Nitrogen Fixation In Methanogens : The Archaeal Perspective

The methanogenic Archaea bring a broadened perspective to the field of nitrogen fixation. Biochemical and genetic studies show that nitrogen fixation in Archaea is evolutionarily related to nitrogen fixation in Bacteria and operates by the same fundamental mechanism. At least six nif genes present in Bacteria (nif H, D, K, E, N and X) are also found in the diazotrophic methanogens. Most nitrogenases in methanogens are probably of the molybdenum type. However, differences exist in gene organization and regulation. All six known nif genes of methanogens, plus two homologues of the bacterial nitrogen sensorregulator glnB, occur in a single operon in Methanococcus maripaludis. nif gene transcription in methanogens is regulated by what appears to be a classical prokaryotic repression mechanism. At least one aspect of regulation, post-transcriptional ammonia switch-off, involves novel members of the glnB family. Phylogenetic analysis suggests that nitrogen fixation may have originated in a common ancestor of the Bacteria and the Archaea.


Introduction
It has long been recognized that nitrogen fixation occurs in bacteria but not eukaryotes.When Archaea were first discovered to be a prokaryotic domain of life separate from Bacteria (1), it was not known that they too contained nitrogen fixing species.In 1984 S. Zinder and L. Daniels independently discovered diazotrophic growth in two different methanogenic Archaea, Methanosarcina barkeri (2) and Methanococcus thermolithotrophicus (3). 15N incorporation from 15 N 2 confirmed that nitrogen fixation occurred in M. barkeri, and acetylene reduction confirmed nitrogenase activity in M. thermolithotrophicus.This discovery of nitrogen fixation in Archaea raised new questions regarding the diversity of nitrogen fixation at the phylogenetic, biochemical, and genetic levels.A review on nitrogen fixation in methanogens appeared in 1992 (4).

Distribution of Nitrogen Fixation in the Archaea
The well-characterized Archaea consist of the strictly anaerobic methanogens, the extreme halophiles, and the extreme thermophiles (5).Some methanogens are also extreme thermophiles.The Archaea are distributed over two main phylogenetic branches (kingdoms) based on 16S rRNA sequence comparisons, the Euryarchaeota and the Crenarchaeota (6).The Euryarchaeota contain the methanogens, the halophiles, and some extreme thermophiles, while the Crenarchaeota contain most of the extreme thermophiles.Within the Archaea, nitrogen fixation has been found only in the methanogenic Euryarchaeota.Within the methanogens, however, nitrogen fixation is widespread, extending to all three orders (7) (Table 1).In the Methanococcales, diazotrophic growth has been reported for Methanococcus thermolithotrophicus (3) and Methanococcus maripaludis (8).M. thermolithotrophicus is the only organism demonstrated to fix nitrogen at 60°C or above.Neither Methanococcus jannaschii (9) nor Methanococcus voltae (10) fix nitrogen despite the presence of nifH homologues (our unpublished results).In M. jannaschii it is clear that other nif genes are not present.Within the Methanomicrobiales, diazotrophic species include Methanosarcina barkeri (2,11) and Methanospirillum hungatei (12).In the Methanobacteriales, nitrogen fixation has been demonstrated for Methanobacterium bryantii (12).Whether Methanobacterium thermoautotrophicum is a diazotrophic species is unclear.There is a report of diazotrophic growth of strain Marburg (13), and both strains Marburg and ∆H have complete or nearly complete nif gene clusters compared to M. maripaludis (below).On the other hand, recent attempts to grow either strain diazotrophically have not been successful (R. Thauer, S. Zinder, personal communication).In addition, diazotrophic growth, 15 N 2 incorporation, or acetylene reduction has been reported for a number of other species of methanogens (4).

How Different is Nitrogen Fixation in the Archaea?
The discovery of nitrogen fixation in methanogens led to a quest to determine whether it resembled nitrogen fixation in Bacteria.Lobo and Zinder (15) presented evidence that diazotrophic growth in M. barkeri was energetically costly and was stimulated by molybdenum.Partial purification of nitrogenase activity revealed that two components were required (16).Subunit analysis suggested the presence of nitrogenase and nitrogenase reductase similar to those in Bacteria, except that the latter appeared to be a homotetramer rather than a homodimer.The putative nitrogenase reductase cross-reacted with antiserum against nitrogenase reductase of Rhodospirillum rubrum.Whole cells, extracts, and purified nitrogenase could reduce acetylene to ethylene, albeit at lower rates than in Bacteria.nifH was cloned from M. thermolithotrophicus using a bacterial probe.Sequencing revealed nifD and part of nifK downstream from nifH (17).These findings suggested that nitrogen fixation in methanogens was evolutionarily related to nitrogen fixation in Bacteria and operated with the same basic mechanism.If nitrogen fixation in Archaea fundamentally resembled nitrogen fixation in Bacteria, it seemed possible that Archaea might feature vanadium or iron-only nitrogenases that are present as "alternative" nitrogenases in certain Bacteria.However, this does not appear to be the case.The predominate nitrogenases in methanogens seem to be molybdenum nitrogenases as is the case in Bacteria (below).

