Journal of Bacteriology, February 2002, p . 1028-1040, Vol . 184, No . 4

Characterization of GlnK₁ from Methanosarcina mazei Strain Gö1: Complementation of an Escherichia coli glnK Mutant Strain by GlnK₁

Claudia Ehlers, Roman Grabbe, Katharina Veit, and Ruth A . Schmitz^*

Institut für Mikrobiologie und Genetik, Universität Göttingen, 37077 Göttingen, Germany

Received 30 August 2001/ Accepted 19 November 2001

It was recently discovered that Escherichia coli and many other members of the Proteobacteria encode two PII-like signal transduction proteins: GlnB and its paralogue GlnK (1, 16, 17, 28, 40, 42, 43, 46, 55) . The glnK genes in enteric bacteria are organized in conjunction with a gene encoding an ammonium transporter (amtB) . In contrast to the glnB gene, enteric glnK genes are under the control of the general nitrogen regulatory system and are therefore only expressed under nitrogen starvation (1, 28, 43, 55) . However, GlnK is covalently modified by uridylylation, as is GlnB, in response to the internal nitrogen status; this occurs at the conserved tyrosine residue located in the T-loop (Y51) by the uridylyltransferase/uridylyl-removing enzyme GlnD (5, 30, 55) . Crystallographic analysis of the two PII-like trimeric proteins revealed that, while GlnK and GlnB are structurally very similar, the conformation of the three loops (the T-, C-, and B-loops) can differ significantly (29, 58) . The T-loop is thought to be the site of interaction with other proteins involved in signal transduction; the conformation of the T-loop can thus determine the specific interactions of the PII-like proteins (2, 33) . Recently, it has been discovered that in several diazotrophic bacteria the GlnK proteins are involved in the regulation of nitrogen fixation (2, 3, 22, 24, 28, 51, 53) . For the diazotroph Klebsiella pneumoniae, genetic experiments indicate that the uridylylation status of GlnK is not essential for its nitrogen signaling function in nitrogen fixation (3, 24), whereas this may be important in other diazotrophic bacteria (39) .

Interestingly, GlnB homologues have also been identified in Archaea, suggesting that GlnB-like proteins are likely to play a key role in nitrogen sensing and regulation in Archaea as well . The first GlnB homologues found in diazotrophic methanogenic archaea are encoded within the nif cluster between the structural genes for nitrogenase, nifH and nifD (9, 10, 35) . These regulatory genes, formerly named glnB_i and glnB_ii, are novel homologues of the glnB family and have been recently renamed into nifI₁ and nifI₂ (1); their regulatory function is presumed to be the switch-off of nitrogen fixation in the presence of ammonium, as shown for Methanococcus maripaludis (34, 36) . In addition, genomic sequencing has revealed additional glnB-like genes in methanogenic archaea that are not associated with nif genes (8, 34, 37, 50) . These glnB-like genes are linked to genes that encode for a putative ammonium transporter . In this respect they most resemble the bacterial glnK genes and may consequently have a regulatory function in nitrogen regulation, as has been shown for the bacterial GlnK proteins (reviewed in reference 1; 24, 28, 39, 41) .

The mesophilic methanogenic archaeon Methanosarcina mazei strain Gö1, which belongs to the methylotrophic methanogens of the order Methanosarcinales, is able to grow on H₂ plus CO₂, methanol, methylamines, or acetate as the sole carbon and energy source (14, 18, 54) . We recently showed that M . mazei is able to use molecular nitrogen as the sole nitrogen source and characterized a single nitrogen fixation (nif) gene cluster in M . mazei Gö1 (C . Ehlers and R . A . Schmitz, unpublished data) . We have now identified, in addition to the two glnB-like genes located between nifH and nifD, two glnK-like genes in M . mazei . In the present study we characterized one of these methanogenic GlnK-like proteins, GlnK₁, and investigated its potential role in nitrogen regulation . This is, to our knowledge, the first biochemical characterization of an archaeal GlnK-like protein .

 
Construction of plasmids. Plasmid pRS149, which contains M . mazei glnK₁ under the control of the tac promoter, was constructed as follows . A 0.4-kbp PCR fragment carrying glnK₁ was generated by using chromosomal Methanosarcina mazei DNA as a template and a set of primers . These were homologous to the glnK₁ flanking 5" and 3" regions with additional EcoRI and HindIII synthetic restriction recognition sites (underlined) as follows: 5"-TAGGATAGAGAATTCCTACTGGTGGTC-3", sense primer (MmGlnK); and 5"-CCATACAGTGTAAGCTTCGTTTATAGCC-3", antisense primer (MmGlnK2) . The 0.4-kbp PCR product was cloned into the EcoRI and HindIII sites of pRS63, and the correct insertion was analyzed by sequencing . pRS155 encoding K . pneumoniae glnK under the control of the tac promoter was constructed by PCR cloning . A PCR-generated EcoRI restriction site upstream of the start codon was used to clone the gene into the expression vector pRS63 restricted with EcoRI and SmaI . pRS161 and pRS154 contain the M . mazei glnK₁ and K . pneumoniae glnK genes, respectively, inserted into the EcoRV and SalI sites of pACYC184 and thereby expressed from the tet promoter .

Cloning and nucleotide sequencing. The complete genomic sequence from M . mazei strain Gö1 was determined by a whole-genome shotgun approach . Chromosomal DNA was isolated according to the method described by Ausubel et al . (6), physically sheared by a high-pressure liquid chromatography pump and fractionated by gel electrophoreses . Then, 2- to 5-kbp fragments were ligated into pTZ19R sequencing vector, and the resulting recombinant plasmids were transformed into E . coli DH5 . More than 20,000 clones from small insert libraries (inserts of ca . 2.5 kbp) representative of the whole genome were purified by using a Qiagen Biorobot 9600 and sequenced in both directions by using LICOR IL 4200 and ABI Prism 377 DNA sequencers . The generated sequences were assembled into contigs with P . Green's Phrap assembling tools and have been edited with GAP which is part of the Staden package software (52) . Sequence analysis was performed with the Genetics Computer Group program package (15) . Plasmid Mmdb57 contains the glnK₁-amtB₁ operon on a 2.2-kbp fragment .

