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Appl Environ Microbiol. Oct 2003; 69(10): 6047–6055.
PMCID: PMC201229

Lysine-2,3-Aminomutase and β-Lysine Acetyltransferase Genes of Methanogenic Archaea Are Salt Induced and Are Essential for the Biosynthesis of Nepsilon-Acetyl-β-Lysine and Growth at High Salinity


The compatible solute Nepsilon-acetyl-β-lysine is unique to methanogenic archaea and is produced under salt stress only. However, the molecular basis for the salt-dependent regulation of Nepsilon-acetyl-β-lysine formation is unknown. Genes potentially encoding lysine-2,3-aminomutase (ablA) and β-lysine acetyltransferase (ablB), which are assumed to catalyze Nepsilon-acetyl-β-lysine formation from α-lysine, were identified on the chromosomes of the methanogenic archaea Methanosarcina mazei Gö1, Methanosarcina acetivorans, Methanosarcina barkeri, Methanococcus jannaschii, and Methanococcus maripaludis. The order of the two genes was identical in the five organisms, and the deduced proteins were very similar, indicating a high degree of conservation of structure and function. Northern blot analysis revealed that the two genes are organized in an operon (termed the abl operon) in M. mazei Gö1. Expression of the abl operon was strictly salt dependent. The abl operon was deleted in the genetically tractable M. maripaludis. Δabl mutants of M. maripaludis no longer produced Nepsilon-acetyl-β-lysine and were incapable of growth at high salt concentrations, indicating that the abl operon is essential for Nepsilon-acetyl-β-lysine synthesis. These experiments revealed the first genes involved in the biosynthesis of compatible solutes in methanogens.

Living cells cope with salt stress by accumulation of osmolytes within their cytoplasm; this prevents loss of water and adjusts the turgor of the cell. The extremely halophilic halobacteria (Archaea) and the anaerobic halophilic Haloanaerobiales (Bacteria) accumulate KCl in their cytoplasm to counterbalance external salt (12, 52). However, this strategy, termed the salt-in-cytoplasm strategy, requires far-reaching adaptations of intracellular machineries to high salt concentrations and limits growth to certain salt concentrations (42). The second strategy, which is used by the majority of living cells, is to accumulate small, soluble, organic molecules that are termed compatible solutes. The ability to respond to an osmotic upshock by accumulating compatible solutes is found in all three lines of descent of life (2, 17, 30, 35). However, the spectrum of compatible solutes used comprises only a limited number of compounds, and these can be divided into two major groups: (i) sugars and polyols and (ii) α- and β-amino acids and their derivatives, including methylamines. This limitation to a rather small number of compounds reflects the fundamental constraints on solutes which are compatible with macromolecular and cellular functions (21).

Most archaeal compatible solutes resemble their bacterial counterparts, with the difference that the majority of them carry a negative charge. This anionic character is conferred to the solutes by the addition of a carboxylate, phosphate, or sulfate group (23, 35). The compatible solutes accumulated by archaea include, depending on the species and the salt concentration, di-myo-1,1′-inositol phosphate, 2-O-d-mannosyl-d-gycerate, diglycerol phosphate, cyclic-2,3-bisphosphoglycerate, α-glucosylglycerate, α-glutamate, and the unusual β-amino acids β-glutamate, β-glutamine, and Nepsilon-acetyl-β-lysine (5, 8, 20, 24-26, 32, 34, 39, 40, 46, 47).

Osmolality is sensed by microorganisms, and the response is on the transcriptional and enzyme activity levels (30, 54). So far, there have been no studies dealing with the signal and the signal transduction chain in archaea. A prerequisite for molecular studies is the identification of genes involved in the accumulation of compatible solutes. However, this is lagging behind, and the first gene cluster, encoding a primary ABC-type transporter for the compatible solute glycine betaine in methanogens, was described only very recently (36). Methanosarcina species were shown before to accumulate the unique compatible solute Nepsilon-acetyl-β-lysine at high salinities. Physiological and enzymatic studies suggested that Nepsilon-acetyl-β-lysine might be synthesized from α-lysine by the action of two enzymes, a lysine-2,3-aminomutase and an β-lysine acetyltransferase (31, 33, 47). We report here the identification and characterization of the lysine-2,3-aminomutase and acetyltransferase genes, ablA and ablB, from different methanoarchaea, and we prove by Northern blot analyses and mutational inactivation that the lysine-2,3-aminomutase and acetyltransferase are essential for the biosynthesis of Nepsilon-acetyl-β-lysine and salt adaptation. These are the first genes that have been identified to be involved in the biosynthesis of compatible solutes in methanoarchaea.


Organism, culture conditions, and growth experiments.