Genetics
nif genes in methanogens were first cloned using nifH genes of Bacteria as hybridization probes (18).It soon became apparent that nifH genes in methanogens were followed by two genes with homology to glnB (below), then nifD and nifK (17,19).In M. maripaludis we used an oligonucleotide designed from a conserved region of nifH to clone a fragment from a DNA library (8).We used transposon insertion mutagenesis, a technique that we developed for M. maripaludis (8), to show that a region extending 8 kb from the beginning of nifH was required for diazotrophic growth (20).Sequencing this region identified the genes nifH through nifX as shown in Figure 1.These genes are clear homologues of the corresponding genes in Bacteria, further demonstrating that nitrogen fixation in Archaea is fundamentally the same as in Bacteria.Five of these genes have functions in Bacteria that are required for and central to nitrogen fixation.nifH encodes nitrogenase reductase, nifD and nifK encode the α and β subunits of nitrogenase itself, and nifE and nifN are required for iron-molybdenum cofactor (FeMoCo) synthesis (21).nifX and the glnB homologues are discussed below.
The nif gene organization found in M. maripaludis is conserved in other methanogens.In M. thermoautotrophicum a cluster with the same eight genes is present, with the sole exception that nifX does not seem to be present in one strain (∆H, ( 14)), although it is found in another strain of the same species (Marburg, GenBank accession number 1854560).A corresponding gene cluster in M. barkeri has been sequenced from nifH through most of nifE (22), and it is likely that nifN and nifX lie 3' to nifE.In M. maripaludis, at least, the entire cluster of eight genes belongs to a single operon, since transposons inserted throughout the cluster always had a polar effect, that is, they always eliminated the synthesis of mRNA from genes 3' to the insertion (20).Furthermore, an identifiable promoter sequence was present only in the region upstream of nifH.nif mRNA observed by Northern blot was usually fragmented into subsets of genes, and this could be attributed to some combination of intergenic termination of transcription and processing of the mRNA.
The organization of the methanogen nif gene cluster reveals differences as well as similarities when compared to bacteria.The order, nifH-nifD-nifK-nifE-nifN-nifX, is common in bacteria (21).However, the presence of novel homologues of glnB (below) between nifH and nifD is unique to the methanogens.In addition, the presence of the six nif genes, nifH, nifD, nifK, nifE, nifN, and nifX in a single operon is unknown in the Bacteria, which tend to separate the first three and the latter three into different operons (21).
Since nifX is not required for nitrogen fixation in several Bacteria ( 21), we checked its function in M. maripaludis, taking advantage of genetic methods developed for Methanococcus species.An in-frame deletion mutant of nifX was not detectably impaired in diazotrophic growth (23).nifX has sequence homology to nifY and to the Cterminus of nifB, and it has been proposed that nifX may have a dispensible function in FeMoCo synthesis that is duplicated by nifB or nifY (21).Whether M. maripaludis contains a functional nifB or nifY gene is unknown.nifB homologues (other than nifX) have been found in other methanogens (9,14), but the homology does not extend to the C-terminal end of bacterial nifB.Therefore, the dispensibility of nifX for nitrogen fixation in methanogens cannot easily be explained by gene duplication.
In contrast to the six nif genes of the nif operon in methanogens, in Klebsiella pneumoniae there are 20 different nif genes (21).It is at present unclear whether additional nif genes function in diazotrophic methanogens.Neither a complete genome sequence, nor saturation mutagenesis, has yet been performed on a bona fide diazotrophic methanogen.It is possible that in methanogens only the six nif genes of the identified nif operon are functional homologues of bacterial nif genes.If this is the case, functions such as electron transport to the nitrogenase complex, nitrogenase protein maturation and stabilization, and homocitrate synthesis, may be carried out by the products of genes that do not have recognizable homology to bacterial nif genes.Genes with similarity to nifH, nifS, nifB, and nifU have been found in nondiazotrophic Archaea (9,24), so the presence of these genes alone does not imply a function in nitrogen fixation.