Protein purification. For GlnK purifications, plasmids pRS149 and pRS155 were transformed into E . coli HS9060 (V . Weiss, unpublished data) with chromosomal deletions in glnB and glnK to avoid contamination by E . coli PII-like proteins . For expression, 1-liter cultures were grown aerobically in Luria-Bertani (LB) medium at 37°C . Expression of proteins was induced with 150 µM isopropyl-ß-D-thiogalactopyranoside (IPTG) when cultures reached a turbidity at 600 nm of 0.6 . Cell extracts were prepared by disruption of cells in 50 mM Tris-HCl (pH 7.6) by using a French pressure cell, followed by centrifugation at 20,000 x g . M . mazei GlnK₁ and K . pneumoniae GlnK were purified from the supernatant by mercapthoethanol precipitation (22% [vol/vol]), followed by intensive dialysis of the supernatant with 50 mM Tris-HCl (pH 7.6) containing 0.1 mM EDTA, and subsequent purification by anion-exchange chromatography . The supernatant was applied to a Q-Sepharose FF (XK26; Pharmacia Biotech) and chromatographed with a linear gradient from 0 to 0.5 M NaCl (total volume, 300 ml) . GlnK fractions eluted at 180 to 255 mM NaCl in a total volume of 40 ml and were reapplied to Q-Sepharose FF (XK16; Pharmacia Biotech) after 1:4 dilution with 50 mM Tris-HCl (pH 7.6) . The K . pneumoniae GlnK fractions, which eluted at 290 mM NaCl, showed apparent homogeneity and were stored at -70°C in 50 mM Tris-HCl (pH 7.6) containing 50% glycerol . M . mazei GlnK₁ fractions from the second Q-Sepharose FF were further purified by gel filtration on Sephacryl S200 (100 cm by 12 mm; Pharmacia Biotech) to achieve homogeneity . A polyclonal rabbit antiserum was raised to the M . mazei GlnK₁ protein by Goetek (Göttingen, Germany), which was specific for GlnK₁ from M . mazei and did not cross-react with either K . pneumoniae or E . coli GlnK .

GlnD" purification. pJES1208 containing glnD" (1 to 1,077 bp) was transformed into the glnD mutant E . coli NCM1686 (23) . The resulting strain was grown in 1 liter of LB medium at 30°C . When the cells reached a turbidity of 0.6 at 600 nm, GlnD" expression was achieved by heat induction at 43°C for 30 min, followed by an incubation at 37°C for 2 h . Cell extract was prepared by disruption of the cells in 50 mM Tris-HCl (pH 7.5) by using a French pressure cell, followed by centrifugation at 20,000 x g . Purification of GlnD" to 95% apparent homogeneity was achieved by anion-exchange chromatography on Q-Sepharose FF (XK26, 60 ml; Pharmacia Biotech) by using a linear gradient from 0 to 1 M KCl (total volume, 500 ml) . The GlnD" protein eluted at 230 mM KCl and was further purified by gel filtration on Sephacryl S200 (Pharmacia Biotech) with 50 mM Tris-HCl (pH 7.5) containing 200 mM KCl . The purified fractions were concentrated and stored at -70°C .

In vitro uridylylation. Purified GlnK₁ protein fractions synthesized under nitrogen sufficiency appeared to be in their unmodified trimeric form as determined by native gel electrophoresis . In vitro uridylylation was performed at 30°C with purified E . coli GlnD" containing only the uridylylase transferase activity . The 200-µl test assay contained 50 mM Tris-HCl (pH 7.5), 100 mM KCl, 10 mM MgCl₂, 1 mM dithiothreitol, 0.285 µM GlnD", 1 mM UTP, 0.1 mM ATP, and either 0.67 µM K . pneumoniae GlnK or 0.67 µM M . mazei GlnK₁ . Uridylylation was initiated by addition of 50 µM 2-ketoglutarate to the reaction . Concentrations of GlnD" are stated in terms of a monomer; the concentration of the GlnK fractions are stated in terms of the trimer . Samples were removed from the assay at various times, treated with 25 mM EDTA to stop the uridylylation reaction, and analyzed by native gel electrophoresis .

Alternatively radioactive labeled UTP was used . The standard uridylylation reactions were performed as described above but contained 0.4 mM UTP and 0.08 MBq of [-³²P]UTP with a specific activity of 110 TBq/mmol and were started with 5 mM 2-ketoglutarate . After 20 s, 10 min, and 60 min, samples were removed from the assay and treated with 25 mM EDTA, separated on a denaturing 12.5% polyacrylamide gel, and analyzed by using a PhosphorImager and ImageQuant 1.2 software (Molecular Dynamics) . The gel was subsequently stained for protein with Coomassie brilliant blue .

Analysis of M . mazei GlnK₁ uridylylation by native gel electrophoresis. For the analysis of GlnK modifications, the different mobilities of the unmodified and uridylylated protein in nondenaturating polyacrylamide gels were investigated (19) . Native gel electrophoresis was performed by using 12.5% polyacrylamide gels (29:1, acrylamide-bisacrylamide) with 5% stacking gels . The buffer for the running gels was 187.5 mM Tris-HCl (pH 8.9), the buffer for the stacking gels was 62.5 mM Tris-HCl (pH 7.5), and the running buffer was 82.6 mM Tris-HCl (pH 9.4) containing 33 mM glycine . Gels were run with a Bio-Rad Miniprotein I electrophoresis apparatus and either stained with Coomassie brilliant blue or subsequently transferred onto nitrocellulose membranes for Western blot analysis . In general, uridylylated forms of PII-like proteins show higher mobilities in nondenaturing polyacrylamide gels resulting in a protein band with an apparent lower molecular mass than the respective nonmodified protein .

Heterotrimerization of M . mazei GlnK₁ with GlnK from K . pneumoniae. Potential heterotrimer formation was analyzed in vitro by denaturation of 0.25 µM M . mazei GlnK₁ in the presence of 0.25 µM K . pneumoniae GlnK with 6 M urea for 20 min on ice . K . pneumoniae GlnK was either unmodified or completely uridylylated (see "In vitro uridylylation" above) . After subsequent renaturation by dialysis for 16 h into 50 mM Tris-HCl (pH 7.6) containing 0.1 mM EDTA trimers were analyzed by native gel electrophoresis and Western blot analysis .

Growth. E . coli glnK mutant strains carrying plasmids encoding M . mazei glnK₁ and K . pneumoniae glnK were grown under aerobic conditions at 37°C in minimal medium supplemented with 0.1 mM tryptophan and 0.5% glucose as the sole energy and carbon source (23) . Precultures for growth experiments on the limiting nitrogen source arginine were grown in medium supplemented with 4 mM glutamine, which was completely utilized during growth of the precultures . The main cultures (30 ml) supplemented with 10 mM arginine as the sole nitrogen source were grown in 100-ml flasks at 37°C with vigorous shaking . Expression of the M . mazei glnK₁ gene was not induced with IPTG . During growth, samples were taken for monitoring growth at 600 nm and for protein expression . Protein expression was monitored by Western blot analyses with polyclonal rabbit antiserum against M . mazei GlnK₁ and K . pneumoniae GlnK . Main cultures of E . coli HS9060 (glnB glnK) carrying M . mazei glnK₁ or K . pneumoniae glnK were grown under the same conditions but with additional 0.02% glycine in the minimal medium .

