Genetics of Nitrogen Regulation in Methanococcus maripaludis

Corresponding author: John A. Leigh, University of Washington, Microbiology, Campus Box 357242, Seattle, WA 98195-7242., leighj{at}u.washington.edu (E-mail)

Communicating editor: W. B. WHITMAN

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

We have used genetic methods in Methanococcus maripaludis to study nitrogen metabolism and its regulation. We present evidence for a "nitrogen regulon" in Methanococcus and Methanobacterium species containing genes of nitrogen metabolism that are regulated coordinately at the transcriptional level via a common repressor binding site sequence, or operator. The implied mechanism for regulation resembles the general bacterial paradigm for repression, but contrasts with well-known mechanisms of nitrogen regulation in bacteria, which occur by activation. Genes in the nitrogen regulons include those for nitrogen fixation, glutamine synthetase, (methyl)ammonia transport, the regulatory protein GlnB, and ammonia-dependent NAD synthetase, as well as a gene of unknown function. We also studied the function of two novel GlnB homologues that are encoded within the nif gene cluster of diazotrophic methanogens. The phenotype resulting from a glnB null mutation in M. maripaludis provides direct evidence that glnB-like genes are involved in "ammonia switch-off," the post-transcriptional inhibition of nitrogen fixation upon addition of ammonia. Finally, we show that the gene nifX is not required for nitrogen fixation, in agreement with findings in several bacteria. These studies illustrate the utility of genetic methods in M. maripaludis and show the enhanced perspective that studies in the Archaea can bring to known biological systems.

THE development of genetic methodology has made Methanococcus maripaludis an ideal archaeon for the study of gene function. Tools include high efficiency transformation (TUMBULA et al. 1994 ), integrative (GERNHARDT et al. 1990 ; SANDBECK and LEIGH 1991 ) and replicative (TUMBULA et al. 1997 ; WHITMAN et al. 1997 ) vectors, selectable markers for antibiotic resistance (GERNHARDT et al. 1990 ; ARGYLE et al. 1996 ), ex situ transposon insertion (BLANK et al. 1995 ), gene insertion and replacement by homologous recombination (BLANK et al. 1995 ; COHEN-KUPIEC et al. 1997 , COHEN-KUPIEC et al. 1999 ; KESSLER et al. 1998 ), and reporter genes (COHEN-KUPIEC et al. 1997 ). In addition, M. maripaludis grows unusually fast for a mesophilic methanogen, yielding liquid cultures overnight and colonies in 3 days.

We are using M. maripaludis as a model organism for the study of nitrogen metabolism and its regulation in Archaea. We have focused mainly on nitrogen fixation, which occurs in a variety of methanogenic Archaea (BELAY et al. 1984 ; MURRAY and ZINDER 1984 ; LOBO and ZINDER 1992 ). Early biochemical and gene sequencing studies in methanogens indicated that the mechanism of nitrogen fixation in methanogens was fundamentally the same as that in bacteria (LOBO and ZINDER 1988 , LOBO and ZINDER 1990 ; SOUILLARD et al. 1988 ; SOUILLARD and SIBOLD 1989 ; SIBOLD et al. 1991 ). However, results so far regarding gene organization and regulation have revealed interesting variations in methanogens. For example, we identified a cluster of eight genes in M. maripaludis (Figure 1), all of which have homologues that function in nitrogen fixation and nitrogen regulation in bacteria (KESSLER et al. 1998 ). Other workers identified portions of a similar cluster in other methanogens (CHIEN and ZINDER 1996 ). Thus, nifH, nifD, nifK, nifE, nifN, and nifX are present in the same order as is common in bacteria (DEAN and JACOBSON 1992 ). But in contrast to bacteria, they are all present in a single operon (KESSLER et al. 1998 ). In addition, two novel homologues of the nitrogen regulatory glnB genes (below) are also present, between nifH and nifD, in the same operon.

View larger version (21K): [in this window] [in a new window] [Download PPT slide]   Figure 1. M. maripaludis nif region. Key constructs are shown without the vector portion. pMMP1.9.2.2 was transformed into M. maripaludis to make the glnB deletion strain. pMMP2.9.1X was transformed into M. maripaludis to make the nifX deletion strain. Important sites used in cloning are indicated. Numbers indicate bases from the left end. Pur refers to the puromycin resistance marker.

We have studied the mechanism of transcriptional regulation of the nif operon in M. maripaludis. Targeted mutagenesis showed that a palindromic (inverted repeat) sequence CGGAAAGAAGCTTCCG, located immediately 3' to the transcriptional start site for the nif operon (KESSLER et al. 1998 ), contains a repressor binding site (COHEN-KUPIEC et al. 1997 ). Therefore, we term this site the nif operator. [In Methanosarcina barkeri, nif regulation occurs by a different negative mechanism (CHIEN et al. 1998 ).] Recently, we characterized a similar sequence (GGAAAGCTATTTCC) in the glnA promoter region of M. maripaludis. glnA, another nitrogen-regulated gene, encodes glutamine synthetase. The sequence was situated immediately 5' to a pair of closely spaced transcription start sites. As with the nif operator, we used targeted mutagenesis to show that this sequence was required for repression (COHEN-KUPIEC et al. 1999 ). In contrast, transcriptional regulation of nif gene expression in the best-studied members of bacteria (the proteobacteria Klebsiella pneumoniae and Azotobacter vinelandii and the proteobacteria Azospirillum brasilense, Rhodobacter capsulatus, and Rhizobium meliloti) always requires NifA, a DNA binding protein that functions as a positive regulator (MERRICK and EDWARDS 1995 ). The level and activity of NifA, in turn, are controlled through a variety of mechanisms, depending on the species.

Recently we have become interested in the regulation of ammonia assimilation as well as nitrogen fixation. Central to the regulation of both processes in bacteria is the GlnB family of proteins (MERRICK and EDWARDS 1995 ; REITZER 1996 ). In Escherichia coli, the levels of glutamine and 2-ketoglutarate serve as indicators of the nitrogen state of the cell. The level of glutamine is communicated to GlnB (the PII protein in E. coli) through covalent uridylylation at a conserved tyrosine residue, while 2-ketoglutarate is bound directly by GlnB (JIANG et al. 1998A , JIANG et al. 1998B , JIANG et al. 1998C ). Recently, additional members of the GlnB family have been discovered, and it is becoming clear that this gene family is involved in many if not all aspects of bacterial nitrogen regulation (ARSENE et al. 1996 ; VAN HEESWIJK et al. 1996 ; ATKINSON and NINFA 1998 ; DE ZAMAROCZY and ELMERICH 1998 ; HE et al. 1998 ; VAN DOMMELEN et al. 1998 ; JACK et al. 1999 ).

