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http://ivyunion.org/index.php/ajcmicrob Hong YG et al. American Journal of Current Microbiology 2014, 2:18-40 Page 1 of 13 Current Microbiology Vo1. 2, Article ID 201400350, 13 pages

Review Article

Anammoxosome in Anaerobic -oxidizing – was It Originated from Endosymbiosis？

Yiguo Hong1,2, Huiluo Cao2,3, Meng Li2,4 and Ji-Dong Gu2*

1State Key Laboratory of Tropical Oceanography, South China Sea Institute of Oceanography, Chinese Academy of Sciences, 164 Xingang Road West, Guangzhou 510301, China 2Laboratory of Environmental Microbiology and Toxicology, School of Biological Sciences, The University of Hong Kong, Pokfulam Road, Hong Kong SAR, China 3Division of Biological Sciences, Hong Kong University of Science and Technology, Clearwater Bay, Kowloon, Hong Kong SAR, China 4Department of Earth and Environmental Sciences, University of Michigan, Ann Arbor, MI 48109, USA

Abstract The anammoxosome is a single membrane-bounded compartment with ATP-generation capability residing in

the yet-to-be purely cultured bacteria, which are responsible for the unique biochemical reaction called anaerobic ammonium oxidation (anammox). In this paper, a plausible mechanism for the origin of anammoxosome is proposed, in which anaerobic archaea with capability of metabolizing ammonium and are thought to gain advantages for survival with reciprocal metabolisms and eventually established as stable endosymbiont under the given environmental conditions by invading into a bacterial cell. Over the long-time specialization and lateral gene transfer during the endosymbiosis establishment, the original

archaea might further devolve into the present anammoxosome inside the host to form the structure of anammox bacteria today. The cytological, biochemical and molecular evidences for this hypothesis are

presented, along with suggestions for further possible experimental verifications. The evolutionary significance of this symbiotic hypothesis is also discussed.

Keywords: Anammoxsome; origin and evolution; endosymbiosis; cycle Peer Reviewer: Adhar C. Manna Manna, PhD, Department of Biological Sciences, Presidency University, India; Chengjian Jiang, PhD, College of Life Science and Technology, Guangxi University, China Received: March 23, 2014; Accepted: July 12, 2014; Published: September 28, 2014 Competing Interests: The authors have declared that no competing interests exist. Copyright: 2014 Gu J-D et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. *Correspondence to: Ji-Dong Gu, School of Biological Sciences, The University of Hong Kong, Pokfulam Road, Hong

Kong SAR, People’s Republic of China; Email: [email protected]

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Introduction

Over the last decades, research on microbial anaerobic ammonium oxidation (anammox) has been extremely active, but pure culture of this group of microorganisms has not been obtained successfully. As reported in several papers, anammox is a microbial process of importance to both the global in the ecosystem and the nitrogen removal in the wastewater treatment plants [1-4]. However, the evolutionary history of the microbes that perform this biochemical process has been a long-standing mystery to be solved by the scientific community. Initially, on the basis of thermodynamics calculation, Broda [5] predicted the existence of the chemolithoautotrophic bacteria capable of generating ATP for growth by forming dinitrogen (N2) directly from coupling ammonium oxidation with (now nitrite) reduction. By 1999, Strous and his colleagues identified the missing as new in an enrichment culture reactor from wastewater treatment plant [6]. These bacteria are named after their distinctive biochemical metabolism as anammox. Capable of such a novel metabolic reaction, these anammox bacteria have many unique features, including the synthesis and metabolization of , an extremely toxic chemical as a catabolic intermediate [7,8], the biosynthesis of [9-12] and the presence of an internal compartmentalization structure as anammoxosome [9,13]. The anammoxosome, unique to anammox planctomycetes, is a single membrane-bounded compartment for ATP-generation associated with the anammox biochemical process [9,13-15]. Thus, anammox bacteria are the firstly identified prokaryote possessing ATP-generation structure inside a host cell in a similar way parallel to mitochondrion in eukaryotes. Based on the electron micrograph observation and immunogold labeling technique, the cellular structure of the anammox bacteria has been illustrated and has been visualized with a three-bilayer membrane. The outermost membrane has been defined as cytoplasmic membrane and the innermost as anammoxosome membrane. The other has been defined as intracytoplasmic membrane, which is located between cytoplasmic membrane and anammoxosome membrane. The anammox cell is thus divided into three compartments by the membranes, from outside to inside, the paryphoplasm, riboplasm and anammoxosome [16]. As an individual compartment, the origination of anammoxosome is still a mystery to be solved, which has great implication to the understanding of early evolution process of life. Understanding the origin of this specialized compartment may provide novel insights into the evolution of cellular structures and clues for discovering potentially new anammox bacteria or archaea before the uncertainty of phylogenetic positions of anammox planctomycetes is ascertained [17]. Generally, the origin of ATP-generation compartment can be explained by two different existing viewpoints. Some take for granted that complex structures might evolve gradually by differentiation of cells de novo (autogenous origin); others argue that they could be formed from the endosymbiosis between two independent individual cells (endosymbiotic origin) [18]. Now, the endosymbiotic origin of mitochondria and chloroplast has been accepted widely [19]. Based on the analyses below, we hypothesize that the anammoxosome might be also originated from an endosymbiosis event between one putative anaerobic archaea and another ancient planctomycetes