K . pneumoniae wild-type strains were grown under anaerobic conditions with N₂ as a gas phase at 30°C in 40 ml of minimal medium supplemented with 0.4% sucrose as the sole carbon source . In strains carrying M . mazei glnK₁, GlnK₁ expression was induced with 10 or 50 µM IPTG . Growth was monitored by determination of the turbidity of the culture at 600 nm, and cell extracts were prepared for Western blot analysis .

M . mazei strain Gö1 was grown without shaking at 37°C in 5-ml closed growth tubes on 150 mM methanol in a minimal medium described previously under a nitrogen gas atmosphere containing 20% CO₂ (13) . For nitrogen-limiting growth conditions, ammonium was omitted from the medium . For the ammonium upshift experiments, 50-ml cultures were grown under nitrogen-limiting conditions until the cells reached a turbidity of 0.4 at 600 nm . The cells were then harvested and resuspended under anaerobic conditions in minimal medium containing 100 µM or 15 mM ammonium without a carbon source and incubated for an additional 1 or 3 h at 37°C . Cell extracts were prepared for Western blot analyses by disruption in 50 mM Tris-HCl (pH 7.6) by sonification .

Western blot analyses. Cells were grown under the different growth conditions described . When exponentially growing cultures reached a turbidity of 0.4 to 0.7 at 600 nm, 1-ml samples were harvested and concentrated 20-fold into sodium dodecyl sulfate (SDS) gel-loading buffer (38) . Samples were separated by SDS-12.5% polyacrylamide gel electrophoresis (PAGE) . For analysis by native gel electrophoresis followed by Western blotting, cell extracts were prepared from 50-ml cultures by disruption in 50 mM Tris-HCl (pH 7.6) by sonification . Proteins were transferred to nitrocellulose membranes as described previously, and the membranes were exposed to polyclonal rabbit antisera directed against the GlnK proteins of K . pneumoniae and M . mazei . Protein bands were detected with secondary antibodies directed against rabbit immunoglobulin G and coupled to horseradish peroxidase (Bio-Rad Laboratories) by using either the Color Development Reagent system (Bio-Rad Laboratories) or the ECL-Plus system (Amersham Pharmacia Biotech) for detection . Purified GlnK from K . pneumoniae, purified GlnK₁ from M . mazei, and prestained protein markers (New England Biolabs) were used as standards .

RNA isolation. M . mazei Gö1 cells (5 ml) from exponentially growing cultures (turbidity at 600 nm of 0.3 to 0.4), grown with N₂ or 10 mM NH₄⁺ as a nitrogen source, were anaerobically centrifuged in the growth tubes at 1,000 x g for 10 min and resuspended in 30 mM sodium acetate (pH 5.2) . After incubation with 1.5% SDS, RNA was extracted by using the RNeasy Kit (Qiagen) according to the protocol of the manufacturer and treated with DNase I . After ethanol precipitation in the presence of 4 M LiCl, the RNA pellet was resuspended in 30 µl of RNase-free water and stored at -70°C .

Northern blot analyses. RNA (9 to 12 µg) was separated by electrophoresis by using a 1% denaturing agarose gel containing 6% formaldehyde and then transferred to nylon membranes (Hybond N; Amersham Pharmacia) by vacuum blotting according to the manufacturer's directions . After 3 min of UV cross-linking, Northern hybridization was performed to locate the mRNA of interest (47) . Filters were hybridized overnight at 55°C with [-³²P]ATP-labeled glnK₁ probe in the presence of 5x SSC (1x SSC is 0.15 M NaCl plus 0.015 M sodium citrate), 0.02% SDS, and 0.1% laurylsarcosine . After hybridization, the membranes were washed twice with 2x SSC-0.1% SDS solution at room temperature for 5 min, twice with 2x SSC-0.1% SDS at 55°C for 30 min, and twice with 0.1x SSC-0.1% SDS at room temperature for 30 min . The hybridized mRNA was detected and analyzed by using a PhosphorImager and the ImageQuant 1.2 software (Molecular Dynamics) . The standard RNA marker used was obtained from New Englands Biolabs .

Generating the glnK₁-DNA probe: glnK₁ was amplified by PCR by using genomic DNA from M . mazei Gö1 as a template and the oligonucleotides MmGlnK and MmGlnK2 (see above) . Reactions were carried out in 100-µl volumes with Vent polymerase (New England Biolabs) and primers at a concentration of 0.12 µM . The annealing temperature was 59°C, and synthesis was carried out for 30 s for 30 cycles . The 415-bp PCR product was purified by gel electrophoresis and extraction by using the QIAQuick extraction kit (Qiagen) . A total of 1 to 1.5 µg of purified PCR product was labeled with [-³²P]ATP by using the random labeling system from Gibco (Random Primers Labeling System) according the protocol of the manufacturer . The specificity of the glnK₁ DNA probe was tested by Southern hybridization (47) with M . mazei DNA completely digested by PstI . Under the conditions used for Northern blot analysis, the hybridization with the labeled glnK₁ probe resulted in only one hybridization signal (1.8 kbp) and did not cross-react with the fragment hybridizing with the labeled glnK₂ probe (3.1 kbp) (Fig . 1). glnK₂ probe (394 bp) was generated and labeled as described for the glnK₁ probe with a set of glnK₂-specific primers .

 
Determination of the transcriptional start site. The glnK₁ transcriptional start site was determined by using the 5" rapid amplification of cDNA ends (5"-RACE) system, as recommended by the supplier (Gibco-BRL) with 1 µg of total RNA (DNA-free) from cells grown under nitrogen starvation and specific primers, glnK₁-GSP1 (5"-CCAACGTAACCGTCACTGCC-3") and glnK₁-GSP2 (5"-GCCTCAATTGTTGGCTCAAG-3"), which hybridize to bases 265 to 245 and bases 226 to 205 of glnK₁, respectively . The obtained PCR product (243 bp) was cloned into pSK⁺ Bluescript (Stratagene, La Jolla, Calif.) and sequenced in both directions by using an ABI Prism 377 DNA sequencer .

Data deposition. The nucleotide sequence for M . mazei glnK₁ amtB₁ has been submitted to GenBank under accession number AF36724 .