GlnB proteins are likely to play a central role in nitrogen sensing and regulation in Archaea as well. In addition to the glnB homologues found in the nif gene cluster of methanogens, genome sequencing has revealed two additional glnB genes, not associated with nif genes, in M. jannaschii (BULT et al. 1996 ) and Methanobacterium thermoautotrophicum (SMITH et al. 1997 ), and three in Archaeoglobus fulgidus (KLENK et al. 1997 ).

Here we present further analyses of the regulatory elements of the nif gene cluster of M. maripaludis. We show by database searches that the nif operator, demonstrated as such by genetic methods in M. maripaludis, may be an example of a common nitrogen regulatory element in Methanococcus and Methanobacterium species. We demonstrate by genetic methods the function of the nif cluster glnB homologues. We also report the phenotype of a nifX mutant. These studies continue to illustrate the utility of genetic methods for determining gene function in M. maripaludis.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Strains, plasmids, and growth of strains:
Strains and plasmids are described in Table 1. M. maripaludis was grown as described (KESSLER et al. 1998 ). McN (WHITMAN et al. 1986 ) and nitrogen-free (BLANK et al. 1995 ) media were as described. For diazotrophic growth, cultures were first grown in McN overnight to an optical density at 660 nm (OD₆₆₀) of 0.4. This culture (0.1 ml) was used to inoculate 5 ml of nitrogen-free medium. Cultures were incubated 5–7 days on a reciprocal shaker at 37°. H₂-CO₂ gas was added every 48 hr to a final pressure of 370 kPa (3.7 atm). To determine the Nif phenotype of mutant strains, growth on N₂ was determined in triplicate cultures and compared to wild-type M. maripaludis and to a nif mutant or to growth in the presence of argon in place of N₂ (BLANK et al. 1995 ).

Acetylene reduction assays:
Four 5-ml cultures were grown diazotrophically to an OD₆₆₀ of 0.25–0.3. These cultures were combined, then returned to the original tubes to obtain four identical cultures. The headspace was flushed with a H₂-CO₂ gas mixture (80–20%) and acetylene was added by syringe to a final concentration of 0.1%. The tubes were pressurized with N₂-CO₂ (80–20%) to 170 kPa, then pressurized with H₂-CO₂ to 370 kPa, and incubated. An anaerobic solution of NH₄Cl was added after 2 hr of incubation where indicated. Ethylene and acetylene concentrations were determined by analysis of the headspace by gas chromatography using a column containing a mixture of Poropak N and Poropak Q attached to a flame ionization detector. Prior to each sampling, H₂-CO₂ was added to each tube to a final pressure of 370 kPa.

Construction of glnB deletion mutant:
Mmp-1 (Table 1, Figure 1) is a clone from the genomic library of M. maripaludis strain LL that contains the nif operon (BLANK et al. 1995 ). PMMP1.0 is a 9.6-kb subclone obtained by cutting at the XbaI sites at the left-end polylinker and near the 3' end of nifE. PMMP1.0 was digested with HindIII followed by ligation. This eliminated a fragment extending from the HindIII site immediately upstream of nifH to a HindIII site in the polylinker at the right end of the insert. The resulting subclone, pMMP1.7, contained 3.4 kb upstream of nifH. The puromycin resistance marker (Pur^r; GERNHARDT et al. 1990 ) was cloned as an EcoRI cassette from pBluePur into the EcoRI site 1.0 kb 5' to nifH to generate pMMP1.7.1. To construct pMMP1.8, a 2.1-kb HindIII fragment (partial digest of pMMP1.0) containing nifH, both glnB genes and the 5' end of nifD were cloned into pBluescript. This plasmid was digested with BseA1 and BseRI, blunted with Klenow, and religated to form pMMP1.8.1. pMMP1.8.1 was sequenced to verify that it contained the first 21 amino acids of glnBi, an in-frame deletion of glnBi/glnBii, and the stop codon of glnBii. The HindIII fragment of pMMP1.8.1 was cloned into the HindIII site of pMMP1.7.1 to yield pMMP1.9. Next, the remainder of nifD, all of nifK, and most of nifE were added by cloning the HindIII fragment of pMMP1.6 (right-hand HindIII site originates from vector and corresponds to XbaI site) into the HindIII site of pMMP1.9 (partial digest) to yield pMMP1.9.2.2. M. maripaludis strain Mm61, which contains a neomycin resistance (Neo^r) marker (ARGYLE et al. 1996 ) in nifD, was made by cloning a PvuII fragment of pMBSN into the EcoRV site of pMMP1.8 to yield pMMP1.8.4 and transforming (TUMBULA et al. 1994 ) M. maripaludis strain LL to obtain double homologous recombination. pMM-P1.9.2.2 was transformed into M. maripaludis strain Mm61, and Pur^r transformants were screened for Neo^s in order to identify transformants in which double homologous recombination had resulted in replacement of the region extending from upstream of the Pur^r insert to downstream of the glnB genes. Southern analysis of chromosomal DNA confirmed the absence of the HindIII site of glnBi, the absence of the StuI site of glnBii, and the expected shortening of the remaining fragments (results not shown). The glnB deletion strain is designated Mm54. A glnB⁺ control strain (Mm53) containing the Pur^r insertion upstream of nifH was made by transforming the wild-type strain LL with pMMP1.7.1.