Metals
A number of workers have used diazotrophic growth assays to determine whether methanogens might contain alternative (vanadium or iron-only) nitrogenases as do some Bacteria.In one study, vanadium as well as molybdenum stimulated M. barkeri (25), while in another study only molybdenum was stimulatory (15).The presence of two genes in M. barkeri that are phylogenetically related to functional nifH genes (19) allows for the possibility that both molybdenum and vanadium nitrogenases are present in this organism, and recently a vanadium nitrogenase has been confirmed (25a).In M. maripaludis, molybdenum was clearly required for diazotrophic growth (26).Vanadium failed to stimulate, and tungsten inhibited, so this species may contain a single nitrogenase of the molybdenum type.The presence of molybdenum nitrogenases in other methanogens is corroborated by nif gene phylogeny (26), and by the apparent absence from the nif gene clusters of a gene corresponding to vnfG or anfG, which encode the δ subunits of alternative nitrogenases in Bacteria (27).Thus, molybdenum nitrogenases seem to predominate in methanogens, but vanadium nitrogenases may be present as well.Nitrogenases have not been purified sufficiently from methanogens to obtain a definitive metal content.

Regulation Transcriptional Regulation
In M. thermolithotrophicus, mRNAs corresponding to nifH and nifD were reported to be present when N 2 was the only nitrogen source but absent when ammonia was provided (17).In vitro transcription found no evidence for regulation (28), and it took in vivo genetic studies in M. maripaludis to identify regulatory features.
The regulation of nif transcription in M. maripaludis occurs by repression.This mechanism contrasts with the regulation of nif transcription in K. pneumoniae and other members of the Proteobacteria, which operates by an activation mechanism involving NifA.M. maripaludis contains two inverted repeats in the promoter region of the nif operon that are similar to repeats previously noted in M. thermolithothophicus (17) (Figure 2).We fused the nif promoter region of M. maripaludis to lacZ, mutagenized the inverted repeats, and showed that the sequence of the first inverted repeat is required for repression in the presence of ammonia (29).Extract from ammonia-grown cells caused a shift in the gel mobility of promoter region DNA that depended on the first inverted repeat sequence.Presumably, a repressor protein in the extract bound to the inverted repeat.Therefore, nif transcription is regulated by repression and the first inverted repeat is the " nif operator".The second inverted repeat does not appear play a major role in regulation; but it could play a secondary role.
An inverted repeat similar to the nif operator of M. maripaludis, and matching the consensus GGAAN 6 TTCC, is a common feature in the promoter regions of nitrogenregulated genes in Methanococcus and Methanobacterium species (23).Genes for nitrogen fixation, ammonia assimilation (glutamine synthetase, glnA), ammonia or methylammonia transport (amtB), and the regulatory GlnB protein, contain this sequence in their promoter regions.In the case of the glnA gene in M. maripaludis, we have confirmed by mutagenesis that the inverted repeat sequence functions in repression (30).Thus, sequences that resemble the nif operator may mark genes that are coordinately regulated by a common repression mechanism.These genes may therefore belong to a "nitrogen regulon" in at least two genera of methanogens belonging to two different orders.Since nif gene transcription in methanogens appears to be regulated directly by this common nitrogen mechanism, it may not have an additional level of regulation specific to nitrogen fixation, as is the case involving NifA in Proteobacteria.However, there does appear to be a post-transcriptional mechanism specific to nitrogen fixation (below).
The repressor protein that binds to the nitrogen operator has not yet been identified, but one may speculate that it has dimeric nature and that each of two identical subunits binds to one half of the inverted repeat on the same face of the helix, as in the paradigm of bacterial repression.The evidence provided by the methanogen nif system, that regulation may resemble bacterial repression, is interesting because the basal transcription apparatus in Archaea resembles the eukaryal apparatus (31,32).
In contrast, there is no evidence for a nitrogen operator sequence in M. barkeri.In that species, nif transcription may be regulated by a different negative mechanism in which a substance present in ammonia-grown cells inhibits binding of a transcription-associated protein or proteins to the promoter region (33).There is no evidence in this case that the inhibiting substance is itself a DNA-binding protein.