Cloning, sequencing, and transcriptional analysis of the glnK₁-amtB₁ operon in M . mazei Gö1. The entire genome of M . mazei Gö1 has been sequenced by the Genomics Laboratory Göttingen . In the course of sequencing, the preliminary data were checked for the presence of glnB-like genes . In addition to nifI₁ and nifI₂, which are localized between nifH and nifD (Ehlers and Schmitz, unpublished), we identified two glnB-like open reading frames, the gene products of which showed high similarity to bacterial GlnB proteins . In contrast to nifI₁ and nifI₂, both of these additional glnB-like genes were organized in an operon in conjunction with a gene, the product of which showed a high similarity to bacterial ammonium transporters . This structural organization prompted us to designate the operons glnK₁-amtB₁ and amtB₂-glnK₂ . We concentrated on characterizing glnK₁-amtB₁ which is located 8 kbp downstream of the nif gene cluster .

Sequence analysis revealed that the glnK₁ gene (342 bp) codes for a polypeptide of 114 amino acids with a predicted molecular mass of 12,744 Da, which showed 44 and 43% identity, respectively, to the bacterial GlnK proteins of E . coli and K . pneumoniae . Interestingly, the conserved tyrosine residue of the T-loop (Y51), the site of modification by uridylylation in the bacterial PII proteins, is also present in the archaeal GlnK₁ protein of M . mazei (Fig . 2). In addition, the amino acid residue in position 54 of M . mazei GlnK₁ is asparagine (N54), which has recently been shown to be a critical amino acid in the T-loop of K . pneumoniae GlnK in distinguishing GlnB and GlnK in respect to the ability of GlnK to regulate nitrogen fixation and is possibly directly involved in the interaction with NifL/NifA (2) . amtB₁ encodes for a polypeptide of a predicted molecular mass of 41,556 Da (396 amino acids), the amino acid sequence of which showed 38% identity to the bacterial AmtB protein of E . coli .

 
Transcriptional analysis of the glnK₁-amtB₁ operon in Northern blot analysis with a homologous glnK₁ probe revealed the presence of one main transcript of ca . 2.3 kb, but only for cultures grown under nitrogen-limiting conditions (Fig . 3) . This indicates that transcription of glnK₁-amtB₁ plus the open reading frame downstream from amtB₁ (orfX) (Fig . 4), coding for a hypothetical protein, is initiated at a single transcriptional start site and is repressed by ammonium, as is the case in bacteria . The two additional smaller transcripts, which appear in very low amounts under nitrogen limitation, may result from termination after amtB₁ (in case of the ca . 1.5-kb transcript) and/or from specific degradation of the 2.3-kb transcript . In order to confirm the presence of a single transcriptional start site and to analyze the promoter region, we determined the glnK₁ transcriptional start site by using the 5"-RACE method and RNA extracted from cells grown under nitrogen starvation as described in Materials and Methods . The transcriptional start site was localized 17 bp upstream of the putative translational start site of glnK₁ . We identified a potential archaeal consensus promoter sequence 65 to 31 bp upstream from the 5" end of the transcriptional start site of the glnK₁-amtB₁ operon . It contains a typical archaeal-factor-B recognition element (BRE) (CGAAA) and a potential TATA box (TTTAGATA) (Fig . 4) . No similar promoter sequences were obtained at an appropriate location 5" of amtB₁ .

 
Purification and characterization of heterologously expressed M . mazei GlnK₁. We cloned glnK₁ from M . mazei, placed it under the control of the tac promoter, and overproduced the corresponding protein in an E . coli background with chromosomal deletions in glnK and glnB to avoid contamination of bacterial PII proteins (RAS40; see Materials and Methods) . Overproduction of the archaeal protein was evaluated by gel electrophoretic analysis of the induced cell extracts . In general, expression resulted in an overproduction of M . mazei GlnK₁ protein up to ca . 4% of the cell extract protein . GlnK₁ expression in RAS41 in the presence of an additional pACYC-based plasmid containing extra copies of seldom tRNA genes did not significantly increase the expression levels of GlnK₁ . Thus, the archaeal protein was overexpressed in RAS40 and purified from cell extracts by mercapthoethanol precipitation, anion-exchange chromatography, and gel filtration to apparent homogeneity as described in Materials and Methods . Subsequent characterization of purified M . mazei GlnK₁ by gel electrophoresis under denaturating and native conditions revealed that GlnK₁ has a molecular mass of ca . 14 kDa, which is constistent with the predicted molecular mass based on the DNA sequence, and is a trimer in its native state .

Analysis of GlnK₁ under different nitrogen availabilities. In the nitrogen regulatory system, bacterial PII proteins are covalently modified by a uridylyltransferase in response to nitrogen limitation at a conserved tyrosine residue (Y51) located in the T-loop . Within the M . mazei genome no open reading frame was identified, the deduced amino acid sequence of which showed significant similarities to the enteric uridylyltransferase (GlnD) . However, E . coli GlnD has been shown to heterologously uridylylate a variety of PII-like proteins at the conserved tyrosine residue (Y51); even tyrosine residue (Y51) of the PII-like protein from Synechococcus sp . strain PCC 7942 is uridylylated by E . coli GlnD, although in vivo the PII protein is modified by phosphorylation at a serine residue (S49) in response to nitrogen limitation (19, 20) . In order to determine whether tyrosine 51 in M . mazei GlnK₁ is modified by E . coli GlnD, we performed in vitro uridylylation assays with purified GlnK₁ protein from M . mazei. Additionally, we analyzed M . mazei GlnK₁ in in vivo ammonium upshift experiments of nitrogen-limited M . mazei cultures to study whether GlnK₁ is modified in response to the internal nitrogen status of the cells .