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Hong YG et al. American Journal of Current Microbiology 2014, 2:18-40 Page 3 of 13 reinforced over time by selection pressure for survival and also reciprocal metabolisms to benefit each other. This paper is divided into three parts. The first part presents our hypothesis based on available evidences in current published literatures, and the second part shows the evolutionary significance of this proposed endosymbiotic hypothesis and the last section suggests some feasible experiments to prove the plausibility of the hypothesis in the future.

Endosymbiotic origin of anammoxosome

Anammoxosome as a unique energy-generating compartment

Most of the present evidences indicate that anammoxosome is an independent energy-generating biological compartment [9,13,15,20]. The anammox metabolism model had been proposed [21] and available evidences indicated that the unique energy-generation process involving nitrite as electron acceptor and ammonium as electron donor occurs on the anammoxosome membrane. Two pivotal points of evidences are available for this model. Firstly, the unique anammoxosome membrane consists of specialized ladderane lipids [22], which are currently only found in this group of microorganisms, forming a proton gradient between the anammoxosome and pirellulosome. It is well known that mitochondria and chloroplast are energy-generating and harvesting , respectively, in eukaryotic cells. Energy conservation of them is carried out through the proton motive force by proton gradient across the cellular membrane; the mechanism was explained by Mitchell’s chemiosmotic theory. In the case of anammox bacteria, anammoxosome and paryphoplasm as two separate compartments are divided by membranes. Proton consumption in the paryphoplasm and generation inside the anammoxosome creates charge separation resulting in an electrochemical proton gradient with unidirectional flow from anammoxosome to the riboplasm, generating proton motive force (PMF) to drive ATP synthesis [15]. This mechanism is consistent with or very similar to the chemiosmotic interpretation of energy generation in mitochondria and chloroplast, indicating that it is actually an energy-generation in bacteria. In some ways, the anammoxosome is similar to the eukaryotic mitochondrion, as both organelles form non-photosynthetic PMFs [23]. Secondly, hydroxylamine (HAO), one of the major specific to the anammox metabolism, is abundant in anammox bacteria and forms up to 9% of the soluble proteins of total anammox biomass [7]. In the genome of Kuenenia stuttgartiensis, eight enigmatic copies are responsible for encoding this [11]. It has also been identified to be located entirely within the anammoxosome compartment by immunogold labeling of thin-sectioned cryosubstituted cells [9]. Recently, two copies of the HAO have been designated with the ability of oxidizing hydrazine, which was named as hydrazine oxidoreductase (HZO) [24]. The location of this crucial enzyme provides the strongest support for the uniqueness of this unique compartment. Similar to the anammox bacterial host cells, the anammoxosomes reproduce through dimidiate division and they always arise through division of pre-existing ones [14,15,17]. This serves as a strong argument that the anammoxosome are foreign body or endosymbiont. Assuming that the anammoxosome was differentiated gradually from anammox bacterial cells de novo, why did not

