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 β- and γ-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 γ-proteobacterial MOB to the AerAOB of the same subphylum, and later to the β-proteobacterial AerAOB (Klotz et al., 2008). This implies that the well-studied β-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 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 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 α- and γ-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 α-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 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 β-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.