Construction of nifX deletion mutant:
A 3.0-kb HindIII fragment from pMMP2.0 was cloned into pGEM7.1 to yield pMMP2.9. pMMP2.9 contains part of nifN, all of nifX, and an additional downstream region (Figure 1). The Pur^r cassette from pBluePur was cloned into the nifX-proximal EcoRI site of pMMP2.9 to yield pMMP2.9E1A. Control strain Mm57 containing the Pur^r insert and intact nifX was produced by transforming Mm305 with pMMP2.9E1A and obtaining gene replacement by double homologous recombination. nifX was deleted from pMMP2.9 by reverse PCR. Primers were designed to hybridize to the nifX upstream region (5'-GAAGATCTACCATCAGTTGATGCAATTGCTAC-3', reverse) and downstream region (5'-GAAGATCTATGACAGGAAATACATTAAAAGACG-3', forward), with a BglII restriction site at the end of each primer. The PCR product was digested with BglII and ligated to produce pMMP2.9X, which was sequenced on both strands to verify the deletion. The Pur^r marker was added as follows: An EcoRI partial digest of pMMP2.9E1A was used to obtain the Pur^r cassette and the adjacent 1.3-kb EcoRI region. pMMP2.9X was digested with EcoRI and this 3.1-kb fragment was inserted. The resulting plasmid, pMMP2.9.1X, was transformed into Mm305 to generate Mm59 by double homologous recombination. Southern analysis of chromosomal digests confirmed the deletion of nifX.

Nitrogen operator sequence search:
The genomic sequences of M. thermoautotrophicum strain H and M. jannaschii were obtained from the Ohio State University and The Institute for Genomic Research websites and divided into contigs of ~350 kb with 50-bp overlaps for analysis by the Genetics Computer Group (GCG) suite of programs. The sequence GGAA(N6)TTCC was sought using the FindPatterns program of GCG. Open reading frames (ORFs) were identified using the Frames program of GCG, and instances of the sequence that occurred between possible ORFs were considered further. Finally, where the reading frame was uncertain, ORFs as designated in the published genome annotations in GenBank were used to identify sequences that occurred upstream of genes.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Distribution of the nitrogen operator:
The nif and glnA operons of M. maripaludis appear to belong to a nitrogen regulon whose members contain similar palindromic repressor binding sites, which we term nitrogen operators. Similar palindromes have also been noted in the promoter regions of the nif operons of M. thermolithotrophicus (SOUILLARD and SIBOLD 1989 ) and Methanobacterium ivanovii (SIBOLD et al. 1991 ), and the glnA gene of M. voltae (POSSOT et al. 1989 ). These sequences suggest the consensus GGAA (N6)TTCC (Figure 2).

View larger version (45K): [in this window] [in a new window] [Download PPT slide]   Figure 2. The nitrogen operator in gene promoter regions. Mc mp, M. maripaludis; Mc tl, M. thermolithotrophicus; Mb ta M, Methanobacterium thermoautotrophicum strain Marburg; Mb iv, Methanobacterium ivanovii; Mc vo, M. voltae; Mc ja, M. jannaschii; Mb ta H, Methanobacterium thermoautotrophicum strain H. MJ and MT numbers refer to the gene number for M. jannaschii (BULT et al. 1996 ; SMITH et al. 1997 ). TATA boxes are shown where known. Distances to putative translation start sites (ATG) are shown. MJ0058 and MJ0059 are divergent and distances to ATG are from the same putative operator.

In the present work we set out to determine the distribution of putative nitrogen operators in the promoter regions of other genes in methanogens. We examined the genome (SMITH et al. 1997 ) of M. thermoautotrophicum strain H to identify all occurrences of the sequence GGAA(N6)TTCC. Of 34 occurrences, only 2 were upstream of ORFs identified in the published genome annotations in GenBank and were therefore likely to have functional significance. The remainder may be considered statistically random occurrences. One of the two sequences was upstream of glnA, and the other was upstream of an apparent amtB/glnB operon (Figure 2). amtB encodes a putative ammonia transporter. glnB and amtB genes are present in a single operon in bacteria as well (HE et al. 1998 ; JACK et al. 1999 ). Surprisingly, the operator sequence was not present in the nif promoter region of the M. thermoautotrophicum strain H but was present in the homologous location in strain Marburg (GenBank accession no. X87971).

Having found putative nitrogen operators for the nif cluster and the genes glnA, glnB, and amtB, we next examined each occurrence of these genes in M. jannaschii. We found three putative nitrogen operators, one upstream of glnA, a second upstream of an apparent glnB/amtB operon, and a third between a second set of glnB and amtB genes that are in divergent orientations with respect to each other (Figure 2). amtB and glnB are also present twice in M. thermoautotrophicum, apparently as operons in each case, but we found a nitrogen operator in front of only one of them. An analysis of the entire M. jannaschii genome for the nitrogen operator sequence revealed a total of 49 occurrences, of which 5 were upstream of ORFs. These 5 included the 3 loci above, a locus 5' to an ammonia-dependent NAD synthetase, nadE (MJ1352) (Figure 2), and a locus upstream of an ORF of unknown function (MJ0224). The nitrogen operator was not found upstream of the M. thermoautotrophicum H nadE gene, nor in the A. fulgidus (KLENK et al. 1997 ) or Pyrococcus horikoshii (KAWARABAYASI et al. 1998 ) homologues of MJ0224.

The nitrogen operator is evidently not present outside of the methanogenic Archaea: searching the regions upstream of the above-listed genes, where known, in A. fulgidus (KLENK et al. 1997 ), Sulfolobus solfataricus (KLENK et al. 1997 ), Pyrococcus woesei (TIBONI et al. 1993 ), and Haloferax volcanii (BROWN et al. 1994 ) revealed no occurrence of a nitrogen operator matching our consensus.

glnB genes in Methanococcus:
As noted above, glnB genes are found in methanogens in two different contexts, the nif gene cluster in diazotrophic methanogens, and adjacent to amtB genes in all methanogens investigated. Recently, we cloned an example of the latter in M. maripaludis (not shown). An alignment of glnB genes (Figure 3) shows that the entire family contains three groups that differ most markedly in their T-loop regions. The T loop in E. coli GlnB contains the site of uridylylation and mediates interactions with other proteins that transmit the nitrogen regulatory signal to various targets (JIANG et al. 1997A , JIANG et al. 1997B , JIANG et al. 1998B , JIANG et al. 1998C ). One group contains the "typical" glnB genes of bacteria and of methanogen glnB-amtB clusters (bottom six, Figure 3). The other two groups are glnBi of the nif cluster (middle five, Figure 3), and glnBii of the nif cluster (top five, Figure 3). These three groups are distinct phylogenetically as well (CHIEN and ZINDER 1996 ). The latter two groups are known almost exclusively in the nif clusters of the methanogenic Archaea. Thus, the nif clusters of diazotrophic methanogens introduce two novel groups of glnB homologues.