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Hong YG et al. American Journal of Current Microbiology 2014, 2:18-40 Page 4 of 13 the anammox bacterial cell differentiate into two or more anammoxosomes in one cell? Previous transmission electron micrographs indicated that there is only one anammoxosome in each anammox bacterial cell [15,17]. Therefore, the presence of only one anammoxosome within a single anammox bacterial cell bears the evidence for the possible independence of this specialized compartment and its symbiotic origination. We thus propose that an ancient anammox microbial cell (we define the microbe as pre-anammoxosome in this paper) invaded or be engulfed into the ancient planctomycete cell in the early phase of life evolution and such association has been maintained the continuity of anammoxosome division, not differentiated gradually by anammox bacterial themselves de novo. Although most DNA in the form of the fibrillar nucleoid is present in the riboplasm, some DNA appearing in the anammoxosome have been confirmed by analysis using anti-DNA antibody [9]. This small amount of DNA within anammoxosome might be speculated to be relic DNA of the ancient pre-anammoxosome cell even after the long-time of co-evolution through adaption and gene exchange with the host genomes, which might include genes encoding some important proteins anchoring on anammoxosome membrane. Therefore, we presume that besides independent ATP-generation, pre-anammoxosome cell should also contain an independent set of genetic systems before the initial formation of the endosymbiosis. Recently, Strous et al. utilized environmental genomics to acquire the genome of the uncultured anammox bacterium K. stuttgartiensis and estimated the more than 98% of the complete genome [11]. However, in their paper, they did not provide any information about the relic DNA in anammoxosome. From their genome analysis, they deduced one candidate gene cluster encoding the unique ladderane biosynthetic pathway proteins and another two responsible for the hydrazine and hydrazine dehydrogenase, separately [11]. However, until now there is no direct experimental evidence to support the presence of these candidate genes and their biochemical functioning in the anammox bacteria. Additionally, the gene homologues to those encoding FtsZ or tubulin, which are pivotal to the cell division and other important cell functions, have not been designated in the genome analysis. The incomplete genome information may be the reason for the incompletion of this crucial and important information at this moment. The deeper sequencing could offer some hints for the origination of anammoxosome in the future.

Reciprocal metabolisms for endosymbiosis

Anammox bacteria can utilize ammonium via anammox biochemical pathway under oxygen-limited conditions, but high concentration of ammonium in cells is very harmful to the organisms, especially to nucleoid in riboplasm. Special biochemical pathway is needed to detoxify this toxic metabolic intermediate and a mechanism to detoxify ammonium in specialized organelles residing in the ancient planctomycetec. The ability of dissimilatory nitrate reduction to ammonium has been demonstrated recently in anammox bacteria [25]. Actually, they are also capable of acquiring ammonium from ammonification [4]. This could resolve the issue about the metabolic source of ammonium (Figure 1).

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Figure 1 Reciprocal metabolization is a basic impetus to the symbiosis between putative anammox Archaea and Planctomycete bacteria. Nas, nitrate reductase；Nir, nitrite reductase； Hh, hydrazine hydrolase；Hzo, hydrazine-oxidizing enzyme.

Klotz and Stein [26] redrew the nitrogen cycle with emphasis on the early incomplete and ammonification as the driving forces for emergence of anammox to form a basis for a complete nitrogen cycle. They also suggested that in the early anaerobic nitrogen metabolism, hydroxylamine (NH2OH) is an intermediate of nitrite metabolized to ammonium and poisonous to organisms. Additionally, on the basis of the genomic analyses of anammox bacterium K. stuttgartiensis, more than 200 genes are related to anammox catabolism and respiration [11]. The extraordinarily high number exceeds that possessed by most other bacteria, in particular more than ten copies of HAO gene present in anammox bacteria. This also indicates that the metabolism and respiration versatility of anammox bacteria is extraordinary. Therefore, we propose that the ancient planctomycetes would be an independent organism capable of utilizing the abundant HAO available to nitrite and then to ammonium via hydroxylamine as an intermediate. Since both ammonium and hydroxylamine are harmful chemicals to the ancient planctomycetes, a unique mechanism or specialized structure is necessary to detoxify them and generate energy efficiently in order to survive. Ancestral pre-anammoxosome could utilize ammonium and hydroxylamine to generate energy and produce N2 gas, and this probable process was proven by experiments of van de Graaf et al [4,8]. In contrast, ancient planctomycets would utilize nitrite through dissimilatory process to ammonium via hydroxylamine as an intermediate. This reciprocal metabolism should be an indispensable force to drive the endosymbiosis because pre-anammoxosome cell invaded into ancestral planctomycetes could detoxify the harmful ammonium in the host of planctomycetes in the early phase of evolution before oxygen was produced and released into the atmosphere. After biologically produced oxygen was generated and released into atmosphere with increasing concentration, they could also survive the damage by forming the co-existence and coevolution from atmospheric oxygen outside. This may be the primary external pressure driving such an endosymbiosis over evolutionary time. After invasion, they would utilize the reciprocal metabolites from each other and evolve into the present metabolic pathways via NO as an intermediate [4,11].