Methanosarcina mazei Gö1 (DSM 3647) was obtained from the Deutsche Sammlung für Mikroorganismen und Zellkulturen and grown under strictly anaerobic conditions as described previously (15). Cells were grown at 37°C in minimal medium, which is the standard Methanosarcina medium (DSMZ 120) without Casitone and yeast extract. Methanol was used as the carbon source at a concentration of 100 mM, and NaCl was added as indicated. Methanococcus maripaludis strain JJ (DSM 2067) was grown in mineral medium (McN) at 37°C with H2 plus CO2 as the carbon source as described previously (53). Growth experiments were performed in 16-ml Hungate tubes containing 5 ml of medium. After inoculation (10%) from an appropriate preculture, cultures were incubated with gentle shaking. The optical density at 578 nm was determined in a type 1101 M photometer (Eppendorf, Hamburg, Germany). All data points given reflect the means from duplicate tubes of one experiment, and diagrams display representative growth curves from at least three independent replications.

Isolation of chromosomal DNA.

Chromosomal DNA of M. mazei Gö1 was isolated essentially as described previously (22). Chromosomal DNA of M. maripaludis JJ was isolated from cells grown to late exponential phase by using a DNeasy tissue kit (Qiagen, Hilden, Germany).

Probe construction and labeling.

ablA was amplified from chromosomal DNA of M. mazei Gö1 by PCR with oligonucleotides lam(BamHI)5′ (5′-CGA GGT GGA TCC GTG AAA TCC-3′) and lam(XbaI)3′ (5′-CCG GAA TTT TCT AGA TTC AGA AAC GC-3′), introducing BamHI and XbaI sites at the 5′ and 3′ ends, respectively. ablB was amplified with oligonucleotides act5′ (5′-ATG GAC TTT ATC GGA CGT TTT GAG G-3′) and act3′ (5′-TCA TAA CAT CCT GCA CCA GAT GTT C-3′). mcrG was amplified with oligonucleotides mcrG1 (5′-TAC GAA TCA CAG TAT TAC-3′) and mcrG2 (5′-GAT CCT CTG TAC CCA TTC-3′). The amplified products were purified from agarose gels by using a QIAEX II gel extraction kit (Qiagen). The DNA fragments were labeled with [α-32P]dATP (Hartmann Analytic, Braunschweig, Germany) by using a random-primed DNA labeling system (Gibco BRL, Eggenstein, Germany) (9). Following 32P labeling, probes were separated from unincorporated nucleotides by using a QIAquick nucleotide removal kit (Qiagen).

Transcript analysis.

Cells of M. mazei Gö1 were grown on methanol (100 mM) in minimal medium with the indicated NaCl concentration to the late exponential growth phase. Nine milliliters of the cultures was harvested by centrifugation and resuspended in Tris-EDTA buffer. Pipetting and vortexing of the cells were sufficient for complete lysis, and RNA was subsequently isolated with a NucleoSpin RNA II kit (Macherey-Nagel GmbH & Co. KG, Düren, Germany). The resulting RNA preparations had an RNA content of 1,312 to 2,660 μg/ml as determined by the absorption at 260 nm. Denaturing agarose gel electrophoresis of RNA in the presence of formaldehyde, transfer to nylon membranes (Amersham Buchler, Braunschweig, Germany), and Northern blot hybridization were performed essentially as described previously (38). Finally, the blots were visualized by autoradiography.

Construction of the integration vector and transformation of M. maripaludis JJ.

The integration vector pIJA03 lacks a suitable replication origin for methanococci. It contains the pac cassette, which encodes puromycin resistance in methanococci, flanked by two multiple cloning sites that allow directed cloning of genomic DNA (14, 27, 48). For construction of pKP1, an internal part of the ablA gene (587 bp) was amplified by PCR from the genomic DNA of M. maripaludis JJ by using the oligonucleotides AMfor1 (5′-TTT TTT GGA TCC GGA ACG ATT ACA AGT GGC-3′) and AMrev1 (5′-TTT TTT TCT AGA TTT GAG GAA GAA CCA CGG-3′) and cloned upstream of the pac cassette of the integrative shuttle vector pIJA03. A 597-bp fragment of the ablB gene was amplified with the oligonucleotides AcTRfor1 (5′-TTT TTT GCT AGC GGA AAA GAC GTA GGC GAA-3′) and AcTRrev1 (5′-TTT TTT AGG TCC TCC CTG TAT CCG TTG TCT T-3′) and cloned downstream of the pac cassette, resulting in plasmid pKP1. The oligonucleotides were based on the sequence of M. maripaludis LL/S2 (18, 53). M. maripaludis JJ was transformed by the polyethylene glycol method of Tumbula et al. (51). Mutants were selected for growth in the presence of puromycin and 400 mM NaCl on H2 plus CO2. Mutants were checked for the correct insertion of the marker-disrupted gene by PCR and Southern hybridizations as described previously (38). For probe construction, the ablA insert and the pac cassette were cut out of plasmid pKP1 by using the restriction endonucleases NheI, BamHI, and XbaI and were subsequently 32P labeled as described above. For PCR analysis, the oligonucleotides lamout-for (5′-GGG ATA CCC CCT CTG TTG-3′) and actout-rev (5′-CCG CTG TAC ATA TAG CCC-3′), which align outside the abl operon, were used.

