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The nitrogen cycle Lisa Y. Stein1,* and Martin G. Klotz2,3 Nitrogen is the fourth most abundant element in cellular biomass, and it comprises the majority of Earth’s atmosphere. The interchange between inert dinitrogen gas (N2) in the extant atmosphere and ‘reactive nitrogen’ (those nitrogen compounds that support, or are products of, cellular metabolism and growth) is entirely controlled by microbial activities. This was not the case, however, in the primordial atmosphere, when abiotic reactions likely played a significant role in the inter-transformation of nitrogen oxides. Although such abiotic reactions are still important, the extant nitrogen cycle is driven by reductive fixation of dinitrogen and an enzyme inventory that facilitates dinitrogen-producing reactions. Prior to the advent of the Haber-Bosch process (the industrial fixation of N2 into ammonia, NH3) in 1909, nearly all of the reactive nitrogen in the biosphere was generated and recycled by microorganisms. Although the Haber-Bosch process more than quadrupled the productivity of agricultural crops, chemical fertilizers and other anthropogenic sources of fixed nitrogen now far exceed natural contributions, leading to unprecedented environmental degradation. The significance of nitrogen to the biosphere and to cellular life is indisputable; however, our fundamental knowledge of the microorganisms and enzymatic processes that transform nitrogen into its various oxidation states (Figure 1) remains incomplete. Here, we outline the major microbial processes of the nitrogen cycle, the microorganisms that perform nitrogen transformations, and the modularity and evolutionary history of the nitrogen cycle, and provide a perspective of this cycle, now and into the future. Major processes of the nitrogen cycle The nitrogen cycle has traditionally been divided into three processes — N2 fixation, nitrification, and denitrification (Figure 2) — and microbes have historically been labeled by their identified participation in one of these R94
processes, that is, ‘nitrogen fixers’, ‘nitrifiers’ and ‘denitrifiers’. This assignment of microbes to specific eco-physiological cohorts based on their association with a single process came under scrutiny when ecologists found evidence for dissimilatory (that is, non-assimilatory) reduction of nitrite to nitric oxides and nitrous oxides in oxic environments (Figure 2, reaction 6A; called ‘nitrifier denitrification’), as well as dissimilatory reduction of nitrite to ammonium (Figure 2, reaction 2; called ‘respiratory ammonification’) and dissimilatory oxidation of ammonium in anoxic environments (Figure 2, reaction 7; called ‘anammox’). Our previous understanding of the nitrogen cycle was complicated by the numerous and diverse scientific approaches and foci — from the compounds that were transformed, to the compounds that were generated, to the environmental status of reactions that constituted the processes. The narrow foci in these approaches — employed for over 100 years in nitrogen-cycle research — have been overcome in the post-genomic era due to much-improved instrumentation, a great wealth of data, and increased interdisciplinary and international collaboration. Thus, our understanding of the nitrogen cycle now consists of five accepted nitrogen-transformation flows: ammonification, including nitrogen fixation, and assimilatory and dissimilatory reduction of nitrite (Figure 2, reactions 1 and 2); nitrification (Figure 2, reactions 3A, 3B and 4); denitrification, including canonical, nitrifier-dependent and methaneoxidation-dependent denitrification (Figure 2, reactions 6A–D); anammox, as a form of coupled nitrification– denitrification (Figure 2, reactions 7A– C); and nitrite–nitrate interconversion (Figure 2, reactions 4 and 5). The general processes of organic matter mineralization (often mislabeled as ‘ammonification’) and assimilation (often incorrectly claimed to include processes that regulate the generation of ammonium and its uptake) by cellular life complete the movement of reactive nitrogen through the biosphere (Figure 2). Ammonification Nitrogen fixation is one of two versions of ammonification and is accomplished by bacteria and archaea that encode nitrogenase enzyme complexes made
Current Biology 26, R83–R101, February 8, 2016 ©2016 Elsevier Ltd All rights reserved
Molecule
Name
Oxidation state
Reduced C-NH2 Organic-N NH3, NH4+ Ammonia, Ammonium -3 More N2H4 Hydrazine -2 electrons NH2OH Hydroxylamine -1 N2 Dinitrogen 0 N2O Nitrous oxide +1 NO Nitric oxide +2 Fewer HNO2, NO2 Nitrous acid, Nitrite +3 electrons NO2 Nitrogen dioxide +4 HNO3, NO3- Nitric acid, Nitrate +5 Oxidized Current Biology
Figure 1. Nitrogen-cycle intermediates. Intermediates, representing nine oxidation states, that donate or accept electrons, thereby contributing to electron flow and conservation of energy in participating microbes.
up of the molybdenum–iron protein dinitrogenase and the vanadium or iron protein dinitrogenase reductase. Although there is momentum to genetically engineer plants to express microbial nitrogenase complexes or to express the signaling pathway to attract nodulating nitrogen-fixing bacteria to colonize cereal crops, biological nitrogen fixation remains an activity solely performed by bacteria and archaea. Nitrogen fixation is extremely oxygen sensitive, requiring microorganisms to develop protective mechanisms, such as spatial decoupling (compartmentalization or forming specialized cells), temporal decoupling, rapid O2 respiration, or maximization of nitrogenase synthesis and turnover. The ammonium produced by nitrogen fixation is either assimilated into biomass or is further respired by aerobic and anaerobic ammonia-oxidizing microbes. This coupling of reaction 1 with either reaction 3 or 7, as shown in Figure 2, occurs either between nitrogen-cycle-active microbes in communities or within single cells, such as in nitrogen fixing, nitrifying methanotrophic bacteria. Anaerobic assimilatory (ANRA) and dissimilatory (DNRA) nitrite reduction to ammonium (Figure 2, reaction 2) is the second version of ammonification and is performed by both bacteria and fungi. This process can be linked to nitrate reduction to nitrite (Figure 2, reaction 5) at the cellular level or between organisms in a community. The acronym DNRA was originally coined to describe an ecophysiological process in which nitrogen in the form of nitrate was traceably removed from soils without either being lost in the form of nitrogenous gases (denitrification) or assimilated
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Magazine into microbial biomass. While the significance of ANRA and DNRA to global nitrogen cycling facilitated by fermenting fungi in soils is not well understood, respiratory ammonification by bacteria and archaea in diverse anoxic habitats is well established. DNRA can generate a cellular protonmotive force and thus conserve energy to support cellular growth, although this feature depends on which enzymes are coupled. A negative redox potential (that is, reducing conditions) is the most important stimulant for DNRA. It should be noted that the ANRA process employs identical chemistry to DNRA but it is facilitated by evolutionarily unrelated nitrite reductases. Nitrification The process of nitrification involves three cohorts of microorganisms: cohort I, ammonia oxidizers that oxidize ammonia to nitrite (nitritation; Figure 2, reactions 3A and 3B); cohort II, nitrite oxidizers that oxidize nitrite to nitrate (nitratation; Figure 2, reaction 4); and cohort III, complete ammonia oxidizers that oxidize ammonia all the way to nitrate (comammox; Figure 2, reactions 3A, 3B and 4). Although comammox microorganisms were discovered only in 2015, their existence was predicted approximately 10 years earlier. Cohorts II and III include only chemolithotrophic microbes that can use nitrite and ammonia, respectively, as sole sources of energy and reductant for cellular growth. Whereas nitrite-oxidizing chemolithotrophs of cohort II are classified within several classes of the Proteobacteria and the phylum Nitrospirae, the comammox bacteria are, so far, restricted to representatives in lineage II of the genus Nitrospira. Nitrifying ammonia-oxidizing microbes of cohort I include chemolithotrophic members of Betaproteobacteria and Gammaproteobacteria, and members of the Thaumarchaeota. In addition to these chemolithotrophs, there are heterotrophic and methanotrophic microorganisms that proficiently oxidize ammonium to nitrite, but they do not gain energy from the process to support growth. The nitrite and nitrate produced by aerobic reactions in the nitrification process can be respired anaerobically (Figure 2, reactions 2 and 5) or the resulting ammonium can be assimilated.