Since PII-like proteins are trimers in their native state, they can appear in the cell in four trimer conformations uridylylated to different amounts: unmodified trimers (PII₃), trimers with one [PII₃-(UMP)] or two monomers uridylylated [PII₃-(UMP)₂], or completely uridylylated trimers [PII₃-(UMP)₃] . The uridylylation status of PII-like proteins can be determined by native gel electrophoresis . In general, uridylylated forms of the PII-like proteins show higher mobilities in nondenaturing polyacrylamide gels, resulting in a protein band with an apparent lower molecular mass than the respective nonmodified protein (Fig . 5A, lanes 2 to 5, which show the K . pneumoniae GlnK trimers uridylylated to different amounts) . In order to determine potential modification by uridylylation, purified M . mazei GlnK₁, which was heterologously synthesized in the E . coli glnB glnK background under nitrogen sufficiency, was analyzed by native gel electrophoresis . In comparison with the unmodified and the modified trimeric forms of K . pneumoniae GlnK, it appeared to be in its unmodified trimeric form (Fig . 5A, compare lane 7 with lanes 2 and 5) . Using purified E . coli GlnD" enzyme, which contains only the uridylylase transferase activity, we analyzed whether M . mazei GlnK₁ synthesized under nitrogen sufficiency can be heterologously modified by uridylylation in vitro . The uridylylation was performed at 30°C in the presence of 2-ketoglutarate, UTP, and ATP, and the grade of uridylylation was analyzed after various incubation times by native gel electrophoresis (see Materials and Methods) . As a control, unmodified K . pneumoniae GlnK protein was modified in a separate uridylylation assay under the same conditions . The time dependence of the respective uridylylation assays is depicted in Fig . 5A . Trimeric unmodified K . pneumoniae GlnK was completely uridylylated within 10 min (Fig . 5A, lane 4), whereas M . mazei GlnK₁ appeared to remain unmodified by characterization of its mobility on native gels even after 60 min of incubation in the uridylylation assay (Fig . 5A, lane 10) . Also, subsequent silver staining of the gel or Western analysis did not indicate the presence of an additional M . mazei GlnK₁ trimer conformation (data not shown) . To rule out the possibility that uridylylation of M . mazei GlnK₁ does not alter the mobility of the trimeric protein in native gels, we used radioactive labeled UTP in the in vitro uridylylation assays (see Materials and Methods) . Analysis of those uridylylation assays of K . pneumoniae GlnK and M . mazei GlnK₁, shown in Fig . 5B, confirmed that M . mazei GlnK₁ is not modified by E . coli GlnD" . This failure to heterologously uridylylate the M . mazei GlnK₁ in vitro may result from the specificity of the E . coli GlnD" protein or from the absence of a metabolic signal specific to Methanosarcina spp .

 
Since the in vitro assay might miss a Methanosarcina-specific metabolic signal, we analyzed potential in vivo modifications of chromosomally expressed GlnK₁ by ammonium upshift experiments with exponentially growing M . mazei cells . In order to monitor chromosomally expressed GlnK₁ under different nitrogen availabilities by immunological means, we raised a specific polyclonal rabbit antiserum against M . mazei GlnK₁ (see Materials and Methods) . M . mazei cells were grown in minimal medium with either molecular nitrogen as the sole nitrogen source (nitrogen limiting) or with 15 mM ammonium . For the ammonium upshift experiments, exponentially growing nitrogen-limited M . mazei cells were harvested under anaerobic conditions, resuspended in anaerobic minimal medium (containing no carbon source but supplemented with 15 mM ammonium), and incubated further at 37°C for 1 or 3 h . Analyses of chromosomally synthesized GlnK₁ protein fractions of the different cell extracts by native gel electrophoresis and subsequent Western blot analysis are shown in Fig . 6 . Expression of GlnK₁ in M . mazei is detectable only under nitrogen limitation with molecular nitrogen as the sole nitrogen source (Fig . 6A, lanes 3 and 4) . Upon the ammonium upshift, no modification of chromosomally expressed GlnK₁ was detected by a change of its migration behavior, as is seen for K . pneumoniae GlnK . M . mazei GlnK₁ under nitrogen-limiting conditions showed the same migration behavior as the unmodified trimeric form of K . pneumoniae GlnK and was not affected by the ammonium upshift (Fig, 6A, lanes 4 and 5, compared to Fig . 6B, lanes 2 and 3) . Also, shifts to much lower ammonium concentrations (100 µM) and further incubation for 1 or 3 h did not result in any changes of the GlnK₁ migration behavior (data not shown) . This finding that M . mazei GlnK₁ is not uridylylated upon an ammonium upshift indicates that the archaeal GlnK₁ protein--if modified at all--may be modified in a different manner in response to the presence of ammonium .

 
Analysis of heterotrimer formation of M . mazei GlnK₁ with K . pneumoniae GlnK. The formation of heterotrimers between different PII-like proteins in one species and also between PII-like proteins from different species has been recently demonstrated (21, 56) . Furthermore, for E . coli and K . pneumoniae it has been proposed that the formation of functionally inactive heterotrimers between GlnK and PII monomers have a significant function in nitrogen regulation upon an ammonium upshift (2, 3, 21) . In order to analyze whether M . mazei GlnK₁ is able to form heterotrimers with K . pneumoniae GlnK, we studied heterologous expression of M . mazei GlnK₁ in K . pneumoniae and heterotrimer formation in vitro .

M . mazei GlnK₁ was heterologously expressed in a K . pneumoniae wild-type strain growing with molecular nitrogen as the sole nitrogen source . It has been shown that GlnK is required for nitrogen fixation in K . pneumoniae (24, 28); thus, growth under those conditions requires functional GlnK trimers . The heterologous expression of M . mazei GlnK₁ in K . pneumoniae (RAS43) did not affect growth or nitrogen fixation of K . pneumoniae (data not shown) . This indicates that heterologous expression either resulted in M . mazei GlnK₁ homotrimers, which do not affect the native K . pneumoniae GlnK trimers, or resulted in the formation of functional heterotrimers between K . pneumoniae GlnK and M . mazei GlnK₁ . Immunological analyses of the trimeric GlnK proteins in cell extracts by using specific polyclonal antibodies directed against M . mazei GlnK₁ or K . pneumoniae GlnK showed that no heterotrimers were formed (Fig . 7A) . The cell extract of RAS43, grown with molecular nitrogen as the sole nitrogen source, contained completely uridylylated homotrimers of K . pneumoniae GlnK, which did not react with the antibodies directed against M . mazei GlnK₁, and unmodified homotrimers of M . mazei GlnK₁, which did not react with the antibodies directed against K . pneumoniae GlnK (Fig . 7A, lanes 5 and 8, respectively) . No heterotrimers were detectable, which one would expect to react with both specific polyclonal antibodies and to migrate between the two homotrimeric forms, i.e., GlnK₃-(UMP)₃ from K . pneumoniae, and (GlnK₁)₃ from M . mazei .

