Off et al., 2010). Furthermore, the Nitrospira carbon fixation pathway is more common to anaerobes, and Nitrospira does not share protection mechanisms to reactive
oxygen species with most aerobes (Lücker et al., 2010).
Hence, following the advent of free oxygen production,
the anaerobic/anoxic ocean with microaerobic zones
would have been conducive to Nitrospira, and make a
likely candidate as pioneering NOB, in agreement with its
early position on the tree of life (Fig. 2).
Complete aerobic ammonia oxidation to nitrate
(comammox) has never been found in one organism,
although this process is thought to have energetic
advantages in certain environments (Costa et al., 2006).
The authors considered that the evolution of sequential
nitritation and nitratation was favoured above comammox, given the more efficient division of the metabolic
labour in the former. Alternatively, the absence of
comammox could also be related to the biogeochemical
history of the Earth. Indeed, if abiotic nitrite production in
the ‘NO’ school ceased only after the advent of oxygenic
photosynthesis, nitrite might have been more abundant
than ammonium, selecting for the emergence of nitratation prior to nitritation. Once both processes were diversified and in place, there would have been no impetus for
comammox to evolve. The plausibility of the such
sequential emergence of denitritation, nitratation and
nitritation might be addressed with a revisited NirK comparison, combining analyses on AerAOB and the NOB
Nitrobacter (Cantera and Stein, 2007) with those on
denitriting bacteria (Jones et al., 2008), and extending
with newly available data from the NOB Nitrococcus
(GenBank Accession No. NZ_AAOF00000000), the NOB
Nitrospira (Lücker et al., 2010) and AOA (Hallam et al.,
2006; Walker et al., 2010).
Nitrospira was shown to be a likely candidate to
pioneer aerobic nitrate formation, and may even have
received its enzymatic repertoire for nitrite oxidation from
the AnAOB through horizontal gene transfer. Indeed, the
high similarity of a small set of proteins encoding nitrite
© 2010 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology
Early microbial nitrogen oxidation 9
oxidation (NXR and NAR) and putative electron
transport/respiration components clearly indicates horizontal gene transfer (Lücker et al., 2010). Since the
genes encoding this set are tightly grouped in the
AnAOB and widely spread in Nitrospira, it is likely that
Nitrospira incorporated the organized gene set from the
AnAOB in a rather fragmentary way in its genome. This
suggests that anammox nitrate production predated
nitratation and that AnAOB were the first nitrate producers on Earth. Both 16S phylogeny, metabolism and cell
structure indeed suggest an ancient origin for the Planctomycetes phylum, in which the AnAOB represent an
early branch (Fig. 2). Planctomycetes along with the
closely related Verrucomicrobia, Chlamydiae, Poribacteria and two other phyla, consitute the so-called PVC
superphylum in the bacterial domain (Wagner and Horn,
2006). In the Planctomycetes, three apparently ancient
features have been described. First, Planctomycetes
along with Chlamydiae, are the only bacteria with proteins instead of peptidoglycan as a major cell wall constituent (Lindsay et al., 2001). Second, the genes of C1
transfer reactions, which are the basis of the carbon
metabolism of the putatively ancient methanogenic Euryarchaeota and of all known MOB (Pol et al., 2007;
Ettwig et al., 2010), have been found in distantly related
Planctomycetes (Chistoserdova et al., 2004), revealing a
potentially ancestral role of the latter. Third, Planctomycetes and Poribacteria cells show a special cell plan
with intracellular membranes dividing the cytoplasm into
different compartments (Fuerst, 2005), a feature that
was recently also discovered in the sister phylum of
the Verrucomicrobia (Lee et al., 2009). Furthermore,
Santarella-Mellwig and colleagues (2010) have recently
shown a link between the endomembrane systems of
the PVC superphylum and that of eukaryotic cells.
Future availability of more genomes from PVC bacteria
should provide insight into the unique features of these
organisms and show if these traits share a common phylogenetic origin (LUCA), if they emerged independently
in Bacteria and Eukaryota, or if they were transferred
horizontally.
In an attempt to reveal the ancestry of anammox,
Klotz and colleagues (2008) performed comparative
sequence analysis on a key cytochrome c protein family
related to HAO, hydrazine oxidoreductase (HZO) and
pentaheme cytochrome c nitrite reductase (NrfA). Interpreted according to the ‘NO’ school, this study hypothesized that both anammox and bacterial nitritation were
derived from dissimilatory nitrite reduction to ammonium.