N2
6C
1
7C N2O N2H4 Assimilation
6D 6B
R~NH2
NH4+/NH3 7B NO
Mineralization 3A
2 NH2OH
7A 6A
NO25
3B 4
-
NO3
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Figure 2. Major processes of the nitrogen cycle. Reactions that comprise the seven major processes of the nitrogen cycle are represented by the numbered circles. Ammonification may be accomplished either by process 1, reduction of dinitrogen (also referred to as ‘nitrogen fixation’ or ‘Nif’), or by process 2, dissimilatory nitrite reduction to ammonium (DNRA). Nitrification is composed of process 3, oxidation of ammonia to nitrite (also referred to as ‘nitritation’), and process 4, oxidation of nitrite to nitrate (also referred to as ‘nitratation’). Process 5, reduction of nitrate to nitrite, can be coupled to processes 2, 6 or 7 in a population or a community. Denitrification is shown as process 6, which is also referred to as ‘nitrogen-oxide gasification’. Anammox is shown as process 7, and is also referred to as coupled ‘nitrification–denitrification’.
Denitrification Denitrification describes the process of anaerobic respiration of nitrite (NO2-), nitric oxide (NO), and nitrous oxide (N2O) to N2 (Figure 2, reactions 6A–C). Heterotrophic microbes that can directly couple these three reactions with the reduction of nitrate to nitrite (Figure 2, reaction 5) and perform denitrification from NO3- to N2 are referred to as classical or canonical denitrifiers. It is nevertheless recognized that many bacteria and archaea express only partial denitrifying inventories, and are missing genes encoding enzymes involved in reactions 5, 6B and/or 6C (Figure 2). Such incomplete pathways can lead to the release of nitrogenous gases such as NO and N2O to the environment, or a failure to deplete NO3-. This group also includes all ammonia-oxidizing chemolithotrophic bacteria, which reduce NO2- to N2O aerobically (Figure 2, reactions 6A and 6B). In addition, several eukaryotes including fungi and foraminifers (a class of protists) can reduce NO2- or NO3- to N2O or N2, respectively. In an unusual twist to the denitrifying
process, a methanotrophic bacterium, Candidatus Methylomirabilis oxyfera, consumes methane as a source of energy, reductant and carbon, and reduces NO2- to N2 by expressing a nitric oxide dismutase. This enzyme dismutates two NO molecules to O2 and N2 without producing the intermediate N2O (Figure 2, reaction 6D); the O2 produced is then used by this bacterium to oxidize methane to methanol while it resides in an anoxic environment, hence the name ‘denitrifying intra-oxygenic methanotroph’. Anammox Anammox, or anaerobic ammonium oxidation, utilizes the pools of NO2and ammonium (NH4+) to form N2 via the intermediates NO and hydrazine (N2H4; Figure 2, reactions 7A–C). The recent literature has also suggested the existence of a modified anammox pathway, in which hydroxylamine (NH2OH) is used instead of NO for the oxidation of NH4+, but additional experimentation will be required to validate this pathway. Anammox chemolithotrophy is performed solely by the Brocadiaceae bacteria in
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Magazine the order Planctomycetales within a specialized organelle called the anammoxosome. Anammox is ecologically beneficial for wastewater treatment as it removes both nitrite and ammonium simultaneously without producing N2O, and has been industrially implemented at full scale. Anammox is also a major nitrogenremoval process in the ocean and in oxygen minimum zones. From organisms to modularity of the nitrogen cycle For hundreds of years, microbiologists have enriched and isolated microorganisms from the environment to connect discrete biological entities to particular processes. The enzymology of the above nitrogen-cycle processes were largely discovered using axenic cultures of microorganisms, leading to the impression that these cultured microbial phylotypes should also be the main players in charge of those processes in the environment. For instance, the first ammonia-oxidizing bacteria were isolated in the 1890s and a great proportion of biochemical, physiological, and genetic knowledge of ammonia oxidation was derived from research performed with these very isolates. For many years, microbial ecologists relied on gene sequences and physiological attributes derived from a small number of isolates to detect and enumerate ammonia oxidizers in the environment. However, in the 2000s, metagenome sequences showed an irrefutable association between ammonia monooxygenase (the first enzyme involved in ammonia chemolithotrophy) and archaeal 16S rRNA genes that shifted the focus from bacterial ammonia-oxidizers to a whole new research venture based on the ammonia-oxidizing thaumarchaeota. Following the discovery of ammoniaoxidizing thaumarchaeota, the first strain isolated into pure culture was Nitrosopumilus maritimus in 2005, enabling physiological and biochemical characterization of a completely different group of ammonia-oxidizing chemolithotrophs. Aside from revealing entirely new phyla of nitrogen-cycling microbes that had evaded age-old cultivation-dependent approaches, genome and metagenome sequencing also revealed that individual modules, independent of complete R96
integrated pathways, are encoded and expressed by microorganisms to perform single segments of nitrogencycle metabolism. For instance, methanotrophic bacteria can express many genes encoding enzymes with a known involvement in the processes of nitrogen fixation, nitrification and denitrification. Intriguingly, there is no predictable association between nitrogen-cycle genes and phylotype among the methanotrophs. Furthermore, the extent of nitrogen-cycling inventory is scattered; some methanotrophs encode nitrite and nitric oxide reductases together, some fix nitrogen and encode a suite of denitrification genes, whereas others may encode only one reductase or none. Hence, environmental factors apparently drive random lateral gene transfer events to perform tasks to enhance fitness of a particular species, thereby leading to niche differentiation. For instance, the acquisition and expression of nitrifying modules could allow certain methanotrophs to survive high ammonium loads in situations where NH3 can compete with methane for access to methane monooxygenase enzymes. Similarly, expression of denitrifying modules can allow a methanotrophic species to grow in nearly anoxic ecosystems. Nitrogen fixation is inherently modular, with gene clusters distributed across numerous bacterial and archaeal phyla, arguing once again that habitat conditions select for this trait. For the majority of history in nitrogencycle research, we have held onto the concept that organisms of particular phylotypes are nitrifiers, denitrifiers, nitrogen fixers, and the like. This organismal concept is now challenged by the knowledge that nitrogen-cycle genes and processes are not necessarily constrained by phylotype, but rather endow organisms with the inventory required to adapt and thrive in ecosystems where nitrogenous molecules undergo dynamic changes in concentration and composition. Envisioning the nitrogen cycle as a continuum of modular evolution is consequently the most parsimonious outlook. Modular evolution of the nitrogen cycle The primordial nitrogen ‘cycle’ was not closed, but rather was dominated by coupled abiotic reactions that provided nitrogen oxides and reduced nitrogen