View larger version (102K): [in this window] [in a new window] [Download PPT slide]   Figure 3. Alignment of glnB homologues. Gene designations, species, and GenBank accession numbers as follows: mbi-i and mbi-ii, Methanobacterium ivanovii nif-cluster genes, X56071; mmp-i and mmp-ii, M. maripaludis nif-cluster genes, U75887; mth1561 and mth1562, Methanobacterium thermoautotrophicum strain H nif-cluster genes, AE000916; msb-i and msb-ii, Methanosarcina barkeri nif-cluster genes, X56072; mtl-i and mtl-ii, M. thermolithotrophicus nif-cluster genes, X13830; eco, E. coli glnB; ecoK, E. coli glnK; mj1344 and mj59, M. jannaschii "typical" glnB, U67574 and U67464; mth662 and mth664, Methanobacterium thermoautotrophicum strain H "typical" glnB, AE000846. The predicted secondary structural elements are shown below the sequences (JIANG et al. 1997A ). (*) The tyrosine residue that is the conserved site of uridylylation in bacteria.

Function of the nif-cluster glnB-like genes:
We have focused on the nif-cluster glnB-like genes of M. maripaludis to determine their function. Because members of the glnB family appear always to function in some aspect of nitrogen regulation, and because two glnB homologues are consistently present in the nif gene cluster in diazotrophic methanogens, we reasoned that the nif-cluster glnB genes of M. maripaludis must have some function in the regulation of nitrogen fixation. Earlier, we had eliminated the possibility that these glnB genes have any major function in transcriptional regulation of the nif operon. In that study (KESSLER et al. 1998 ), we generated transposon insertions upstream of the glnB genes that eliminated glnB expression due to polarity. These insertions had no effect on transcription from the nifH promoter as determined by mRNA levels. Therefore, we hypothesized that the glnB genes regulate nitrogen fixation, positively or negatively, at a post-transcriptional level. Post-transcriptional regulation by reversible covalent ADP-ribosylation of the NifH protein is known in Rhodospirillum rubrum (LIANG et al. 1991 ) and A. brasilense (ZHANG et al. 1993 ), but it is not known whether glnB genes are involved in those cases.

To test the function of the nif-cluster glnB genes, we made a construct containing an in-frame deletion that eliminated most of both glnB-like genes. Use of an in-frame deletion rather than a polar insertion mutation assured that the nif genes would remain functional. We introduced the deletion construct into M. maripaludis using a selectable marker for puromycin resistance, and confirmed by Southern blot that the glnB deletion mutation replaced the wild-type genes by double homologous recombination.

If one or both glnB genes function positively, we expect to observe a decrease in diazotrophic growth. However, diazotrophic growth was normal (not shown). An alternative hypothesis was that the glnB genes function negatively to decrease nitrogen fixation when the cell encounters a nitrogen source more favorable than N₂, such as ammonia. This phenomenon is termed ammonia switch-off. We added ammonia to acetylene-reducing cultures of M. maripaludis. In the glnB⁺ control strain (Figure 4A), acetylene reduction quickly stopped. It resumed after a time interval related to the amount of ammonia added, perhaps after its depletion. In contrast, in the glnB mutant (Figure 4B), acetylene reduction was unaffected by ammonia addition for the duration of the assay. These results show that one or both of the nif-cluster glnB genes is required for switch-off of nitrogenase activity in M. maripaludis.

View larger version (18K): [in this window] [in a new window] [Download PPT slide]   Figure 4. Acetylene reduction assays to test ammonia switch-off. (A) glnB+ strain Mm53. (B) glnB mutant Mm54. Ammonia was added at the time indicated by arrows. Quantity of ethylene corresponds to a sample of 0.1 ml at atmospheric pressure (100 kPa) taken from a headspace of 20 ml at 370 kPa (3.7 atm).

Function of nifX:
nifX is present in K. pneumoniae and many other diazotrophic bacteria. It is also present in the M. thermoautotrophicum strain Marburg (GenBank accession no. X87971), but not strain H (SMITH et al. 1997 ), in the same position (end of the nif gene cluster) as in M. maripaludis. The function of nifX in bacteria is unclear, but it seems to participate in the synthesis of the iron-molybdenum cofactor (FeMoCo) of nitrogenase (SHAH et al. 1999 ). It is not required for normal diazotrophic growth in K. pneumoniae, A. vinelandii, R. capsulatus, or Bradyrhizobium japonicum (DEAN and JACOBSON 1992 ). nifX is homologous to nifY and to the C-terminal portion of nifB, and any essential function of nifX in nitrogen fixation in bacteria might be duplicated by one of these genes.

Although nifX is not essential for nitrogen fixation in bacteria, it seemed plausible that it would be in M. maripaludis. In M. maripaludis, we have not identified a nifY or a nifB gene. In K. pneumoniae, nifB is required for biosynthesis of FeMoCo and for diazotrophic growth (DEAN and JACOBSON 1992 ; DEAN et al. 1993 ). A nifB homologue is present in the related species M. jannaschii, but we noticed that the homology is limited to the N-terminal portion of bacterial nifB, and M. jannaschii seems to lack the C-terminal portion of the gene. If M. maripaludis lacks the nifY or nifB gene or its C-terminal portion, one might expect nifX to take on its function and to be essential for nitrogen fixation. To test this hypothesis, we generated an in-frame deletion mutant of nifX using genetic methods similar to those we used to make the glnB mutant. However, the mutant was Nif⁺ (not shown), indicating that nifX is not essential for nitrogen fixation in M. maripaludis.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

We have used genetic methods to investigate several aspects of nitrogen metabolism and its regulation in methanogens. This work illustrates the utility of genetic methods in M. maripaludis. It also demonstrates the perspective that the Archaea bring to our understanding of biological systems. We have presented evidence that the methanogens contain a nitrogen regulon containing many of the same regulated genes that one finds in the various bacteria. Yet the regulation of the system has contrasting as well as similar features. Regulation by repression involving a palindromic operator is the defining characteristic in our current knowledge of the methanogen nitrogen regulon. This paradigm for repression, though common in bacteria in general, is not known in the bacterial nitrogen regulon (MERRICK and EDWARDS 1995 ; MAGASANIK 1996 ). The glnB gene family provides another example of a familiar-yet-contrasting regulatory feature. The methanogen nif cluster contains members of that family that are novel, yet conform to the rule that glnB genes regulate some aspect of nitrogen metabolism, in this case ammonia switch-off of nitrogenase activity. Finally, our finding that nifX is not required for nitrogen fixation corroborates results from bacteria.

