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Hong YG et al. American
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Vo1. 2, Article ID 201400350, 13 pages
Review Article
Anammoxosome in Anaerobic
Ammonium-oxidizing Bacteria –
was It Originated from Endosymbiosis？
Yiguo Hong1,2, Huiluo Cao2,3, Meng Li2,4 and Ji-Dong Gu2*
1
State Key Laboratory of Tropical Oceanography, South China Sea Institute of Oceanography,
Chinese Academy of Sciences, 164 Xingang Road West, Guangzhou 510301, China
2
Laboratory of Environmental Microbiology and Toxicology, School of Biological Sciences, The
University of Hong Kong, Pokfulam Road, Hong Kong SAR, China
3
Division of Biological Sciences, Hong Kong University of Science and Technology, Clearwater Bay,
Kowloon, Hong Kong SAR, China
4
Department 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 anammox 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
nitrite 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; nitrogen 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 nitrogen cycle 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 nitrate (now nitrite) reduction. By 1999, Strous
and his colleagues identified the missing lithotrophs as new planctomycetes 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 hydrazine, an
extremely toxic chemical as a catabolic intermediate [7,8], the biosynthesis of ladderane lipids
[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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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 organelles,
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 organelle in bacteria. In some ways,
the anammoxosome is similar to the eukaryotic mitochondrion, as both organelles form
non-photosynthetic PMFs [23].
Secondly, hydroxylamine oxidoreductase (HAO), one of the major enzymes 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 enzyme [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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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 hydrolase 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
denitrification 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 (NH2 OH) 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 N 2 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 lipid 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
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
Cytoplasmic membrane
Intraytoplasmic membrane
Anammoxosome membrane
Riboplasm
Nucleoid
ⅰ
ⅱ
ⅲ
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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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 nitrites 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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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 biology 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).
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