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in the form of NH4+, which is required for nitrogen assimilation into biomass. The nitrogen oxide reservoir in the form of NO3- vastly exceeded available NH4+, which favored the evolution of a reductase protein inventory involved in nitrate–nitrite inter-conversion (Figure 2, reactions 4 and 5) along with numerous nitrite reductases (Figure 2, reactions 2, and 6A/7A). The suite of reductase enzymes relied on the bioavailability of transition metals under anoxic conditions such as molybdenum (as found in molybdopterin) and iron (found in siroheme or heme cytochrome c). The function and evolution of these protein complexes and their encoding genes have been comprehensively reviewed by Simon and Klotz (2013) with an emphasis on bioenergetics. With the exception of reactions labeled 3A, 3B, 4, 7B and 7C in Figure 2, the reactions that interconvert nitrogen oxides of different oxidation states (Figure 1) are reductions, which require the availability and accessibility of electrons. These electrons are derived from the quinone pool, facilitated by quinone-reactive proteins (QRPs) directly, or via redox carrier proteins. Therefore, the evolution of proteins that participate in the nitrogen cycle is intrinsically connected to the evolution of QRPs. While QRP complexes that contain cytochrome b proteins have been well characterized, those that include Fe–S clusters (NapH/NosH family), cytochrome c proteins (NrfH/NapC/TorC family) or the enigmatic transmembrane protein NrfD/PsrC are significantly less well understood. Interestingly, the evolution and implementation of these QRPs in catabolic electron flow are also modular, interconnected and function to provide electrons to a variety of catalytic redox proteins involved in the sulfur and nitrogen cycles by oxidation of quinol. Exceptions are the composite MFc complex that evolved into an ‘alternative complex III’ for branched electron transfer (quinol oxidase not serving electrons to enzymes), the cM552 (CycB) protein, which functions as a quinone reductase in ammoniaoxidizing chemolithotrophs, and the copper-containing analog in ammoniaoxidizing Thaumarchaeota. Reactions 3A, 3B, 4, 7B and 7C in Figure 2 are oxidations, with reactions 3B and 7C being dehydrogenations performed by homologous multiheme
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Magazine cytochrome c (MMC) proteins. Interestingly, these dehydrogenases evolved from MMC reductases that perform reactions 2 and 6A/7A in Figure 2. Reactions 6A/7A and leaky dehydrogenase 3B were likely the main biotic generators of the NO radical, the source of nitrosative stress in cells and otherwise of abiotic origin, which served as the primordial oxidant before the advent of molecular oxygen. Abundant production of NO radicals led to the emergence of a large complement of evolutionarily unrelated enzymes capable of NO detoxification, most of them being reductases (reaction 6B in Figure 2), such as the soluble tetraheme cytochrome c’ and c’-beta proteins, which were later complemented by numerous other NO reductases, the main biotic source of N2O. We proposed in 2008 that the key innovation for the closure of the nitrogen cycle was likely the emergence of N2H2 hydrolase (reaction 7B in Figure 2) and its subsequent integration into a redox gradient generated by reactions 7A and 7C (Figure 2), which reversed electron flow through the protein complex and created what is now known as N2H2 synthase, the key enzyme in the anammox process. It is remarkable that this protein complex is restricted only to the Brocadiaceae that facilitate the anammox process. This innovation created a connection between reduced, oxidized, and inert nitrogen and thus constituted the closure and formation of a real cycle. The onset of nitrogen fixation is presently still debated, but there is growing momentum for placing the evolution of this pathway before the great oxygenation event, potentially as a contemporary invention to the emergence of the anammox pipeline. Copper was not bioavailable in anoxic environments, which is the reason why many enzymes with copper as the redox-active metal emerged after the great oxygenation event, including N2O, NO and NO2- reductases, and functional ammonia monooxygenase. While there is an increasingly clear picture on the evolutionary relationships between copper-containing membrane-bound monooxygenases (Cu-MMOs), including those that oxidize ammonia and short alkanes such as methane, the origins of the enzyme subunit proteins remain elusive. This explosion of nitrogen-
cycle inventory occurred in parallel to the explosion of aerobic lifestyles, which provided more efficient electron flow into and out of the quinone pool, while providing additional means for energy conservation (generation of proton-motive force). Hence, the taxonomically unpredictable and broad distribution of diverse nitrogencompound-transformation inventory emerged gradually over evolutionary time. This evolution followed diverse environmental and functional pressures, resulting in the acquisition, maintenance or loss of functionally linked inventory, thereby generating a nitrogen cycle of modular design, similar to the modular designs of the cellular central pathways (glycolysis, TCA, pentose phosphate pathway) or the Calvin cycle. It thus comes as no surprise to those who accept the modular nature of the nitrogen cycle that anammox bacteria can oxidize NO2- anaerobically with an enzyme homologous to the NO2--oxidizing enzyme of the aerobic Nitrospira even though the genomic background (phylotype) of these two bacterial groups is very distantly related. Bioenergetically speaking, the dissimilatory reactions of the nitrogen cycle make it the most advanced biogeochemical nutrient cycle because, out of nine possible intermediates, the nitrogen cycle uniquely includes representative reactions for seven oxidation states between -3 and +5 (Figure 1). While the sulfur and carbon cycles also span over nine oxidation states, they provide only three states (0, +2, +4) between -2 and +6 and three states (-2, 0, +2) between -4 and +4, respectively, that are available for electron-transfer reactions. This bioenergetics picture is congruent with our understanding of the timeline for the emergence of a complete nutrient cycle, a picture in which the extant nitrogen cycle represents a more evolved cycle, cemented by the finding that significant nitrogen-cycle inventory evolved from inventory active in the sulfur and/or carbon cycles.