The nitrogen regulon:
Our findings in Methanococcus and Methanobacterium species suggest a common set of genes that belong to the nitrogen regulon as defined by the presence of a consensus operator sequence in the promoter region. nif genes, glnA, glnB, amtB, nadE, and a gene of unknown function are included (Figure 2). Genes belonging to the nitrogen regulon may be regulated by a common repressor protein that recognizes the operator sequence. The binding of the repressor to the operator may interfere in some way with the function of TATA box binding protein, other transcription factors, or RNA polymerase in the initiation of transcription (LEIGH 1999 ). The association of nitrogen regulatory features with these genes is consistent with their functions. GlnA is glutamine synthetase, universally the primary ammonia assimilation enzyme. AmtB functions in the transport of methylammonia or ammonia into the cell (SOUPENE et al. 1998 ). Nif proteins function in nitrogen fixation. Finally, GlnB regulates these processes. The homologous genes are also known to be regulated by nitrogen in E. coli (e.g., VAN HEESWIJK et al. 1996 ).

NadE in Salmonella typhimurium functions in the synthesis of NAD, apparently using ammonia as a substrate (SCHNEIDER and REITZER 1998 ). Mutants are defective in growth at low ammonia concentrations or on alternative nitrogen sources, apparently because NAD becomes limiting. If nadE is indeed regulated by nitrogen in Methanococcus, this could be a mechanism to increase NAD synthesis when it is limited by ammonia.

Thus, the regulation by nitrogen of all the genes we have identified as belonging to the nitrogen regulon in methanogens is expected. However, the repression mechanism in methanogens contrasts with nitrogen regulation in well-characterized bacterial systems, where activation using NifA or NtrC occurs (MERRICK and EDWARDS 1995 ; MAGASANIK 1996 ).

GlnB:
As in bacteria, methanogens contain "typical" glnB genes that are adjacent to amtB homologues. In addition, we and others (CHIEN and ZINDER 1996 ) have shown that the diazotrophic methanogens contain representatives of two novel groups of glnB homologues within the nif gene cluster. This discovery is a unique contribution of the Archaea.

We have shown that one or both of the nif-cluster glnB genes in M. maripaludis functions in post-transcriptional ammonia switch-off of nitrogenase activity. This conclusion is supported by the observation that a glnB⁺ strain loses nitrogenase activity within an hour after the addition of ammonia, while a glnB mutant does not (Figure 4). The effect is post-transcriptional because transposon insertion mutants showed that the glnB genes have no effect on the regulation of mRNA levels (KESSLER et al. 1998 ). Ammonia switch-off occurs in R. rubrum (LIANG et al. 1991 ) and A. brasilense (ZHANG et al. 1992 ), but in neither case is it known whether glnB genes are involved. Switch-off also appears to occur in the diazotrophic methanogen M. barkeri by an unknown mechanism (LOBO and ZINDER 1988 , LOBO and ZINDER 1990 ). Our results from M. maripaludis provide the first direct evidence for the involvement of glnB homologues in post-transcriptional regulation of nitrogen fixation. Determining which of the two nif-cluster glnB genes is involved, or whether both are needed, will require additional genetic work. It will also be interesting to learn whether additional components are involved. In R. rubrum and A. brasilense, switch-off occurs when dinitrogenase reductase ADP-ribosyl transferase (DRAT) catalyzes ADP ribosylation of nitrogenase reductase (NifH). Dinitrogenase reductase-activating glycohydrolase (DRAG) removes the modification. A homologue of DRAG has been found in the genome of M. jannaschii (BULT et al. 1996 ). To determine whether homologues of DRAT and DRAG exist in M. maripaludis, and whether they perform the same function, will require additional genetic experimentation.

The typical (non-nif cluster) GlnB proteins of methanogens almost certainly regulate some aspect of nitrogen metabolism as well, following precedent for all known GlnB functions. An involvement in transcriptional regulation of the nitrogen regulon is a possibility, and additional genetic experiments will test this hypothesis. If the typical GlnB proteins are involved in transcriptional regulation of the nitrogen regulon, they may control the activity of the nitrogen repressor that we hypothesize binds to the nitrogen operator.

The GlnB (PII) (CARR et al. 1996 ) and GlnK (XU et al. 1998 ) proteins of E. coli are homotrimers in which each subunit contains three protruding loops. The three groups of glnB genes differ primarily in the T loop (Figure 3). The conservation in the rest of the protein suggests that the basic structure of the protein is always the same. In typical GlnB proteins the T loop contains the site of uridylylation. The nif cluster glnB homologues contain no recognizable uridylylation site so modification must occur by other means or not at all. In M. barkeri modification evidently does not occur (BRABBAN and ZINDER 1998 ; CHIEN et al. 1998 ). The T loop also mediates interactions with other proteins that transmit the regulatory signal to various targets. In enteric bacteria these proteins include adenylyl transferase (JIANG et al. 1998C ), NtrB (JIANG et al. 1998B ), and apparently NifA (HE et al. 1998 ; JACK et al. 1999 ). Mutations in the T loop can affect all or a subset of these interactions (JIANG et al. 1997A , JIANG et al. 1997B ). While minor differences in the T loop of typical GlnB proteins seem to account for differences in the proteins that receive the signal, it is striking that in each of the methanogen nif cluster GlnB proteins, the T loop is entirely different. The interactions of these proteins that lead to regulation by ammonia switch-off will be novel indeed.

	ACKNOWLEDGMENTS

This work was supported by grant 96-35305-3891 from the National Research Initiative Competitive Grants Program of the U. S. Department of Agriculture, and National Institutes of Health grant GM-55255.