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Anammoxosome membrane and Archaea

The unique ladderane lipids in anammox bacterial anammoxosome are fundamentally different from those of cytoplasmic membrane of bacteria [10,12], implying that the distinctive origins and biosynthetic pathways of this kind of lipids. Because the special chemical constitutes of ladderane lipids is much similar to those of archaea, which is composed of ether-linked fatty acids, these two kinds of lipids are proposed to originate from a possibly common ancestor followed by diverging into two different biochemical pathways. Another possibility about the origin of this kind of unique lipids in anammoxosome is that they were synthesized by ancient ‘anammox’ archaea. Based on the simple rule when creating a novel organism, it is seldom to synthesize a novel kind of lipids in the original organism as this may needs another novel synthesis pathway and much more unique proteins. Thus, the most feasible origin of the ladderane lipids is likely that an ancient anammox microorganism was recruited into the anaerobic host cell, and then incorporated these two biosynthetic pathways into the present unique anammox planctomycetes through evolution to form a stable relationship for co-existence. The most promising candidate genes to encode the unique lipid biosynthesis pathway have been proposed, however no experiments have been conducted to prove their biochemical roles [11]. In our blast analyses, no highly homologous proteins and genes to those suggested by Strous et al. responsible for biosynthesis of ladderane lipids could be retrieved from the GenBank [11]. We proposed that these genes could be from the anaerobic ammonium-oxidizing archaea though we have not confirmed this directly as the larger differences exist on genetic level.

Anammox catabolism genes

ATPase gene clusters: In the K. stuttgartiensis genome, more than 200 genes related to anammox catabolism and respirations have been annotated [11]. The number of genes far exceeds the number possessed by most of other bacteria, e.g., Geobacter sulfurreducens and Shewanella oneidensis [27,28]. Selective genes are present in multiple copies. For example, four ATPase gene clusters were identified in the K. stuttgartiensis genome [11]. Through analyses of gene arrangement (a, c, b, δ, α, γ, β, ε) and similarity, one ATP synthase gene cluster resembles the “standard” ATP-synthase (F-ATPase) commonly found in all Bacteria [29,30]. Another two clusters have different operon structures similar to Pirellula sp. strain 1 (a typical F-ATPases) and the archaea Methanosarcina barkeri (V-ATPase). Immunogold localization showed that the typical F-ATPase was predominantly located on both the outermost and anammoxosome membrane and to a lesser extent on the middle membrane. This is consistent with our hypothesis that the host bacterial cell would be an independent living microorganism, which can generate energy by itself initially. The occurrence of ATPase in the anammoxosome membrane suggests that an anammoxosome cell had devolved into an organelle similar to those found in eukaryotes, a membrane-bounded compartment with a specific cellular function for energy metabolism [31]. Hydroxylamine oxidoreductase (HAO)/hydrazine-oxidizing enzyme (HZO): In the annotated K. stuttgartiensis genome, besides the multicopies of ATP-synthase genes, more than ten hydroxylamine oxidoreductase (HAO) gene copies have been identified through homologous blast [11]. All proteins expressed by these homologous copies form up to 9% of the soluble protein of

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Hong YG et al. American Journal of Current Microbiology 2014, 2:18-40 Page 7 of 13 anammox biomass [7,24], indicating the extraordinary catalytic capability of hydroxylamine and also provides potential possibilities to the evolution from HAO to HZO. Considering genes homologous to HZO, these multiheme HAO proteins must have a very interesting history. However, these copies with lower similarity between them, only two copies (kuste1340 and kustc0694 annotated in the genome of K. stuttgartiensis), have been proved homologous to hydrazine-oxidizing enzyme (HZO) which have been designated through purified proteins encoded by this gene [24]. This multiheme protein enzyme has hydrazine-oxidizing activity in vitro might have a critical role in anaerobic ammonium oxidation. However, whether these two gene copies of HZO can metabolize hydrazine still needs further experimental support in vivo. Others except for these two copies still have not been identified to be capable of metabolizing hydrazine by bench work only conserved motifs and homology are used to form presumably confirmation. However, the two copies of HAO in K. stuttgartiensis were not included in the best possible hydrazine dehydrogenase candidate when annotation was carried out for the genome [11]. From phylogenetic trees constructed based on HAO/HZO partial amino acid sequences [32], hzo genes from all of the anammox bacteria are clustered together and formed one strongly supported clade with a long branch from other clades. The present phylogenetic trees suggest an evolutionary history and indicate that the emergence of HZO in anammox should be a later evolutionary event. In early anaerobic environment, it is possible that HAO was employed to catalyze hydroxylamine extensively in pre-anammox organisms. Then, with the emergence of oxygen by early blue-green algae and cyanobacteria and invasion of pre-anammox organism into planctomycetes, HAO became specifically adapt to the new anaerobic environment inside bacterial cells and evolved into HZO eventually.