intertwined with our ability to control the nitrogen cycle. On one side, the Green Revolution of the 20th century averted a global food crisis, in part by introducing widespread synthetic fertilizer usage. To put this in perspective, the Haber-Bosch process is responsible for feeding approximately 48% of the global human population, equivalent to supporting four billion births since 1908. On the other side, with our extremely rapid population growth and continued increases in fertilizer usage, we have pushed the nitrogen cycle beyond sustainability where now NO3- pollution is responsible for increasing dead zones in coastal areas and N2O is considered the key greenhouse gas to mitigate in the 21st century and beyond. Control of the nitrogen cycle is a tricky business, in part because of our success with fertilizer-dependent food production, and also because we are still discovering how the microbial processes of the nitrogencycle work. For instance, the relatively recent discoveries of the anammox and comammox processes, as well as the discovery of ammoniaoxidizing thaumarchaeota (described above) have revealed organisms and processes that profoundly impacted how we view and study nitrogen transformations and the environmental factors that control them. Furthermore, metabolic and environmental controls on organisms and processes that release and accumulate major pollutants, like NO3and N2O, remain largely mysterious. While it is tempting to use genetic manipulation to engineer nitrogenfixing cereal crops or to improve nitrification inhibitors, technological fixes will continue to be limited by our understanding of the underlying microbiology. It is therefore incumbent on science and society to pay attention to the nitrogen cycle and take our cues from the microbes that ultimately control it. ACKNOWLEDGEMENTS
Modern day nitrogen cycle and societal challenges It has become widely recognized, particularly in analyzing studies of food production and global climate change, that the fate of humanity is
Nitrogen-cycle research in the Stein lab is supported by the Natural Sciences and Engineering Research Council of Canada. Nitrogen-cycle research in the Klotz lab has been supported during the last two decades
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Arp, D.J., and Bottomley, P.J. (2006). Nitrifiers: More than 100 years from isolation to genome sequences. Microbe 1, 229–234. Daims, H., Lebedeva, E.V., Pjevac, P., Han, P., Herbold, C., Albertsen, M., Jehmlich, N., Palatinszky, M., Vierheilig, J., Bulaev, A., et al. (2015). Complete nitrification by Nitrospira bacteria. Nature 528, 555–559. Erisman, J.W., Sutton, M.A., Galloway, J., Klimont, Z., and Winiwarter, W. (2008). How a century of ammonia synthesis changed the world. Nat. Geosci. 1, 636–639. Ettwig, K.F., Butler, M.K., Le Paslier, D., Pelletier, E., Mangenot, S., Kuypers, M.M.M., Schreiber, F., Dutilh, B.E., Zedelius, J., de Beer, D., et al. (2010). Nitrite-driven anaerobic methane oxidation by oxygenic bacteria. Nature 464, 543–548. Hoffman, B.M., Lukoyanov, D., Yang, Z.-Y., Dean, D.R., and Seefeldt, L.C. (2014). Mechanism of nitrogen fixation by nitrogenase: the next stage. Chem. Rev. 114, 4041–4062. Kartal, B., Maalcke, W.J., de Almeida, N.M., Cirpus, I., Gloerich, J., Geerts, W., den Camp, H., Harhangi, H.R., Janssen-Megens, E.M., Francoijs, K.J., et al. (2011). Molecular mechanism of anaerobic ammonium oxidation. Nature 479, 127–130. Klotz, M.G., Schmid, M.C., Strous, M., op den Camp, H.J., Jetten, M.S., and Hooper, A.B. (2008). Evolution of an octahaem cytochrome c protein family that is key to aerobic and anaerobic ammonia oxidation by bacteria. Environ. Microbiol. 10, 3150–3163. Klotz, M.G., and Stein, L.Y. (2008). Nitrifier genomics and evolution of the nitrogen cycle. FEMS Microbiol. Lett. 278, 146–456. Schleper, C., and Nicol, G.W. (2010). Ammoniaoxidising archaea—physiology, ecology and evolution. Adv. Microb. Physiol. 57, 1–41. Simon, J. (2002). Enzymology and bioenergetics of respiratory nitrite ammonification. FEMS Microbiol. Rev. 26, 285–309. Simon, J., and Klotz, M.G. (2013). Diversity and evolution of bioenergetic systems involved in microbial nitrogen compound transformations. Biochim. Biophys. Acta 1827, 114–135. Stein, L.Y. (2011). Heterotrophic nitrification and nitrifier denitrification. In Nitrification, B.B. Ward, D.J. Arp and M.G. Klotz, eds. (Washington, D.C.: ASM Press), pp. 95–114. Stein, L.Y., and Klotz, M.G. (2011). Nitrifying and denitrifying pathways of methanotrophic bacteria. Biochem. Soc. Tran. 39, 1826–1831. Ward, B.B. (2011). Nitrification: an introduction and overview of the state of the field. In Nitrification, B.B. Ward, D.J. Arp and M.G. Klotz, eds. (Washington, DC: ASM Press), pp. 3–8. Zhu-Barker, X., Cavazos, A.R., Ostrom, N.E., Horwath, W.R., and Glass, J.B. (2015). The importance of abiotic reactions for nitrous oxide production. Biogeochemistry 126, 251–267.
1
Department of Biological Sciences, University of Alberta, Edmonton, AB, Canada. 2 Department of Biology and School of Earth & Environmental Sciences, Queens College of the City University of New York, Flushing, NY, USA. 3Institute of Marine Microbes & Ecospheres and State Key Laboratory of Marine Environmental Science, Xiamen University, Xiamen, China. *E-mail: [email protected]
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Clémence Poirotte1,*, Peter M. Kappeler2, Barthelemy Ngoubangoye3, Stéphanie Bourgeois4, Maick Moussodji5, and Marie J.E. Charpentier1 Parasites are sometimes capable of inducing phenotypic changes in their hosts to improve transmission [1]. Toxoplasma gondii, a protozoan that infects a broad range of warm-blooded species, is one example that supports the so-called ‘parasite manipulation hypothesis’: it induces modifications in rodents’ olfactory preferences, converting an innate aversion for cat odor into attraction and probably favoring trophic transmission to feline species, its only definitive hosts [2]. In humans, T. gondii induces behavioral modifications such as personality changes, prolonged reaction times and decreased long-term concentration [3]. However, modern humans are not suitable intermediate hosts because they are no longer preyed upon by felines. Consequently, behavioral modifications in infected people are generally assumed to be side effects of toxoplasmosis or residual manipulation traits that evolved in appropriate intermediate hosts. An alternative hypothesis, however, states that these changes result from parasite manipulative abilities that evolved when human ancestors were still under significant feline predation [3,4]. As such, T. gondii also alters olfactory preferences in humans; infected men rate cat urine, but not tiger urine, as pleasant while non-infected men do not [5]. To unravel the origin of Toxoplasmainduced modifications in humans, we performed olfactory tests on a living primate still predated by a feline species. We found in our closest relative, the chimpanzee (Pan troglodytes troglodytes), that Toxoplasmainfected (TI) animals lost their innate
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Investigations
Morbid attraction to leopard urine in Toxoplasmainfected chimpanzees
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Correspondence
Chimpanzee's behavioral responses (Least Square Means and SEM)
by the NSF, DOE, USDA, the Gordon and Betty Moore Foundation and institutional incentive funds from the University of Louisville and UNC Charlotte.
-1
p = 0.008 p = 0.048
-2 -3 -4 -5 -6
p = 0.003 p = 0.047
Figure 1. Comparison of the behavioral responses of 33 chimpanzees towards urine of human, leopard (natural predator) and tiger and lion (non-natural predators) during behavioral tests based on olfactory cues. (A) Olfactory investigations performed towards urine source (for each urine type, n = 9 TI and n = 24 TN chimpanzees). (B) Approaches displayed towards urine source (for each urine type, n = 40 scans per individual). Least Square Means (LSM) and SEM are represented. White bars, TN chimpanzees; black bars, TI chimpanzees. Only significant differences in LSM (p < 0.05) are shown for biologically relevant two-by-two comparisons (intra-treatment comparisons: same urine type, different parasite status; and intra-parasite status comparisons: same parasite status, different urine type; see the Supplemental Information for a full set of biologically relevant comparisons.) For approach data, high negative values indicate fewer approaches towards urine source than low negative values.