Manuscript received March 16, 1999; Accepted for publication May 14, 1999.

	LITERATURE CITED

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

ARGYLE, J. L., D. L. TUMBULA, and J. A. LEIGH, 1996  Neomycin resistance as a selectable marker in Methanococcus maripaludis.. Appl. Environ. Microbiol. 62:4233-4237[Abstract].

ARSENE, F., P. A. KAMINSKI, and C. ELMERICH, 1996  Modulation of NifA activity by PII in Azospirillum brasilense: evidence for a regulatory role of the NifA N-terminal domain. J. Bacteriol. 178:4830-4838[Abstract/Free Full Text].

ATKINSON, M. R. and A. J. NINFA, 1998  Role of the GlnK signal transduction protein in the regulation of nitrogen assimilation in Escherichia coli.. Mol. Microbiol. 29:431-447[Medline].

BELAY, N., R. SPARLING, and L. DANIELS, 1984  Dinitrogen fixation by a thermophilic methanogenic bacterium. Nature 312:286-288[Medline].

BLANK, C. E., P. S. KESSLER, and J. A. LEIGH, 1995  Genetics in methanogens: transposon insertion mutagenesis of a Methanococcus maripaludis nifH gene. J. Bacteriol. 177:5773-5777[Abstract/Free Full Text].

BRABBAN, A. D., and S. H. ZINDER, 1998 Nitrogen state regulation and osmolyte production in the methanogenic archaeon Methanosarcina barkeri 227, pp. 316 in Abstracts, 98th General Meeting of the American Society for Microbiology, Atlanta.

BROWN, J. R., Y. MASUCHI, F. T. ROBB, and W. F. DOOLITTLE, 1994  Evolutionary relationships of bacterial and archaeal glutamine synthetase genes. J. Mol. Evol. 38:566-576[Medline].

BULT, C. J., O. WHITE, G. J. OLSEN, L. ZHOU, and R. D. FLEISCHMANN et al., 1996  Complete genome sequence of the methanogenic archaeon, Methanococcus jannaschii.. Science 273:1058-1073[Abstract].

CARR, P. D., E. CHEAH, P. M. SUFFOLK, S. G. VASUDEVAN, and N. E. DIXON et al., 1996  X-ray structure of the signal transduction protein PII from Escherichia coli at 1.9Å. Acta Crystallogr. Sect. D 52:93-104.

CHIEN, Y. and S. H. ZINDER, 1996  Cloning, functional organization, transcript studies, and phylogenetic analysis of the complete nitrogenase structural genes (nifHDK2) and associated genes in the Archaeon Methanosarcina barkeri.. J. Bacteriol. 178:143-148[Abstract/Free Full Text].

CHIEN, Y., J. D. HELMANN, and S. H. ZINDER, 1998  Interactions between the promoter regions of nitrogenase structural genes (nifHDK2) and DNA-binding proteins from N₂- and ammonium-grown cells of the archaeon Methanosarcina barkeri 227. J. Bacteriol. 180:2723-2728[Abstract/Free Full Text].

COHEN-KUPIEC, R., C. BLANK, and J. A. LEIGH, 1997  Transcriptional regulation in Archaea: in vivo demonstration of a repressor binding site in a methanogen. Proc. Natl. Acad. Sci. USA 94:1316-1320[Abstract/Free Full Text].

COHEN-KUPIEC, R., C. J. MARX, and J. A. LEIGH, 1999  Function and regulation of glnA in the methanogenic archaeon Methanococcus maripaludis.. J. Bacteriol. 181:256-261[Abstract/Free Full Text].

DEAN, D. R., and M. R. JACOBSON, 1992 Biochemical genetics of nitrogenase, pp. 763–834 in Biological Nitrogen Fixation, edited by G. STACEY, R. H. BURRIS and H. J. EVANS. Chapman & Hall, New York.

DEAN, D. R., J. T. BOLIN, and L. ZHENG, 1993  Nitrogenase metalloclusters: structures, organization, and synthesis. J. Bacteriol. 175:6737-6744[Free Full Text].

DE ZAMAROCZY, M., and C. ELMERICH, 1998 Regulatory roles of the structural homologues PII and PZ proteins in Azospririllum brasilense, pp. 111–114 in Biological Nitrogen Fixation for the 21st Century, edited by C. ELMERICH, A. KONDOROSI and W. E. NEWTON. Kluwer Academic Publishers, Dordrecht, The Netherlands.

GERNHARDT, P., O. POSSOT, M. FOGLINO, L. SIBOLD, and A. KLEIN, 1990  Construction of an integration vector for use in the archaebacterium Methanococcus voltae and expression of a eubacterial resistance gene. Mol. Gen. Genet. 221:273-279[Medline].

HE, L., E. SOUPENE, A. NINFA, and S. KUSTU, 1998  Physiological role for the GlnK protein of enteric bacteria: relief of NifL inhibition under nitrogen-limiting conditions. J. Bacteriol. 180:6661-6667[Abstract/Free Full Text].

JACK, R., M. DE ZAMAROCZY, and M. MERRICK, 1999  The signal transduction protein GlnK is required for NifL-dependent nitrogen control of nif gene expression in Klebsiella pneumoniae.. J. Bacteriol. 181:1156-1162[Abstract/Free Full Text].

JIANG, P., P. ZUCKER, M. R. ATKINSON, E. S. KAMBEROV, and W. TIRASOPHON et al., 1997a  Structure/function analysis of the PII signal transduction protein of Escherichia coli: genetic separation of interactions with protein receptors. J. Bacteriol. 179:4342-4353[Abstract/Free Full Text].

JIANG, P., P. ZUCKER, and A. J. NINFA, 1997b  Probing interactions of the homotrimeric PII signal transduction protein with its receptors by use of PII heterotrimers formed in vitro from wild-type and mutant subunits. J. Bacteriol. 179:4354-4360[Abstract/Free Full Text].

JIANG, P., J. A. PELISKA, and A. J. NINFA, 1998a  Enzymological characterization of the signal-transducing uridylyltransferase/uridylyl-removing enzyme (EC 2.7.7.59) of Escherichia coli and its interaction with the PII protein. Biochemistry 37:12782-12794[Medline].