Homologues of the nif Gene Cluster
The glnB family of nitrogen sensory-regulatory genes is widespread.New glnB homologues have recently been discovered in Bacteria and Archaea, and it is beginning to appear likely that GlnB proteins are involved in all aspects of nitrogen regulation in prokaryotes.Genome sequencing has revealed glnB homologues, closely related to bacterial glnB genes, in methanogens (9,14).In addition to these genes, two unique subfamilies of glnB homologues reside in the nif gene clusters of methanogenic Archaea (see for example Figure 1).Compared to "typical" glnB genes of Bacteria and Archaea, and compared to each other, these nif-cluster glnB genes differ markedly in the T-loop region (Figure 3).In the well-characterized GlnB protein, PII of Escherichia coli, the T-loop has been shown to contain the sites of interaction with other proteins that modulate the regulatory activity of PII or that transmit the nitrogen signal to regulatory targets (34).Therefore, the novel sequences of the regions corresponding to the T-loop indicate that the methanogen nif-cluster GlnB proteins are likely to have novel interactions with other proteins.
We have recently determined the function of the nifcluster glnB genes of M. maripaludis.Any function at the level of nif gene transcription was ruled out by the results of insertion mutagenesis across the nif cluster (20).(However, a function in transcriptional regulation for "typical" glnB genes encoded elsewhere in the genome is still possible).To test for other possible functions of the nifcluster glnB genes, we made an in-frame deletion mutation that eliminated most of both genes.We found ( 23) that the glnB mutant was deficient in ammonia switch-off, that is, in the immediate post-transcriptional inhibition of nitrogen fixation that occurs in some species (35,36) upon addition of ammonia to nitrogen-fixing cultures.This finding shows that one or both of the nif-cluster glnB genes acts negatively to regulate nitrogen fixation at a post-transcriptional level.Switch-off also occurs in Methanosarcina barkeri (15,16) and certain Bacteria (Rhodospirillum rubrum (35) and Azospirillum brasilense (36)), and while an involvement of glnB homologues is likely, it has not yet been demonstrated in these cases.It is known that ammonia switch-off in Bacteria is achieved through covalent ADP-ribosylation of nitrogenase reductase, controlled by a specific ADP-ribosyl transferase and a specific glycohydrolase.Similar covalent modification of nitrogenase does not appear to occur in methanogens (16, our unpublished results).
Figure 3. Alignment of glnB homologues in Archaea and Bacteria.The top group of five represents the second glnB gene in the nif operon of methanogens (see for example Figure 1).The next group of five represents the first glnB gene in the nif operon of methanogens.The last group of six contains the "typical" glnB genes of Bacteria and methanogens.Species designations are as follows: mbi, Methanobacterium ivanovii; mmp, Methanococcus maripaludis; mth, Methanobacterium thermoautotrophicum strain DH; msb, Methanosarcina barkeri; mtl, Methanococcus thermolithotrophicus; eco, Escherichia coli; mj, Methanococcus jannachii.Arabic numerals indicate gene designations relative to whole genome analyses (9,14).The predicted T-loop is indicated (39,40).* indicates the tyrosine reside which is the conserved site of uridylylation in Bacteria.