Note that anammox can consume nitrite while being
physically separated from its production source (van der
Star et al., 2007), rendering anammox plausible in the
absence of a neighbouring nitrite producer, as can be
expected in the ‘NO’ school. Regardless of the outcome
of the great ‘NO-ON’ time debate, anammox is a likely
candidate process for the first ever nitrate production.
However, in the ‘NO’ school of thought, abiotic nitrate
production would still have been the major nitrate
source, as shown in the following best-case estimation
for anammox nitrate production. Assuming that a part of
the abiotically produced nitric oxide was oxidized to 20%
nitrite and 80% nitrate (see The ‘NO’ school) and that
AnAOB had access to all this nitrite with minor competition from abiotic reduction or biological denitritation, the
anammox stoichiometry (Eq. 1) suggests that 5% (= 4/
84) of the overall nitrate production could have been
biological. Additionally, if the nitrite produced from denitratation was also consumed by anammox, up to 24%
(= 20/84) of the total nitrate production could have been
due to anammox.
Phages and prophages
Besides genomic and structural information relevant to
the metabolism of nitrogen cycle prokaryotes, data on
their parasites can also shed light on the evolutionary
establishment of microbial nitrogen oxidation. Phages
are obligatory viral parasites to prokaryotes and outnumber prokaryotes in aqueous ecosystems, where they
infect significant fractions of the microbial community
(Weinbauer, 2004). Furthermore, the genome of most
culturable prokaryotes contains complete or defective
prophages, i.e. integrated phage genes (Ackermann,
2007). The evolutionary relevance of phages derives
from two findings. First, although most prokaryotes have
more than one specific phage, the host range of a
phage is often quite narrow (Weinbauer, 2004). Second,
recent findings show that all viruses share a common
ancestor which likely emerged before LUCA, preceding
the diversification of cellular life (Bamford et al.,
2005).
For the microbial groups relevant to this review
(Fig. 2), direct observation of infected cells was limited to
type II MOB and b-proteobacterial AerAOB (Ackermann,
2007; Vlaeminck et al., 2010). For other groups, indirect
phage infection has been shown by prophages identified
in the genomes of the type X MOB Methylococcus, the
AerAOB Nitrosococcus, the NOB Nitrobacter and the
AOA Nitrosopumilus (Ward et al., 2004; Klotz et al.,
2006; Starkenburg et al., 2008; Walker et al., 2010).
Despite the widespread occurrence of phages over the
prokaryotic phyla (Ackermann, 2007), to date, their
detection in the nitrogen cycle is very limited because
direct observation is understudied and few full prokaryotic genomes are available. It is therefore expected that
future genomic and structural characterization of
(pro)phages will shed new light on the evolution of
microbial nitrogen oxidation.
© 2010 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology
10 S. E. Vlaeminck, A. G. Hay, L. Maignien and W. Verstraete
Conclusions
Nature has developed multiple mechanisms to generate
nitrite and nitrate. It is likely that their accumulation on the
early Earth provided emerging life forms with additional
redox couples that allowed for the exploitation of new
niches and helped to spur diversification. Specifically, the
presence of these compounds permitted the development
of various extant dissimilatory and assimilatory pathways,
which play a critical role in today’s nitrogen cycle. This
review has, for the first time, discussed possible ancestral
and primordial roles of NC10 MOB and AOA, respectively,
for early nitrite production, and of AnAOB followed by the
NOB Nitrospira for early nitrate production. Although
nitrite and nitrate obviously were and are major multifacetted driving forces in biological evolution, the true picture
of nitrogen cycle evolution remains murky. The availability
of additional genomic and structural information on the
prokaryotic protagonists and on their phages, as well as
the continued search for novel nitrogen cycling processes
will lead to greater clarity and provide insights that may
resolve the ongoing ‘NO-ON’ time debate. The ‘ON’
school would be particularly supported by demonstrating
that denitratation was the last process to emerge, or
that nitritation evolved later than either anammox, nitrite
reduction to ammonium or denitritation (Fig. 3). In contrast, more definitive evidence for the reverse would be a
strong argument in favour of the ‘ON’ school.
Acknowledgements
S.E.V. was supported as a postdoctoral fellow from the
Research Foundation Flanders (FWO-Vlaanderen), L.M. was
recipient of a PhD grant from the Institute for the Promotion of
Innovation by Science and Technology in Flanders (IWTVlaanderen, number SB-53575) and A.G.H was funded as a
visiting foreign researcher by the Special Research Fund
from Ghent University (BOF-UGent, number VBO017,
01T01709 B/10902/02). The authors gratefully thank Haydée
De Clippeleir, Nico Boon and Peter De Schryver for the
inspiring scientific discussions.
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