aversion towards the urine of leopards (Panthera pardus), their only natural predator. By contrast, we observed no clear difference in the response of TI and Toxoplasma-non-infected (TN) animals towards urine collected from other definitive feline hosts that chimpanzees do not encounter in nature. Although the adaptive value of parasitically induced behavior should be assessed carefully, we suggest that the behavioral modification we report could increase the probability of chimpanzee predation by leopards for the parasite’s own benefit. This possible parasite adaptation would hence suggest that Toxoplasma-induced modifications in modern humans are an ancestral legacy of our evolutionary past. We performed collective olfactory tests on 33 chimpanzees (9 TI and 24 TN), living in five captive groups in
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The nitrogen cycle Lisa Y. Stein1,* and Martin G. Klotz2,3 Nitrogen is the fourth most abundant element in cellular biomass, and it comprises the majority of Earth’s atmosphere. The interchange between inert dinitrogen gas (N2) in the extant atmosphere and ‘reactive nitrogen’ (those nitrogen compounds that support, or are products of, cellular metabolism and growth) is entirely controlled by microbial activities. This was not the case, however, in the primordial atmosphere, when abiotic reactions likely played a significant role in the inter-transformation of nitrogen oxides. Although such abiotic reactions are still important, the extant nitrogen cycle is driven by reductive fixation of dinitrogen and an enzyme inventory that facilitates dinitrogen-producing reactions. Prior to the advent of the Haber-Bosch process (the industrial fixation of N2 into ammonia, NH3) in 1909, nearly all of the reactive nitrogen in the biosphere was generated and recycled by microorganisms. Although the Haber-Bosch process more than quadrupled the productivity of agricultural crops, chemical fertilizers and other anthropogenic sources of fixed nitrogen now far exceed natural contributions, leading to unprecedented environmental degradation. The significance of nitrogen to the biosphere and to cellular life is indisputable; however, our fundamental knowledge of the microorganisms and enzymatic processes that transform nitrogen into its various oxidation states (Figure 1) remains incomplete. Here, we outline the major microbial processes of the nitrogen cycle, the microorganisms that perform nitrogen transformations, and the modularity and evolutionary history of the nitrogen cycle, and provide a perspective of this cycle, now and into the future. Major processes of the nitrogen cycle The nitrogen cycle has traditionally been divided into three processes — N2 fixation, nitrification, and denitrification (Figure 2) — and microbes have historically been labeled by their identified participation in one of these R94
processes, that is, ‘nitrogen fixers’, ‘nitrifiers’ and ‘denitrifiers’. This assignment of microbes to specific eco-physiological cohorts based on their association with a single process came under scrutiny when ecologists found evidence for dissimilatory (that is, non-assimilatory) reduction of nitrite to nitric oxides and nitrous oxides in oxic environments (Figure 2, reaction 6A; called ‘nitrifier denitrification’), as well as dissimilatory reduction of nitrite to ammonium (Figure 2, reaction 2; called ‘respiratory ammonification’) and dissimilatory oxidation of ammonium in anoxic environments (Figure 2, reaction 7; called ‘anammox’). Our previous understanding of the nitrogen cycle was complicated by the numerous and diverse scientific approaches and foci — from the compounds that were transformed, to the compounds that were generated, to the environmental status of reactions that constituted the processes. The narrow foci in these approaches — employed for over 100 years in nitrogen-cycle research — have been overcome in the post-genomic era due to much-improved instrumentation, a great wealth of data, and increased interdisciplinary and international collaboration. Thus, our understanding of the nitrogen cycle now consists of five accepted nitrogen-transformation flows: ammonification, including nitrogen fixation, and assimilatory and dissimilatory reduction of nitrite (Figure 2, reactions 1 and 2); nitrification (Figure 2, reactions 3A, 3B and 4); denitrification, including canonical, nitrifier-dependent and methaneoxidation-dependent denitrification (Figure 2, reactions 6A–D); anammox, as a form of coupled nitrification– denitrification (Figure 2, reactions 7A– C); and nitrite–nitrate interconversion (Figure 2, reactions 4 and 5). The general processes of organic matter mineralization (often mislabeled as ‘ammonification’) and assimilation (often incorrectly claimed to include processes that regulate the generation of ammonium and its uptake) by cellular life complete the movement of reactive nitrogen through the biosphere (Figure 2). Ammonification Nitrogen fixation is one of two versions of ammonification and is accomplished by bacteria and archaea that encode nitrogenase enzyme complexes made
Current Biology 26, R83–R101, February 8, 2016 ©2016 Elsevier Ltd All rights reserved
Molecule
Name
Oxidation state
Reduced C-NH2 Organic-N NH3, NH4+ Ammonia, Ammonium -3 More N2H4 Hydrazine -2 electrons NH2OH Hydroxylamine -1 N2 Dinitrogen 0 N2O Nitrous oxide +1 NO Nitric oxide +2 Fewer HNO2, NO2 Nitrous acid, Nitrite +3 electrons NO2 Nitrogen dioxide +4 HNO3, NO3- Nitric acid, Nitrate +5 Oxidized Current Biology
Figure 1. Nitrogen-cycle intermediates. Intermediates, representing nine oxidation states, that donate or accept electrons, thereby contributing to electron flow and conservation of energy in participating microbes.
up of the molybdenum–iron protein dinitrogenase and the vanadium or iron protein dinitrogenase reductase. Although there is momentum to genetically engineer plants to express microbial nitrogenase complexes or to express the signaling pathway to attract nodulating nitrogen-fixing bacteria to colonize cereal crops, biological nitrogen fixation remains an activity solely performed by bacteria and archaea. Nitrogen fixation is extremely oxygen sensitive, requiring microorganisms to develop protective mechanisms, such as spatial decoupling (compartmentalization or forming specialized cells), temporal decoupling, rapid O2 respiration, or maximization of nitrogenase synthesis and turnover. The ammonium produced by nitrogen fixation is either assimilated into biomass or is further respired by aerobic and anaerobic ammonia-oxidizing microbes. This coupling of reaction 1 with either reaction 3 or 7, as shown in Figure 2, occurs either between nitrogen-cycle-active microbes in communities or within single cells, such as in nitrogen fixing, nitrifying methanotrophic bacteria. Anaerobic assimilatory (ANRA) and dissimilatory (DNRA) nitrite reduction to ammonium (Figure 2, reaction 2) is the second version of ammonification and is performed by both bacteria and fungi. This process can be linked to nitrate reduction to nitrite (Figure 2, reaction 5) at the cellular level or between organisms in a community. The acronym DNRA was originally coined to describe an ecophysiological process in which nitrogen in the form of nitrate was traceably removed from soils without either being lost in the form of nitrogenous gases (denitrification) or assimilated
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Magazine into microbial biomass. While the significance of ANRA and DNRA to global nitrogen cycling facilitated by fermenting fungi in soils is not well understood, respiratory ammonification by bacteria and archaea in diverse anoxic habitats is well established. DNRA can generate a cellular protonmotive force and thus conserve energy to support cellular growth, although this feature depends on which enzymes are coupled. A negative redox potential (that is, reducing conditions) is the most important stimulant for DNRA. It should be noted that the ANRA process employs identical chemistry to DNRA but it is facilitated by evolutionarily unrelated nitrite reductases. Nitrification The process of nitrification involves three cohorts of microorganisms: cohort I, ammonia oxidizers that oxidize ammonia to nitrite (nitritation; Figure 2, reactions 3A and 3B); cohort II, nitrite oxidizers that oxidize nitrite to nitrate (nitratation; Figure 2, reaction 4); and cohort III, complete ammonia oxidizers that oxidize ammonia all the way to nitrate (comammox; Figure 2, reactions 3A, 3B and 4). Although comammox microorganisms were discovered only in 2015, their existence was predicted approximately 10 years earlier. Cohorts II and III include only chemolithotrophic microbes that can use nitrite and ammonia, respectively, as sole sources of energy and reductant for cellular growth. Whereas nitrite-oxidizing chemolithotrophs of cohort II are classified within several classes of the Proteobacteria and the phylum Nitrospirae, the comammox bacteria are, so far, restricted to representatives in lineage II of the genus Nitrospira. Nitrifying ammonia-oxidizing microbes of cohort I include chemolithotrophic members of Betaproteobacteria and Gammaproteobacteria, and members of the Thaumarchaeota. In addition to these chemolithotrophs, there are heterotrophic and methanotrophic microorganisms that proficiently oxidize ammonium to nitrite, but they do not gain energy from the process to support growth. The nitrite and nitrate produced by aerobic reactions in the nitrification process can be respired anaerobically (Figure 2, reactions 2 and 5) or the resulting ammonium can be assimilated.