 
In addition to these in vivo studies, we analyzed heterotrimer formation in vitro by denaturation by 6 M urea and subsequent renaturation of GlnK trimers by dialysis (see Materials and Methods) . M . mazei GlnK₁ at 0.25 µM was denatured and renatured in the presence of equal amounts of K . pneumoniae GlnK . In addition to unmodified K . pneumoniae GlnK (GlnK₃), we used completely uridylylated K . pneumoniae GlnK [GlnK₃-(UMP)₃] to distinguish between homotrimers and heterotrimers based on their different migration behavior in native gel electrophoresis . Heterotrimers should be detectable with both specific polyclonal antibodies and should migrate between the two homotrimeric forms, GlnK₃-(UMP)₃ in the case of K . pneumoniae and (GlnK₁)₃ in the case of M . mazei. The immunological analysis of the resulting trimers confirmed our in vivo data: no heterotrimers between M . mazei GlnK₁ and uridylylated K . pneumoniae GlnK were detectable (Fig . 7B, lanes 5 and 8) . These data strongly indicate that M . mazei GlnK₁ is not able to oligomerize with enteric PII-like proteins .

Functional analysis of M . mazei GlnK₁ in an E . coli glnK mutant strain. Archaeal PII proteins are likely to play a crucial role in nitrogen sensing and regulation comparable to that of the bacterial PII proteins . Because of the similarities in structural organization and transcriptional regulation, we studied the possibility that M . mazei GlnK₁ can functionally substitute for bacterial E . coli GlnK . Since the E . coli glnK mutant strain NCM1971 (24) showed a small but significant growth phenotype on NB solid medium, we studied aerobic growth of this strain in minimal medium in the presence of the limiting nitrogen source arginine . Under these growth conditions, the glnK mutant strain showed significantly lower growth rates and lower cell densities than the respective parental strain, NCM1529 (Fig . 8A) . Since some components of the arginine catabolism in E . coli are expressed in an NtrC-dependent manner (59) and GlnK is required for fine control of NtrC phosphorylation (4), the observed growth defect on arginine apparently results from decreased arginine degradation in the absence of GlnK . Unexpectedly, the expression of M . mazei GlnK₁, either from the tet promoter on a low-copy plasmid (RAS39) or from the tac promoter (RAS37), was able to restore the wild-type growth phenotype of the E . coli glnK mutant strain on arginine as the sole nitrogen source . Restored growth was comparable to that seen with the addition of K . pneumoniae glnK on the plasmid (RAS38) and showed the same growth rate as determined for the wild type (Fig . 8A) . To demonstrate that the observed complementation is based on expression of M . mazei glnK₁ in E . coli glnK mutant strains, we monitored E . coli GlnK and M . mazei GlnK₁ in the respective strains by immunological means . By using specific polyclonal antibodies, M . mazei GlnK₁ was detected in RAS39 and RAS37, whereas E . coli GlnK was not detectable in any of the complemented glnK mutant strains (data not shown) . These results strongly suggest that the archaeal M . mazei GlnK₁ is able to functionally complement E . coli GlnK for growth on arginine as the sole nitrogen source and most likely acts in E . coli in the same way as the bacterial nitrogen-regulatory protein GlnK . The fact that M . mazei GlnK₁ is apparently not modified by uridylylation in response to nitrogen availability further suggests that the GlnK nitrogen signaling function in E . coli is independent of the uridylylation state of the trimeric protein, as is apparently the case for K . pneumoniae GlnK regarding its control in regulating NifLA activity (3, 24) .

 
To rule out that the restoration of growth on arginine by M . mazei GlnK₁ is not based on M . mazei GlnK₁ affecting GlnB functions, we studied the growth on arginine of an E . coli glnB glnK double-mutant strain (HS9060) and derivatives containing M . mazei glnK₁ (RAS40) or K . pneumoniae glnK (RAS42) under the control of the tac promoter . Since the E . coli glnB glnK double-mutant strain was not able to grow in the minimal medium used for the glnK mutant strain, the respective medium was additionally supplemented with 0.02% glycine . No IPTG was added to ensure very low induction levels of K . pneumoniae glnK and M . mazei glnK₁ . The expression of M . mazei GlnK₁ was able to restore the wild-type growth rate of the glnK glnB double-mutant strain on arginine, as was the expression of K . pneumoniae GlnK . Cultures containing M . mazei GlnK₁ (RAS40) did not reach the same maximum turbidity obtained for the double-mutant strain expressing K . pneumoniae GlnK (RAS42) (Fig . 8B) .

Effects of M . mazei GlnK₁ on nitrogen fixation in K . pneumoniae. M . mazei glnK₁ is able to complement an E . coli glnK mutant strain for growth on arginine . The amino acid sequence of the M . mazei GlnK₁ T-loop shows 52% similarity to that of K . pneumoniae (Fig . 2) . However, it contains the conserved amino acid residue asparagine in position 54 (N54), which has been recently shown to be a critical residue in distinguishing GlnB and GlnK with respect to the ability of GlnK to regulate nitrogen fixation (2) . We therefore studied the ability of M . mazei glnK₁ to complement a K . pneumoniae glnK mutant strain for growth on molecular nitrogen as the sole nitrogen source .

In order to monitor the effect of GlnK₁ on nitrogen fixation by directly analyzing growth on molecular nitrogen as the sole nitrogen source, we constructed a chromosomal glnK deletion in the wild-type strain K . pneumoniae M5a1 by transducing the glnK null allele of the glnK mutant strain UNF3433 (28) as described in Materials and Methods . The resulting strain was designated RAS31 . As expected from previous studies of nif inductions determined with lacZ-reporter fusions (24, 28), anaerobic growth on minimal medium with molecular nitrogen as the sole nitrogen source was completely abolished in the glnK mutant strain RAS31 (Fig . 9) . Additional expression of M . mazei GlnK₁ from a low-copy plasmid (RAS33) did not affect the mutant growth phenotype of RAS31 on molecular nitrogen . Unphysiological high induction of M . mazei glnK₁ in the presence of up to 50 µM IPTG (RAS32) did result in slow but significant growth (Fig . 9) . The doubling time of RAS32 on molecular nitrogen in the presence of 50 µM IPTG was calculated to be 32 h compared to 9 h for the parental strain . Thus, M . mazei GlnK₁ appeared to weakly complement a K . pneumoniae glnK mutant for diazotrophic growth . However, since the expression level of GlnK₁ was unphysiologically high, these findings indicate that M . mazei GlnK₁ is not able to complement for functions of K . pneumoniae GlnK with respect to its regulatory function in nitrogen fixation .

 
We recently showed that the mesophilic methanogenic archaeon M . mazei is able to use molecular nitrogen as the sole nitrogen source (Ehlers and Schmitz, unpublished) . In addition to the two glnB-like genes within the nif cluster, we have now identified two additional glnB-like genes in M . mazei at two different locations on the chromosome by analyzing the genome sequence . Based on their structural organization these genes have been designated glnK₁ and glnK₂ . In order to determine whether M . mazei GlnK proteins may have a function in nitrogen regulation, we studied the glnK₁-amtB₁ operon, which is located 8 kbp upstream of the nif operon, and characterized native and purified heterologously synthesized GlnK₁ .