Evolution of Nitrogen Fixation
The availability of nif gene sequences from Archaea expands our ability to trace the evolution of nitrogen fixation with phylogenetic methods.Expanding on the results of Chien and Zinder (22), we carried out a phylogenetic analysis of nifD and nifE (Figure 4).A separate analysis by parsimony gave essentially the same results as the distance matrix analysis.nifD and nifE are evidently paralogous, that is, related via an ancient gene duplication.Consequently, nifD genes provide a root for the nifE tree and vice versa.Each tree (nifD and nifE) contains two main branches.One branch is predominately bacterial, and the other branch contains nif genes of Archaea.This observation suggests that nitrogen fixation had its origin in a common ancestor of the two domains.Anabaena variabilis vnfE is anomalous, and is distant from both nifD and nifE of other species (37).
The overall pattern places the nif genes of Archaea and Bacteria into separate groups, but there are two exceptions.One exception is that for both nifE and nifD, Methanosarcina barkeri and Clostridium pasteurianum are closely related within a separate branch.In the case of nifE the M. barkeri -C.pasturianum clade groups with the Archaea with 100% bootstrap support.The simplest explanation is that C. pasteurianum acquired the nif genes from an ancestor of M. barkeri by horizontal gene transfer.The nifD tree would suggest that the M. barkeri -C.pasteurianum clade groups with the Bacteria, but this is not supported by bootstrap analysis.The other exception to the Archaea-Bacteria split is that the alternative (vanadium and iron-only) nitrogenase subunit genes vnfD and anfD (but not vnfE) of the Bacteria form a separate branch within the Archaea.vnfD and anfD could have arisen through an ancient gene duplication that predates the Archaea-Bacteria split.Alternatively, they could have Figure 4. Phylogenetic analysis of nifD and nifE by distance matrix.Expert analysis kindly provided by A. Rodrigo.The computer program PROTDIST was used to estimate distances between all pairs of aligned amino acid sequences, using the Dayhoff PAM matrix.A phylogenetic tree was reconstructed using the computer program NEIGHBOR, which builds a tree by neighbor-joining (41).Both programs are part of the PHYLIP suite of programs (42).To determine the degree of support for each branch of the neighbor-joining phylogenetic tree, bootstrapping (43) was performed.1000 pseudoreplicate sequence datasets were generated using SEQBOOT (also part of the PHYLIP suite), and for each pseudoreplicate, pairwise distances were estimated and the phylogenetic tree reconstructed.The number of trees which contained a given branch was counted and expressed as a percentage.Only those branches supported by more than 70% (44,45) of the bootstrap trees are shown.Alternative nitrogenase genes are designated vnf (vanadium type) and anf (iron-only type).Species designations are as follows: Anasp, Anabaena species; Anava, Anabaena variabilis; Azovi, Azotobacter vinelandii; Azsbr, Azospirillum brasilense; Braja, Bradyrhizobium japonicum; Clopa, Clostridium pasteurianum; Frasp, Frankia species; Klepn, Klebsiella pneumoniae; Mtbth, Methanobacterium thermoautotrophicum strain Marburg; Mtcma, Methanococcus maripaludis; Mtctl, Methanococcus thermolithotrophicus; Mtsba, Methanosarcina barkeri; Plebo, Plectonema boryanum; Rhisp, Rhizobium species; Rhoca, Rhodobacter capsulatus; Synsp, Synechococcus species; Thife, Thiobacillus ferrooxidans.evolved in the methanogen line and were passed by horizontal transfer to the Bacteria.
A parsimony analysis of nifK and nifN (not shown) gave results that were consistent with the conclusions from the nifD-nifE tree.Like nifD and nifE, nifK and nifN are paralogs.On the nifK side of the tree, bootstrap analysis strongly supported the discrete groups, bacterial nifK, bacterial vnfK and anfK, and methanogen nifK.C. pasteurianum and M. barkeri nifK formed a separate clade but with poor bootstrap support.On the nifN side, bacterial nifN formed a robust group that excluded methanogen nifN.In this case, bacterial vnfN clustered with nifN from the same species.
Similar conclusions can also be drawn from the nifH tree.In these analyses (26,38), there were four major branches.Three branches contained nifH genes that function in nitrogen fixation, while the fourth branch contained genes that evidently participate in a process unrelated to nitrogen fixation.Bacterial and archaeal nifH genes fell in two separate major branches.C. pasteurianum and M. barkeri nifH were closely related and formed a third major branch.anfH genes of bacteria fell in the archaeal branch.(vnfH genes, however, clustered with bacterial nifH).
These analyses all support a scenario in which nitrogen fixation had an ancient origin that preceded the divergence of Archaea and Bacteria.In addition, ancient gene duplication or horizontal transfer participated in the evolution of the alternative nitrogenase genes anfH, vnfD, anfD, vnfK and anfK, which remarkably are equally divergent from typical bacterial nif genes as are the methanogen nif genes.On the other hand, vnfH, vnfE, and vnfN evolved recently from the corresponding nif genes of Bacteria.Finally, horizontal gene transfer apparently occurred between ancestors of M. barkeri and C. pasteurianum.

Conclusions and Future Directions
Since the discovery of nitrogen fixation in methanogenic Archaea, it has become clear that the process is genetically and evolutionarily related to nitrogen fixation in Bacteria.However, phylogenetic analysis suggests that the relationship is as distant as are the two domains themselves.Given this distance, one expects to find in it the extremities in variations to the fundamental mechanism, making those aspects that are conserved all the more notable.The presence of nifH, D, K, E, N, and X, their gene order, and even whether they are essential for nitrogen fixation, is conserved.However, more work is needed to tell whether additional nif genes, less central to the process but nevertheless necessary in Bacteria, also exist in Archaea.The predominance of molybdenum as a metal of the nitrogenase cofactor also seems to extend across domains.Regulation varies across domains but also contains conserved features.The regulation of nif gene transcription in some methanogenic species involves repression, but the components of the nitrogen sensoryregulatory apparatus have yet to be identified.glnB homologues are present in both domains and may turn out to represent components of the regulatory apparatus in methanogens.We know already that divergent members of the glnB family exist in methanogens as well as less divergent members, and the former function in posttranscriptional regulation of nitrogenase activity.Further work on nitrogen fixation in Archaea is sure to reveal additional variations on familiar paradigms.

Figure 2 .
Figure 2. nif promoter region of Methanococcus maripaludis.The TATA box, ribosome binding site, and translation start are underlined.Inverted repeats are indicated with arrows above.

Table 1 .
Nitrogen Fixing Species in the Archaea