JIANG, P., J. A. PELISKA, and A. J. NINFA, 1998b  Reconstitution of the signal-transduction bicyclic cascade responsible for the regulation of Ntr gene transcription in Escherichia coli.. Biochemistry 37:12795-12801[Medline].

JIANG, P., J. A. PELISKA, and A. J. NINFA, 1998c  The regulation of Escherichia coli glutamine synthetase revisited: role of 2-ketoglutarate in the regulation of glutamine synthetase adenylylation state. Biochemistry 37:12802-12810[Medline].

KAWARABAYASI, Y., M. SAWADA, H. HORIKAWA, Y. HAIKAWA, and Y. HINO et al., 1998  Complete sequence and gene organization of the genome of a hyper-thermophilic archaebacterium, Pyrococcus horikoshii OT3. DNA Res. 5:55-76[Abstract].

KESSLER, P. S., C. BLANK, and J. A. LEIGH, 1998  The nif gene operon of the methanogenic archaeon Methanococcus maripaludis.. J. Bacteriol. 180:1504-1511[Abstract/Free Full Text].

KLENK, H. P., R. A. CLAYTON, J. F. TOMB, O. WHITE, and K. E. NELSON et al., 1997  The complete genome sequence of the hyperthermophilic, sulphate-reducing archaeon Archaeoglobus fulgidus.. Nature 390:364-370[Medline].

LEIGH, J. A., 1999  Transcriptional regulation in Archaea. Curr. Opin. Microbiol. 2:131-134[Medline].

LIANG, J. H., G. M. NIELSEN, D. P. LIES, R. H. BURRIS, and G. P. ROBERTS et al., 1991  Mutations in the draT and draG genes of Rhodospirillum rubrum result in loss of regulation of nitrogenase by reversible ADP-ribosylation. J. Bacteriol. 173:6903-6909[Abstract/Free Full Text].

LOBO, A. L. and S. H. ZINDER, 1988  Diazotrophy and nitrogenase activity in the archaebacterium Methanosarcina barkeri 227. Appl. Environ. Microbiol. 54:1656-1661[Abstract/Free Full Text].

LOBO, A. L. and S. H. ZINDER, 1990  Nitrogenase in the archaebacterium Methanosarcina barkeri 227. J. Bacteriol. 172:6789-6796[Abstract/Free Full Text].

LOBO, A. L., and S. H. ZINDER, 1992 Nitrogen fixation by methanogenic bacteria, pp. 191–211 in Biological Nitrogen Fixation, edited by G. STACEY, R. H. BURRIS and H. J. EVANS. Chapman and Hall, New York.

MAGASANIK, B., 1996 Regulation of nitrogen utilization, pp. 1344–1356 in Escherichia coli and Salmonella: Cellular and Molecular Biology, edited by F. C. NEIDHARDT. ASM Press, Washington, DC.

MERRICK, M. J. and R. A. EDWARDS, 1995  Nitrogen control in bacteria. Microbiol. Rev. 59:604-622[Abstract/Free Full Text].

MURRAY, P. A. and S. H. ZINDER, 1984  Nitrogen fixation by a methanogenic archaebacterium. Nature 312:284-286.

POSSOT, O., L. SIBOLD, and J. P. AUBERT, 1989  Nucleotide sequence and expression of the glutamine synthetase gene, glnA, of the archaebacterium Methanococcus voltae.. Res. Microbiol. 140:355-371[Medline].

REITZER, L. J., 1996 Ammonia assimilation and the biosynthesis of glutamine, glutamate, aspartate, asparagine, L-alanine and D-alanine, pp. 391–407 in Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology, edited by F. C. NEIDHARDT. ASM Press, Washington, DC.

SANDBECK, K. A. and J. A. LEIGH, 1991  Recovery of an integration shuttle vector from tandem repeats in Methanococcus maripaludis.. Appl. Environ. Microbiol. 57:2762-2763[Abstract/Free Full Text].

SCHNEIDER, B. L. and L. J. REITZER, 1998  Salmonella typhimurium nit is nadE: defective nitrogen utilization and ammonia-dependent NAD synthetase. J. Bacteriol. 180:4739-4741[Abstract/Free Full Text].

SHAH, V. K., P. RANGARAJ, R. CHATTERJEE, R. M. ALLEN, and J. T. ROLL et al., 1999  Requirement of NifX and other nif proteins for in vitro biosynthesis of the iron-molybdenum cofactor of nitrogenase. J. Bacteriol. 181:2797-2801[Abstract/Free Full Text].

SIBOLD, L., M. HENRIQUET, O. POSSOT, and J. P. AUBERT, 1991  Nucleotide sequence of nifH regions from Methanobacterium ivanovii and Methanosarcina barkeri 227 and characterization of glnB-like genes. Res. Microbiol. 142:5-12[Medline].

SMITH, D. R., L. A. DOUCETTE-STAMM, C. DELOUGHERY, H. LEE, and J. DUBOIS et al., 1997  Complete genome sequence of Methanobacterium thermoautotrophicum deltaH: functional analysis and comparative genomics. J. Bacteriol. 179:7135-7155[Abstract/Free Full Text].

SOUILLARD, N. and L. SIBOLD, 1989  Primary structure, functional organization and expression of nitrogenase structural genes of the thermophilic archaebacterium Methanococcus thermolithotrophicus.. Mol. Microbiol. 3:541-551[Medline].

SOUILLARD, N., M. MAGOT, O. POSSOT, and L. SIBOLD, 1988  Nucleotide sequence of regions homologous to nifH (nitrogenase Fe protein) from the nitrogen fixing archaebacteria Methanococcus thermolithotrophicus and Methanobacterium ivanovii: evolutionary implications. J. Mol. Evol. 27:65-76[Medline].

SOUPENE, E., L. HE, D. YAN, and S. KUSTU, 1998  Ammonia acquisition in enteric bacteria: physiological role of the ammonium/methylammonium transport B (AmtB) protein. Proc. Natl. Acad. Sci. USA 95:7030-7034[Abstract/Free Full Text].

TIBONI, O., P. CAMMARANO, and A. M. SANANGELANTONI, 1993  Cloning and sequencing of the gene encoding glutamine synthetase I from the archaeum Pyrococcus woesei: anomalous phylogenies inferred from analysis of archaeal and bacterial glutamine synthetase I sequences. J. Bacteriol. 175:2961-2969[Abstract/Free Full Text].