Analyses of M . mazei GlnK₁ in comparison to bacterial PII-like proteins. Transcriptional analysis by Northern blotting showed that the glnK₁-amtB₁ operon from M . mazei is transcriptionally regulated through ammonium availability and only transcribed in the absence of a combined nitrogen source (Fig . 3), as is known for enteric glnK₁-amtB₁ operons . This regulation of GlnK₁ expression was confirmed by analyzing protein synthesis of GlnK₁ in M . mazei under different nitrogen availabilities (Fig . 6) . The mechanisms in M . mazei resulting in transcriptional regulation of the glnK₁-amtB₁ operon in response to nitrogen availability, however, have yet to be elucidated . Concerning potential mechanisms of transcriptional regulation in response to ammonium in methanogenic archaea, a common repressor binding site sequence [GGAA(N₆)TTCC] has been identified in Methanococcus maripaludis in the promoter region of the nif and glnA operon, indicating a coordinated transcriptional regulation in Methanococcus maripaludis via an operator site (34) . This repressor binding site sequence was further found in the genome of Methanobacterium and Methanococcus species, located in front of several genes involved in nitrogen metabolism including an amtB-glnB operon (8, 12, 50) . However, in M . mazei, neither the nif operon nor the glnK₁-amtB₁ operon showed this repressor binding site sequence (Ehlers and Schmitz, the present study) . In Methanosarcina barkeri 227, the nif2 operon is also missing the operator sequence but is transcriptionally regulated through ammonium availability (10, 11) . Thus, these findings indicate the mechanism of transcriptional regulation through ammonium availability in M . mazei and M . barkeri may differ from the mechanisms in Methanococcus and Methanobacterium spp .

Amino acid sequence analysis showed that M . mazei GlnK₁ contained the conserved tyrosine (Y51) located in the T-loop structure of the protein which, in enteric GlnB and GlnK proteins, is modified by uridylylation in response to the nitrogen status of the cell (1, 5, 41) . Two lines of evidence strongly indicate that the archaeal GlnK protein, unlike the enteric GlnK proteins, is not modified by uridylylation in response to the nitrogen status of the cells . First, in vitro uridylylation assays with GlnD" from E . coli showed that a covalent modification by uridylylation was not detectable for the purified, heterologously expressed M . mazei GlnK₁ (Fig . 5) . This failure to uridylylate the M . mazei GlnK₁ protein in vitro may be due to the specificity of the E . coli GlnD protein . If this is the case, it is of special importance since E . coli GlnD has been shown to heterologously uridylylate a variety of bacterial PII-like proteins . In this respect, it is of interest that even the PII-like protein from Synechoccoccus sp . strain PCC 7942, which has been shown to be modified by phosphorylation at a serine residue (Ser-49), can be heterologously uridylylated by the E . coli GlnD protein at the conserved tyrosine residue (Tyr-51) (19, 20) . This indicates that the archaeal GlnK protein differs from bacterial PII-like proteins in a manner that does not allow recognition by the E . coli uridylyltransferase GlnD, even though the conserved tyrosine residue is present . Alternatively, a Methanosarcina-specific metabolic signal may be required for covalent modification of the archaeal GlnK protein in response to nitrogen availability . Consequently, we secondly looked for covalent modifications in response to nitrogen availability in vivo, with ammonium upshift experiments of M . mazei cultures in which GlnK₁ was chromosomally expressed . These in vivo ammonium upshift experiments clearly confirmed the in vitro results by heterologous uridylylation (Fig . 6) . This is consistent with our finding that no open reading frame was identified in the M . mazei genome with a deduced amino acid sequence similar to the enteric uridylyltransferase (GlnD) . Furthermore, we can also exclude that the M . mazei GlnK₁ is in vivo modified by phosphorylation in response to nitrogen availability—as occurs with the Synechococcus PII protein—since an increase in the electrophoretic mobility of the phosphorylated form of the protein would also result (19, 20) . These findings strongly indicate that if there is a covalent modification in response to nitrogen availability in M . mazei GlnK₁, the methanogenic protein is modified in a manner different from the bacterial GlnB-like proteins .

In addition to the absence of modification in response to the internal nitrogen status, we obtained in vivo and in vitro evidence that M . mazei GlnK₁ does not form heterotrimers with GlnK from K . pneumoniae (Fig . 7) or E . coli (data not shown) . In this respect the archaeal GlnK protein again differs from most bacterial PII-like proteins, for which heterotrimerization is observed even among species which are phylogenetically distant (19) . The two additional amino acids in the M . mazei GlnK₁ B-loop region, which or are not found in PII-like proteins in enteric bacteria (Fig . 2) and which are either part of the second -helix or part of the B-loop, might be of importance for differences in the overall structure and thus for the failure to form heterotrimers . These findings indicate that the structure of the bacterial and archaeal GlnK core proteins, which are presumed to be responsible for interaction between GlnK monomers, differs significantly .

Regulatory function of M . mazei GlnK₁ in nitrogen regulation. To date, no genetic system has been established in M . mazei for introducing chromosomal deletions in order to perform functional analysis of M . mazei proteins . Thus, in order to study the potential regulatory function of GlnK₁ in nitrogen regulation, we studied the ability of M . mazei GlnK₁ to restore function to an E . coli glnK mutant . In a result both interesting and unexpected, expression of M . mazei GlnK₁ restored growth of an E . coli glnK mutant on the limiting nitrogen source arginine (Fig . 8A) . This is, to our knowledge, the first report of a functional complementation of a bacterial glnK mutant by an archaeal GlnK protein . Since M . mazei GlnK₁ differs from bacterial PII-like proteins in its modification in response to nitrogen availability and in its ability to form heterotrimers, the ability to restore growth of an E . coli glnK mutant on arginine is of significant interest . The functional complementation of the E . coli glnK mutant strongly indicates that M . mazei GlnK₁ is involved in nitrogen regulation . It further suggests that the uridylylation status of the protein is not of importance for the regulatory function of GlnK in E . coli, since M . mazei GlnK₁ is not uridylylated in response to nitrogen availability . This is consistent with the results obtained for the function of GlnK uridylylation on K . pneumoniae NifLA regulation: uridylylation is apparently not required for relief of NifL inhibition (3, 24) . However, uridylylation is important for GlnB function, thus M . mazei GlnK₁ cannot substitute for GlnB functions in an E . coli glnB glnK double mutant to the same amount as does K . pneumoniae GlnK, which is uridylylated under nitrogen-limiting conditions (Fig . 8B) .