N2
6C
1
7C N2O N2H4 Assimilation
6D 6B
R~NH2
NH4+/NH3 7B NO
Mineralization 3A
2 NH2OH
7A 6A
NO25
3B 4
-
NO3
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Figure 2. Major processes of the nitrogen cycle. Reactions that comprise the seven major processes of the nitrogen cycle are represented by the numbered circles. Ammonification may be accomplished either by process 1, reduction of dinitrogen (also referred to as ‘nitrogen fixation’ or ‘Nif’), or by process 2, dissimilatory nitrite reduction to ammonium (DNRA). Nitrification is composed of process 3, oxidation of ammonia to nitrite (also referred to as ‘nitritation’), and process 4, oxidation of nitrite to nitrate (also referred to as ‘nitratation’). Process 5, reduction of nitrate to nitrite, can be coupled to processes 2, 6 or 7 in a population or a community. Denitrification is shown as process 6, which is also referred to as ‘nitrogen-oxide gasification’. Anammox is shown as process 7, and is also referred to as coupled ‘nitrification–denitrification’.
Denitrification Denitrification describes the process of anaerobic respiration of nitrite (NO2-), nitric oxide (NO), and nitrous oxide (N2O) to N2 (Figure 2, reactions 6A–C). Heterotrophic microbes that can directly couple these three reactions with the reduction of nitrate to nitrite (Figure 2, reaction 5) and perform denitrification from NO3- to N2 are referred to as classical or canonical denitrifiers. It is nevertheless recognized that many bacteria and archaea express only partial denitrifying inventories, and are missing genes encoding enzymes involved in reactions 5, 6B and/or 6C (Figure 2). Such incomplete pathways can lead to the release of nitrogenous gases such as NO and N2O to the environment, or a failure to deplete NO3-. This group also includes all ammonia-oxidizing chemolithotrophic bacteria, which reduce NO2- to N2O aerobically (Figure 2, reactions 6A and 6B). In addition, several eukaryotes including fungi and foraminifers (a class of protists) can reduce NO2- or NO3- to N2O or N2, respectively. In an unusual twist to the denitrifying
process, a methanotrophic bacterium, Candidatus Methylomirabilis oxyfera, consumes methane as a source of energy, reductant and carbon, and reduces NO2- to N2 by expressing a nitric oxide dismutase. This enzyme dismutates two NO molecules to O2 and N2 without producing the intermediate N2O (Figure 2, reaction 6D); the O2 produced is then used by this bacterium to oxidize methane to methanol while it resides in an anoxic environment, hence the name ‘denitrifying intra-oxygenic methanotroph’. Anammox Anammox, or anaerobic ammonium oxidation, utilizes the pools of NO2and ammonium (NH4+) to form N2 via the intermediates NO and hydrazine (N2H4; Figure 2, reactions 7A–C). The recent literature has also suggested the existence of a modified anammox pathway, in which hydroxylamine (NH2OH) is used instead of NO for the oxidation of NH4+, but additional experimentation will be required to validate this pathway. Anammox chemolithotrophy is performed solely by the Brocadiaceae bacteria in
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Magazine the order Planctomycetales within a specialized organelle called the anammoxosome. Anammox is ecologically beneficial for wastewater treatment as it removes both nitrite and ammonium simultaneously without producing N2O, and has been industrially implemented at full scale. Anammox is also a major nitrogenremoval process in the ocean and in oxygen minimum zones. From organisms to modularity of the nitrogen cycle For hundreds of years, microbiologists have enriched and isolated microorganisms from the environment to connect discrete biological entities to particular processes. The enzymology of the above nitrogen-cycle processes were largely discovered using axenic cultures of microorganisms, leading to the impression that these cultured microbial phylotypes should also be the main players in charge of those processes in the environment. For instance, the first ammonia-oxidizing bacteria were isolated in the 1890s and a great proportion of biochemical, physiological, and genetic knowledge of ammonia oxidation was derived from research performed with these very isolates. For many years, microbial ecologists relied on gene sequences and physiological attributes derived from a small number of isolates to detect and enumerate ammonia oxidizers in the environment. However, in the 2000s, metagenome sequences showed an irrefutable association between ammonia monooxygenase (the first enzyme involved in ammonia chemolithotrophy) and archaeal 16S rRNA genes that shifted the focus from bacterial ammonia-oxidizers to a whole new research venture based on the ammonia-oxidizing thaumarchaeota. Following the discovery of ammoniaoxidizing thaumarchaeota, the first strain isolated into pure culture was Nitrosopumilus maritimus in 2005, enabling physiological and biochemical characterization of a completely different group of ammonia-oxidizing chemolithotrophs. Aside from revealing entirely new phyla of nitrogen-cycling microbes that had evaded age-old cultivation-dependent approaches, genome and metagenome sequencing also revealed that individual modules, independent of complete R96
integrated pathways, are encoded and expressed by microorganisms to perform single segments of nitrogencycle metabolism. For instance, methanotrophic bacteria can express many genes encoding enzymes with a known involvement in the processes of nitrogen fixation, nitrification and denitrification. Intriguingly, there is no predictable association between nitrogen-cycle genes and phylotype among the methanotrophs. Furthermore, the extent of nitrogen-cycling inventory is scattered; some methanotrophs encode nitrite and nitric oxide reductases together, some fix nitrogen and encode a suite of denitrification genes, whereas others may encode only one reductase or none. Hence, environmental factors apparently drive random lateral gene transfer events to perform tasks to enhance fitness of a particular species, thereby leading to niche differentiation. For instance, the acquisition and expression of nitrifying modules could allow certain methanotrophs to survive high ammonium loads in situations where NH3 can compete with methane for access to methane monooxygenase enzymes. Similarly, expression of denitrifying modules can allow a methanotrophic species to grow in nearly anoxic ecosystems. Nitrogen fixation is inherently modular, with gene clusters distributed across numerous bacterial and archaeal phyla, arguing once again that habitat conditions select for this trait. For the majority of history in nitrogencycle research, we have held onto the concept that organisms of particular phylotypes are nitrifiers, denitrifiers, nitrogen fixers, and the like. This organismal concept is now challenged by the knowledge that nitrogen-cycle genes and processes are not necessarily constrained by phylotype, but rather endow organisms with the inventory required to adapt and thrive in ecosystems where nitrogenous molecules undergo dynamic changes in concentration and composition. Envisioning the nitrogen cycle as a continuum of modular evolution is consequently the most parsimonious outlook. Modular evolution of the nitrogen cycle The primordial nitrogen ‘cycle’ was not closed, but rather was dominated by coupled abiotic reactions that provided nitrogen oxides and reduced nitrogen