TUMBULA, D. L., R. A. MAKULA, and W. B. WHITMAN, 1994  Transformation of Methanococcus maripaludis and identification of a PstI-like restriction system. FEMS Microbiol. Lett. 121:309-314.

TUMBULA, D. L., T. L. BOWEN, and W. B. WHITMAN, 1997  Characterization of pURB500 from the archaeon Methanococcus maripaludis and construction of a shuttle vector. J. Bacteriol. 179:2976-2986[Abstract/Free Full Text].

VAN DOMMELEN, A., V. KEIJERS, J. VANDERLEYDEN, and M. DE ZAMAROCZY, 1998  Methyl)ammonium transport in the nitrogen-fixing bacterium Azospirillum brasilense.. J. Bacteriol. 180:2652-2659[Abstract/Free Full Text].

VAN HEESWIJK, W. C., S. HOVING, D. MOLENAAR, B. STEGEMAN, and D. KAHN et al., 1996  An alternative PII protein in the regulation of glutamine synthetase in Escherichia coli.. Mol. Microbiol. 21:133-146[Medline].

WHITMAN, W. B., J. SHIEH, S. SEEHYANG, D. S. CARAS, and U. PREMACHANDRAN, 1986  Isolation and characterization of 22 mesophilic methanococci. Syst. Appl. Microbiol. 7:235-240.

WHITMAN, W. B., D. L. TUMBULA, J. P. YU, and W. KIM, 1997  Development of genetic approaches for the methane-producing archaebacterium Methanococcus maripaludis.. Biofactors 6:37-46[Medline].

XU, Y., E. CHEAH, P. D. CARR, W. C. VAN HEESWIJK, and H. V. WESTERHOFF et al., 1998  GlnK, a PII-homologue: structure reveals ATP binding site and indicates how the T-loops may be involved in molecular recognition. J. Mol. Biol. 282:149-165[Medline].

ZHANG, Y., R. H. BURRIS, and G. P. ROBERTS, 1992  Cloning, sequencing, mutagenesis, and functional characterization of draT and draG genes from Azospirillum brasilense.. J. Bacteriol. 174:3364-3369[Abstract/Free Full Text].

ZHANG, Y., R. H. BURRIS, P. W. LUDDEN, and G. P. ROBERTS, 1993  Posttranslational regulation of nitrogenase activity by anaerobiosis and ammonium in Azospirillum brasilense.. J. Bacteriol 175:6781-6788[Abstract/Free Full Text].

This article has been cited by other articles:

				T. J. Lie and J. A. Leigh Genetic Screen for Regulatory Mutations in Methanococcus maripaludis and Its Use in Identification of Induction-Deficient Mutants of the Euryarchaeal Repressor NrpR Appl. Envir. Microbiol., October 15, 2007; 73(20): 6595 - 6600. [Abstract] [Full Text] [PDF]

				J. Enkh-Amgalan, H. Kawasaki, and T. Seki Molecular evolution of the nif gene cluster carrying nifI1 and nifI2 genes in the Gram-positive phototrophic bacterium Heliobacterium chlorum Int J Syst Evol Microbiol, January 1, 2006; 56(1): 65 - 74. [Abstract] [Full Text] [PDF]

				T. J. Lie, G. E. Wood, and J. A. Leigh Regulation of nif Expression in Methanococcus maripaludis: ROLES OF THE EURYARCHAEAL REPRESSOR NrpR, 2-OXOGLUTARATE, AND TWO OPERATORS J. Biol. Chem., February 18, 2005; 280(7): 5236 - 5241. [Abstract] [Full Text] [PDF]

				P. Cabello, M. D. Roldan, and C. Moreno-Vivian Nitrate reduction and the nitrogen cycle in archaea Microbiology, November 1, 2004; 150(11): 3527 - 3546. [Abstract] [Full Text] [PDF]

				E. L. Hendrickson, R. Kaul, Y. Zhou, D. Bovee, P. Chapman, J. Chung, E. Conway de Macario, J. A. Dodsworth, W. Gillett, D. E. Graham, et al. Complete Genome Sequence of the Genetically Tractable Hydrogenotrophic Methanogen Methanococcus maripaludis J. Bacteriol., October 15, 2004; 186(20): 6956 - 6969. [Abstract] [Full Text] [PDF]

				J. Raymond, J. L. Siefert, C. R. Staples, and R. E. Blankenship The Natural History of Nitrogen Fixation Mol. Biol. Evol., March 1, 2004; 21(3): 541 - 554. [Abstract] [Full Text] [PDF]

				G. B.G. Moorhead and C. S. Smith Interpreting the Plastid Carbon, Nitrogen, and Energy Status. A Role for PII? Plant Physiology, October 1, 2003; 133(2): 492 - 498. [Full Text] [PDF]

				T. J. Lie and J. A. Leigh Regulatory Response of Methanococcus maripaludis to Alanine, an Intermediate Nitrogen Source J. Bacteriol., October 1, 2002; 184(19): 5301 - 5306. [Abstract] [Full Text] [PDF]

				D. E. Martin and B. Reinhold-Hurek Distinct Roles of PII-Like Signal Transmitter Proteins and amtB in Regulation of nif Gene Expression, Nitrogenase Activity, and Posttranslational Modification of NifH in Azoarcus sp. Strain BH72 J. Bacteriol., April 15, 2002; 184(8): 2251 - 2259. [Abstract] [Full Text]

				C. Ehlers, R. Grabbe, K. Veit, and R. A. Schmitz Characterization of GlnK1 from Methanosarcina mazei Strain Go1: Complementation of an Escherichia coli glnK Mutant Strain by GlnK1 J. Bacteriol., February 15, 2002; 184(4): 1028 - 1040. [Abstract] [Full Text] [PDF]

Y. Zhang, E. L. Pohlmann, P. W. Ludden, and G. P. Roberts Functional Characterization of Three GlnB Homologs in the Photosynthetic Bacterium Rhodospirillum rubrum: Roles in Sensing Ammonium and Energy Status J. Bacteriol., November 1, 2001; 183(21): 6159 - 6168. [Abstract] [Full Text] [PDF]