Ancestral Planctomycete

Ribosome Parypholasm Cytopl asmic membr ane Intraytopl asmic membr ane

Anammoxos ome membr ane Riboplasm

Nucleoid

ⅰ ⅲ ⅱ ⅳ

Hypothetic Anammox Archaea Figure 2 A schemic model of endosymbiosis pathway for illuminating the proposed origin and evolution of anammoxosome. This model includes four phases: (i) The hypothetic anammox archaea and the ancestral planctomycete exist independently; (ii) hypothetic anammox archaea invaded into the bacterial cell of ancestral planctomycete accidentally; (iii) establishment of a symbiosis between these two; (iv) the original archaea might further evolve into the present anammoxosome.

Based on the evidence presented above from cytological, biochemical and molecular information, the anammoxosome should be regarded as an independent ATP-producing compartment. Anaerobic ammonium-oxidizing bacteria (ANAB) appear to have originated from an endosymbiont event

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Hong YG et al. American Journal of Current Microbiology 2014, 2:18-40 Page 8 of 13 through invasion by a putative anammox archaea into ancient bacteria, possibly planctomycetes. Through a long time of specialization and optimization of reciprocal metabolism under symbiotic condition, the original anammox archaea evolved into the present anammoxosome and formed part of the new type organism (Figure 2). Despite we have detected numerous novel phylogenetic lineages of archaea in nature ecosystem through PCR amplification of environmental genomic DNA, the results of amplification ultimately depend on the specificity of the PCR primers designed on the basis of the available archaeal 16S rRNA gene sequences and other functional genes-encoding protein information. Therefore, surprisingly high frequencies of mismatch for some archaeal PCR primers result in amplification bias against the corresponding archaeal lineages [33-35]. Thus, we believe one kind of archaea capable of utilizing ammonium and under anaerobic environment may exist in special habitat where the condition may mirror those of early earth environment. Discovery of anammox archaea would improve our understanding to the evolution of anammox bacteria greatly in the future.

The potential evolutionary significance of this symbiotic hypothesis

Symbioses widely spread in the living organisms, not only prokaryotes, but also eukaryotes, and are pivotal to ecology and evolution [36-39]. Endosymbiosis also play a key role in the evolution of the whole biological world, especially in the formation of all kinds of specialized compartments and structures. Based on the endosymbiotic theory in general, a large cell as a host engulfs the free-living prokaryotic or eukaryotic microorganisms, which are named as endosymbionts. Through adaptation and exchange of genetic materials between the two over a long period of time, these endosymbiotic microorganisms gradually evolved into intracellular organelles, such as the chloroplasts and mitochondria as accepted examples to form a stable relationship between the two in a single organism of today [40]. Regarding the origin of eukaryotic cell, it is generally accepted that symbiotic interactions between various cells of different species leading to the first eukaryotic cell. Symbioses also have been proposed to explain the origin of mitochondrion and plastid. Until now, the widely accepted explanation about the origin of eukaryotic cell is that they originated from the endosymbiosis between bacteria and archaea. However, Bacteria could invade into Archaea, and vice versa. Though the chimeric eukaryotic genome structure that is archeal-type for informational genes, and bacterial-type for metabolic genes has been designated, we still could not confirm the invasive direction either from a bacterium to archaea or the other way around [41-43]. Martin and Müller [44] proposed a new hypothesis for the origin of eukaryotic cells which merged from archaebacterium as the host and eubacterium as symbiont based on the comparative biochemistry analysis of energy metabolism while others have the contrary conclusion based on their own analyses [45]. This indicates the complexity of endosymbiosis and provides the uncertainty involved using phylogenetic inferences. In this paper, we proposed another endosymbiont hypothesis about the invasion of archaea to bacteria forming a unique anammox bacterium. The present hypothesis offered the plausible evidence that the enigmatic phylogenetic position of Planctomycetes was raised by the invasion