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in the form of NH4+, which is required for nitrogen assimilation into biomass. The nitrogen oxide reservoir in the form of NO3- vastly exceeded available NH4+, which favored the evolution of a reductase protein inventory involved in nitrate–nitrite inter-conversion (Figure 2, reactions 4 and 5) along with numerous nitrite reductases (Figure 2, reactions 2, and 6A/7A). The suite of reductase enzymes relied on the bioavailability of transition metals under anoxic conditions such as molybdenum (as found in molybdopterin) and iron (found in siroheme or heme cytochrome c). The function and evolution of these protein complexes and their encoding genes have been comprehensively reviewed by Simon and Klotz (2013) with an emphasis on bioenergetics. With the exception of reactions labeled 3A, 3B, 4, 7B and 7C in Figure 2, the reactions that interconvert nitrogen oxides of different oxidation states (Figure 1) are reductions, which require the availability and accessibility of electrons. These electrons are derived from the quinone pool, facilitated by quinone-reactive proteins (QRPs) directly, or via redox carrier proteins. Therefore, the evolution of proteins that participate in the nitrogen cycle is intrinsically connected to the evolution of QRPs. While QRP complexes that contain cytochrome b proteins have been well characterized, those that include Fe–S clusters (NapH/NosH family), cytochrome c proteins (NrfH/NapC/TorC family) or the enigmatic transmembrane protein NrfD/PsrC are significantly less well understood. Interestingly, the evolution and implementation of these QRPs in catabolic electron flow are also modular, interconnected and function to provide electrons to a variety of catalytic redox proteins involved in the sulfur and nitrogen cycles by oxidation of quinol. Exceptions are the composite MFc complex that evolved into an ‘alternative complex III’ for branched electron transfer (quinol oxidase not serving electrons to enzymes), the cM552 (CycB) protein, which functions as a quinone reductase in ammoniaoxidizing chemolithotrophs, and the copper-containing analog in ammoniaoxidizing Thaumarchaeota. Reactions 3A, 3B, 4, 7B and 7C in Figure 2 are oxidations, with reactions 3B and 7C being dehydrogenations performed by homologous multiheme
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Magazine cytochrome c (MMC) proteins. Interestingly, these dehydrogenases evolved from MMC reductases that perform reactions 2 and 6A/7A in Figure 2. Reactions 6A/7A and leaky dehydrogenase 3B were likely the main biotic generators of the NO radical, the source of nitrosative stress in cells and otherwise of abiotic origin, which served as the primordial oxidant before the advent of molecular oxygen. Abundant production of NO radicals led to the emergence of a large complement of evolutionarily unrelated enzymes capable of NO detoxification, most of them being reductases (reaction 6B in Figure 2), such as the soluble tetraheme cytochrome c’ and c’-beta proteins, which were later complemented by numerous other NO reductases, the main biotic source of N2O. We proposed in 2008 that the key innovation for the closure of the nitrogen cycle was likely the emergence of N2H2 hydrolase (reaction 7B in Figure 2) and its subsequent integration into a redox gradient generated by reactions 7A and 7C (Figure 2), which reversed electron flow through the protein complex and created what is now known as N2H2 synthase, the key enzyme in the anammox process. It is remarkable that this protein complex is restricted only to the Brocadiaceae that facilitate the anammox process. This innovation created a connection between reduced, oxidized, and inert nitrogen and thus constituted the closure and formation of a real cycle. The onset of nitrogen fixation is presently still debated, but there is growing momentum for placing the evolution of this pathway before the great oxygenation event, potentially as a contemporary invention to the emergence of the anammox pipeline. Copper was not bioavailable in anoxic environments, which is the reason why many enzymes with copper as the redox-active metal emerged after the great oxygenation event, including N2O, NO and NO2- reductases, and functional ammonia monooxygenase. While there is an increasingly clear picture on the evolutionary relationships between copper-containing membrane-bound monooxygenases (Cu-MMOs), including those that oxidize ammonia and short alkanes such as methane, the origins of the enzyme subunit proteins remain elusive. This explosion of nitrogen-
cycle inventory occurred in parallel to the explosion of aerobic lifestyles, which provided more efficient electron flow into and out of the quinone pool, while providing additional means for energy conservation (generation of proton-motive force). Hence, the taxonomically unpredictable and broad distribution of diverse nitrogencompound-transformation inventory emerged gradually over evolutionary time. This evolution followed diverse environmental and functional pressures, resulting in the acquisition, maintenance or loss of functionally linked inventory, thereby generating a nitrogen cycle of modular design, similar to the modular designs of the cellular central pathways (glycolysis, TCA, pentose phosphate pathway) or the Calvin cycle. It thus comes as no surprise to those who accept the modular nature of the nitrogen cycle that anammox bacteria can oxidize NO2- anaerobically with an enzyme homologous to the NO2--oxidizing enzyme of the aerobic Nitrospira even though the genomic background (phylotype) of these two bacterial groups is very distantly related. Bioenergetically speaking, the dissimilatory reactions of the nitrogen cycle make it the most advanced biogeochemical nutrient cycle because, out of nine possible intermediates, the nitrogen cycle uniquely includes representative reactions for seven oxidation states between -3 and +5 (Figure 1). While the sulfur and carbon cycles also span over nine oxidation states, they provide only three states (0, +2, +4) between -2 and +6 and three states (-2, 0, +2) between -4 and +4, respectively, that are available for electron-transfer reactions. This bioenergetics picture is congruent with our understanding of the timeline for the emergence of a complete nutrient cycle, a picture in which the extant nitrogen cycle represents a more evolved cycle, cemented by the finding that significant nitrogen-cycle inventory evolved from inventory active in the sulfur and/or carbon cycles.