M . mazei GlnK₁ contains the conserved asparagine 54 in the T-loop, which has been shown to be a crucial amino acid which discriminates GlnK from GlnB in K . pneumoniae in respect to the GlnK ability to regulate nitrogen fixation (2) . However, M . mazei GlnK₁ is not able to substitute for K . pneumoniae GlnK with respect to its signaling function in the nitrogen fixation regulatory system (Fig . 9) . This indicates that M . mazei GlnK₁ is missing the specific GlnK function necessary for transducing the nitrogen signal to the bacterial nif system . The finding that M . mazei GlnK₁ is not uridylylated cannot account for the failure, since the uridylylation state of K . pneumoniae GlnK is not essential for the signal transduction (3, 24, 28) . It is more likely that the failure is based on the specificity of the regulatory system of nitrogen fixation in K . pneumoniae itself, in which GlnK transduces the nitrogen signal to the nif system by interacting with the transcriptional activator NifA or with its antagonist NifL (2, 24, 28, 49) . The transcriptional regulation of methanogenic nif genes, however, appears to be based in general on a negative mechanism (7, 11, 12, 34, 35) . Thus, if GlnK₁ functions in the transcriptional regulation of nitrogen fixation in M . mazei at all, specific interacting partners and metabolic signals may be required for GlnK₁ . Additionally, we cannot exclude that the second operon, amtB₂-glnK₂, in M . mazei (data not shown) may be important for nif transcriptional regulation .

In summary, our findings strongly indicate that GlnK₁ is involved in nitrogen regulation in M . mazei . However, the archaeal GlnK protein differs from most bacterial PII-like proteins in a manner that (i) does not allow recognition by the E . coli uridylyltransferase GlnD, even though the conserved tyrosine 51 is present, and (ii) does not allow heterotrimerization with bacterial GlnK proteins, suggesting that the structure of the bacterial and archaeal GlnK core proteins differ significantly . Hence, it can be speculated that archaeal GlnK proteins which are presumed to be involved in nitrogen regulation, evolved differently from bacterial PII-like proteins concerning their structural features depending on the archaeon-specific interacting partners and requirements .

This work was supported by the Deutsche Forschungsgemeinschaft and the Fonds der Chemischen Industrie .

1. Arcondéguy, T., R . Jack, and M . Merrick. 2001 . P_II signal transduction proteins, pivotal players in microbial nitrogen control . Microbiol . Mol . Biol . Rev . 65:80-105.
2. Arcondeguy, T., D . Lawson, and M . Merrick. 2000 . Two residues in the T-loop of Klebsiella pneumoniae GlnK determine NifL-dependent nitrogen control of nif gene expression . J . Biol . Chem . 275:38452-38456.
3. Arcondeguy, T., W . C . van Heeswijk, and M . Merrick. 1999 . Studies on the roles of GlnK and GlnB in regulating Klebsiella pneumoniae NifL-dependent nitrogen control . FEMS Microbiol . Lett . 2:263-270.
4. 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.
5. Atkinson, M . R., and A . J . Ninfa. 1999 . Characterization of the GlnK protein of Escherichia coli . Mol . Microbiol . 32:301-313.
6. Ausubel, F., R . Brent, R . E . Kingston, D . D . Moore, J . G . Seidmann, J . A . Smith, and K . Struhl. 1987 . Current protocols in molecular biology . John Wiley & Sons, Inc., New York, N.Y.
7. 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.
8. Bult, C . J., O . White, G . J . Olsen, L . Zhou, R . D . Fleischmann, G . G . Sutton, J . A . Blake, L . M . FitzGerald, R . A . Clayton, J . D . Gocayne, A . R . Kerlavage, B . A . Dougherty, J . F . Tomb, M . D . Adams, C . I . Reich, R . Overbeek, E . F . Kirkness, K . G . Weinstock, J . M . Merrick, A . Glodek, J . L . Scott, N . S . M . Geoghagen, and J . C . Venter. 1996 . Complete genome sequence of the methanogenic archaeon Methanococcus jannaschii. Science 273:1058-1073.
9. Chien, Y . T., and S . H . Zinder. 1994 . Cloning, DNA sequencing, and characterization of a nifD-homologous gene from the archaeon Methanosarcina barkeri 227 which resembles nifD1 from the eubacterium Clostridium pasteurianum . J . Bacteriol . 176:6590-6598.
10. Chien, Y . T., 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 227 . J . Bacteriol . 178:143-148.
11. Chien, Y . T., 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.
12. 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.
13. Deppenmeier, U., M . Blaut, A . Mahlmann, and G . Gottschalk. 1990 . Reduced coenzyme F₄₂₀:hetereodisulfide oxidoreductase, a proton-translocating redox system in methanogenic bacteria . Proc . Natl . Acad . Sci . USA 87:9449-9453.
14. Deppenmeier, U., V . Müller, and G . Gottschalk. 1996 . Pathways of energy conservation in methanogenic archaea . Arch . Microbiol . 165:149-163.
15. Devereux, J., P . Haeberli, and O . Smithies. 1984 . A comprehensive set of sequence analysis programs for the VAX . Nucleic Acids Res . 12:387-395.
16. deZamaroczy, M. 1998 . Structural homologoues P_II and P_Z of Azospirillum brasilense provide intracellular signalling for selective regulation of various nitrogen-dependent functions . Mol . Microbiol . 29:449-463.
17. deZamaroczy, M., A . Paquelin, G . Peltre, K . Forchhammer, and C . Elmerich. 1996 . Coexistence of two structurally similar but functionally different PII proteins in Azospirillum brasilense. J . Bacteriol . 178:4143-4149.
18. Ferry, J . G. 1999 . Enzymology of one-carbon metabolism in methanogenic pathways . FEMS Microbiol . Rev . 23:13-38.
19. Forchhammer, K., and A . Hedler. 1997 . Phosphoprotein PII from cyanobacteria: analysis of functional conservation with the PII signal-transduction protein from Escherichia coli . Eur . J . Biochem . 244:869-875.
20. Forchhammer, K., and N . Tandeau de Marsac. 1995 . Functional analysis of the phosphoprotein PII (glnB gene product) in the cyanobacterium Synechococcus sp . strain PCC 7942 . J . Bacteriol . 177:5812-5817.
21. Forchhammer, K., A . Hedler, H . Strobel, and V . Weiss. 1999 . Heter