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Hong YG et al. American Journal of Current Microbiology 2014, 2:18-40 Page 9 of 13 from archaea to bacteria, as well. Planctomycetes have generally been accepted as an early group branched out from bacteria [46,47]. Anammox bacteria are also determined as a distinct, deeply branching group within the Planctomycetes based on 16S, 23S rRNA genes and a concatenated dataset consisting of 49 protein sequences [11,48-54]. The present hypothesis about the symbiosis of different species forming one whole anammox bacteria can also provide further information for the classification of enigmatic phylogenetic relationships about Planctomycetes and anammox bacteria. Another significant point for studying the origin of anammoxosome is that we can obtain some clues about the evolution and the origins of eukaryotic nucleus and cell organization. The significance for cell lies in their possession of intracellular membrane compartmentalization, a shared cell strategy within the cell cytoplasm is divided into compartment by one or more membranes including a major cell compartment with the nucleoid inside [17,55]. Anammox bacteria must provide clues and evidences to researchers about the origin of organelle and cell strategy for reason of the unique organelle, anammoxosome in it from its uniqueness standing point. Another special branch of Planctomycetes is Gemmata obscuriglobus in which the nucleoid is enveloped in two membranes to form a nuclear body analogous to the structure of a eukaryotic nucleus [9,56,57]. The origin of nuclei is still a debatable issue, although the origin of eukaryotic cell nuclei by symbiosis of archaea in bacteria was proposed on the basis of homology-hit analysis [42]. The similar organelle origin from the endosymbiosis between archaea and bacteria presented in this hypothesis would provide some clues to understand the origin of nuclei.

Further feasible experiments to prove this hypothesis

If the anammoxosome was actually originated from the invagination of cytoplasm, the anammox bacteria should possess the capability of continually differentiating anammoxosome. In other words, the anammoxosome, if indeed differentiated from the cytoplasm, must have become inheritable. A cell in ancient times possessing the ability to differentiate anammoxosome from its cytoplasm could not have just suddenly arisen. We assume that this capability developed gradually after many many generations under the pressure of the environmental conditions, especially availability of substrates to support growth and metabolisms and the increasing concentration of O2 in the atmosphere. If so, the capability to differentiate protoplasm must have become inheritable, for only then could each step towards manifestation of this ability have been supplemented and improved by the next. Logically, based on the endosymbiosis hypothesis presented above, if the anammoxosome was eliminated from anammox bacterium through some special means, it could not arise de novo, and notwithstanding the anammox bacteria can reproduce and grow through other energy-generating pathways as they are metabolic versatile. Small amount of DNA have been detected by anti-DNA antibody within anammoxosome [9]. In the future, culture and enrichment of anammox bacteria are still needed in light of a new cultivation system for anammox bacteria developed by van der Sar et al [58]. Then successful isolation of anammoxosome will make a major advancement. Refining and sequencing the relic DNA in anammoxosome will reveal more important evidences leading to the proving or disproving of this

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hypothesis. It will be interesting to find some functionally important proteins or enzymes related to the anammox metabolism encoded by these DNA sequences. Actually, Kuenen indicated that many partial genomes of anammox bacteria are currently being sequenced and identified [4]. Based on our analyses, some anammox Archaea may still exist in ocean sediment, because of the abundance of ammonium and nitrate as well as niche similar to early earth environment where molecular oxygen is limited. The anammox archaea should be the prototype of symbiont evolved into the present anammoxosome. Discovery of this microorganism can offer supporting evidence for this endosymbiotic hypothesis. Recently, Kuenen also proposed potential researches about anammox bacteria including the discovery of other Bacteria or even Archaea at the expense of anammox process [4]. One pivotal issue is the efficiency and specificity of PCR primers used to amplify target gene sequences from the environmental samples with unknown range of anammox bacteria (Han and Gu, 2013). Here we propose that except for 16S rRNA gene, both hzo and hzs should be appropriate gene markers to unravel the existence of additional unknown anammox archaea, for HZO and HZS are essential enzymes center to the nitrogen transformation in synthesis and oxidation of hydrazine in anammox bacteria [24].

Acknowledgments

This research was supported by national Natural Science Foundation of China (31270163, 41076095) (YGH) and Hong Kong SAR RGC GRF grant HKU701913P (J-DG).

References

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