intertwined with our ability to control the nitrogen cycle. On one side, the Green Revolution of the 20th century averted a global food crisis, in part by introducing widespread synthetic fertilizer usage. To put this in perspective, the Haber-Bosch process is responsible for feeding approximately 48% of the global human population, equivalent to supporting four billion births since 1908. On the other side, with our extremely rapid population growth and continued increases in fertilizer usage, we have pushed the nitrogen cycle beyond sustainability where now NO3- pollution is responsible for increasing dead zones in coastal areas and N2O is considered the key greenhouse gas to mitigate in the 21st century and beyond. Control of the nitrogen cycle is a tricky business, in part because of our success with fertilizer-dependent food production, and also because we are still discovering how the microbial processes of the nitrogencycle work. For instance, the relatively recent discoveries of the anammox and comammox processes, as well as the discovery of ammoniaoxidizing thaumarchaeota (described above) have revealed organisms and processes that profoundly impacted how we view and study nitrogen transformations and the environmental factors that control them. Furthermore, metabolic and environmental controls on organisms and processes that release and accumulate major pollutants, like NO3and N2O, remain largely mysterious. While it is tempting to use genetic manipulation to engineer nitrogenfixing cereal crops or to improve nitrification inhibitors, technological fixes will continue to be limited by our understanding of the underlying microbiology. It is therefore incumbent on science and society to pay attention to the nitrogen cycle and take our cues from the microbes that ultimately control it. ACKNOWLEDGEMENTS
Modern day nitrogen cycle and societal challenges It has become widely recognized, particularly in analyzing studies of food production and global climate change, that the fate of humanity is
Nitrogen-cycle research in the Stein lab is supported by the Natural Sciences and Engineering Research Council of Canada. Nitrogen-cycle research in the Klotz lab has been supported during the last two decades
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Arp, D.J., and Bottomley, P.J. (2006). Nitrifiers: More than 100 years from isolation to genome sequences. Microbe 1, 229–234. Daims, H., Lebedeva, E.V., Pjevac, P., Han, P., Herbold, C., Albertsen, M., Jehmlich, N., Palatinszky, M., Vierheilig, J., Bulaev, A., et al. (2015). Complete nitrification by Nitrospira bacteria. Nature 528, 555–559. Erisman, J.W., Sutton, M.A., Galloway, J., Klimont, Z., and Winiwarter, W. (2008). How a century of ammonia synthesis changed the world. Nat. Geosci. 1, 636–639. Ettwig, K.F., Butler, M.K., Le Paslier, D., Pelletier, E., Mangenot, S., Kuypers, M.M.M., Schreiber, F., Dutilh, B.E., Zedelius, J., de Beer, D., et al. (2010). Nitrite-driven anaerobic methane oxidation by oxygenic bacteria. Nature 464, 543–548. Hoffman, B.M., Lukoyanov, D., Yang, Z.-Y., Dean, D.R., and Seefeldt, L.C. (2014). Mechanism of nitrogen fixation by nitrogenase: the next stage. Chem. Rev. 114, 4041–4062. Kartal, B., Maalcke, W.J., de Almeida, N.M., Cirpus, I., Gloerich, J., Geerts, W., den Camp, H., Harhangi, H.R., Janssen-Megens, E.M., Francoijs, K.J., et al. (2011). Molecular mechanism of anaerobic ammonium oxidation. Nature 479, 127–130. Klotz, M.G., Schmid, M.C., Strous, M., op den Camp, H.J., Jetten, M.S., and Hooper, A.B. (2008). Evolution of an octahaem cytochrome c protein family that is key to aerobic and anaerobic ammonia oxidation by bacteria. Environ. Microbiol. 10, 3150–3163. Klotz, M.G., and Stein, L.Y. (2008). Nitrifier genomics and evolution of the nitrogen cycle. FEMS Microbiol. Lett. 278, 146–456. Schleper, C., and Nicol, G.W. (2010). Ammoniaoxidising archaea—physiology, ecology and evolution. Adv. Microb. Physiol. 57, 1–41. Simon, J. (2002). Enzymology and bioenergetics of respiratory nitrite ammonification. FEMS Microbiol. Rev. 26, 285–309. Simon, J., and Klotz, M.G. (2013). Diversity and evolution of bioenergetic systems involved in microbial nitrogen compound transformations. Biochim. Biophys. Acta 1827, 114–135. Stein, L.Y. (2011). Heterotrophic nitrification and nitrifier denitrification. In Nitrification, B.B. Ward, D.J. Arp and M.G. Klotz, eds. (Washington, D.C.: ASM Press), pp. 95–114. Stein, L.Y., and Klotz, M.G. (2011). Nitrifying and denitrifying pathways of methanotrophic bacteria. Biochem. Soc. Tran. 39, 1826–1831. Ward, B.B. (2011). Nitrification: an introduction and overview of the state of the field. In Nitrification, B.B. Ward, D.J. Arp and M.G. Klotz, eds. (Washington, DC: ASM Press), pp. 3–8. Zhu-Barker, X., Cavazos, A.R., Ostrom, N.E., Horwath, W.R., and Glass, J.B. (2015). The importance of abiotic reactions for nitrous oxide production. Biogeochemistry 126, 251–267.
1
Department of Biological Sciences, University of Alberta, Edmonton, AB, Canada. 2 Department of Biology and School of Earth & Environmental Sciences, Queens College of the City University of New York, Flushing, NY, USA. 3Institute of Marine Microbes & Ecospheres and State Key Laboratory of Marine Environmental Science, Xiamen University, Xiamen, China. *E-mail: [email protected]
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Clémence Poirotte1,*, Peter M. Kappeler2, Barthelemy Ngoubangoye3, Stéphanie Bourgeois4, Maick Moussodji5, and Marie J.E. Charpentier1 Parasites are sometimes capable of inducing phenotypic changes in their hosts to improve transmission [1]. Toxoplasma gondii, a protozoan that infects a broad range of warm-blooded species, is one example that supports the so-called ‘parasite manipulation hypothesis’: it induces modifications in rodents’ olfactory preferences, converting an innate aversion for cat odor into attraction and probably favoring trophic transmission to feline species, its only definitive hosts [2]. In humans, T. gondii induces behavioral modifications such as personality changes, prolonged reaction times and decreased long-term concentration [3]. However, modern humans are not suitable intermediate hosts because they are no longer preyed upon by felines. Consequently, behavioral modifications in infected people are generally assumed to be side effects of toxoplasmosis or residual manipulation traits that evolved in appropriate intermediate hosts. An alternative hypothesis, however, states that these changes result from parasite manipulative abilities that evolved when human ancestors were still under significant feline predation [3,4]. As such, T. gondii also alters olfactory preferences in humans; infected men rate cat urine, but not tiger urine, as pleasant while non-infected men do not [5]. To unravel the origin of Toxoplasmainduced modifications in humans, we performed olfactory tests on a living primate still predated by a feline species. We found in our closest relative, the chimpanzee (Pan troglodytes troglodytes), that Toxoplasmainfected (TI) animals lost their innate
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Investigations
Morbid attraction to leopard urine in Toxoplasmainfected chimpanzees
Toxoplasma-infected
A
Non-infected 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0
B
0
Approaches
FURTHER READING
Correspondence
Chimpanzee's behavioral responses (Least Square Means and SEM)
by the NSF, DOE, USDA, the Gordon and Betty Moore Foundation and institutional incentive funds from the University of Louisville and UNC Charlotte.
-1
p = 0.008 p = 0.048
-2 -3 -4 -5 -6
p = 0.003 p = 0.047
Figure 1. Comparison of the behavioral responses of 33 chimpanzees towards urine of human, leopard (natural predator) and tiger and lion (non-natural predators) during behavioral tests based on olfactory cues. (A) Olfactory investigations performed towards urine source (for each urine type, n = 9 TI and n = 24 TN chimpanzees). (B) Approaches displayed towards urine source (for each urine type, n = 40 scans per individual). Least Square Means (LSM) and SEM are represented. White bars, TN chimpanzees; black bars, TI chimpanzees. Only significant differences in LSM (p < 0.05) are shown for biologically relevant two-by-two comparisons (intra-treatment comparisons: same urine type, different parasite status; and intra-parasite status comparisons: same parasite status, different urine type; see the Supplemental Information for a full set of biologically relevant comparisons.) For approach data, high negative values indicate fewer approaches towards urine source than low negative values.
aversion towards the urine of leopards (Panthera pardus), their only natural predator. By contrast, we observed no clear difference in the response of TI and Toxoplasma-non-infected (TN) animals towards urine collected from other definitive feline hosts that chimpanzees do not encounter in nature. Although the adaptive value of parasitically induced behavior should be assessed carefully, we suggest that the behavioral modification we report could increase the probability of chimpanzee predation by leopards for the parasite’s own benefit. This possible parasite adaptation would hence suggest that Toxoplasma-induced modifications in modern humans are an ancestral legacy of our evolutionary past. We performed collective olfactory tests on 33 chimpanzees (9 TI and 24 TN), living in five captive groups in