NMR spectroscopy.

The pool of compatible solutes of M. maripaludis and M. mazei Gö1 was analyzed by nuclear magnetic resonance (NMR) spectroscopy. Cells of M. maripaludis were grown in 300 ml of minimal medium with H2 plus CO2 as the carbon and energy source, whereas cells of M. mazei Gö1 were grown in 500 ml of minimal medium with methanol as the substrate, to the late exponential growth phase. Subsequently, cells were harvested by centrifugation, resuspended in 6 ml fresh minimal medium, and freeze-dried. The intracellular solutes were extracted with boiling 80% ethanol as previously described by Martins and Santos (26). Freeze-dried extracts were dissolved in distilled water and analyzed by NMR. NMR spectra were acquired in a Bruker AMX300 spectrometer with a 5-mm inverse detection probe head at 25°C, with presaturation of the water signal, a 60° flip angle, and a repetition delay of 60 s. A known amount of sodium formate was added and used as a concentration standard. The cell protein content in the samples used for solute analysis was determined with a 23225 kit from Pierce (Rockford, Ill.) after cell lysis by sonication (B. Braun-Biotech SA) and overnight treatment with 0.1 M NaOH at 37°C.

Nucleotide sequence accession numbers.

The sequences of the ablA and ablB genes have been deposited in GenBank under accession numbers NP_632958 and NP_632959.


Construction of a pathway for the biosynthesis of Nepsilon-acetyl-β-lysine based on genomic sequences.

A survey of the genomic sequences of M. mazei Gö1 and other methanogens revealed that the biosynthesis of α-lysine is via the diaminopimelate pathway. The first step toward lysine formation is the condensation of pyruvate and aspartate semialdehyde catalyzed by the activity of dihydrodipicolinate synthetase, resulting in dihydrodipicolinate, which is subsequently reduced by dihydropicolinate reductase. Tetrahydrodipicolinate then is N acylated by the action of tetrahydrodipicolinate succinylase, which forces the ring to open. The 2-oxo group of succinyl-epsilon-oxo-α-aminopimelate is transaminated by the action of succinyl diaminopimelate aminotransferase, followed by the hydrolysis of the acyl group catalyzed by succinyl diaminopimelate desuccinylase. The resulting l,l-α,epsilon-diaminopimelate is subsequently epimerized to the meso form by diaminopimelate epimerase. The last step in lysine formation is the decarboxylation of meso-diaminopimelate by diaminopimelate decarboxylase, resulting in α-lysine. Of these seven enzymes involved in α-lysine formation, four orthologs are found in the genome of M. mazei Gö1. Genes coding for proteins with homology to the dihydrodipicolinate synthetase (MM1201), dihydropicolinate reductase (MM1202), succinyl diaminopimelate aminotransferase (MM2649), and diaminopimelate decarboxylase (MM1885) could be identified. α-Lysine is proposed to be converted to Nepsilon-acetyl-β-lysine by the action of two enzymes, a lysine-2,3-aminomutase and an acetyltransferase (31, 33). To identify the encoding genes, we did BLAST searches on the genome of M. mazei Gö1 with the lysine-2,3-aminomutase sequence from Clostridium subterminale as the query sequence (37). By using this approach, an open reading frame termed ablA whose product was very similar to the lysine-2,3-aminomutase of C. subterminale (55.8% identity) was identified. Directly downstream of ablA was another open reading frame, ablB, whose product is similar to acetyltransferases from other organisms. A hypothetical pathway for Nepsilon-acetyl-β-lysine synthesis is shown in Fig. Fig.11.

FIG. 1.
Hypothetical pathway of Nepsilon-acetyl-β-lysine synthesis in methanogenic archaea. α-Lysine is converted by the activity of a lysine-2,3-aminomutase to β-lysine, which is then acetylated to Nepsilon-acetyl-β-lysine. ...

Genetic organization of ablA and ablB.

In M. mazei Gö1 the two genes are tandemly organized in the order 5′-ablA-ablB-3′, with a short intergenic region of at least 204 bp (Fig. (Fig.2).2). Upstream of ablA is a large, apparently noncoding region of 963 bp, followed by the start of the next gene, kefC. A putative promoter structure including the BRE element and BoxA was found 170 bp upstream of ablA, but not upstream of ablB, indicating that the two genes might form a transcriptional unit. An unusual start codon (GTG) seems to be used in the case of ablA. Each gene is preceded by a well-conserved and well-placed Shine-Dalgarno sequence. A possible transcription termination signal in the form of a stem-loop was found 49 bp downstream of the stop codon of ablB. At 21 bp downstream of ablB was the end of the next gene coding for a conserved protein. The genetic organizations of ablA and ablB are similar in M. mazei Gö1, Methanosarcina acetivorans, M. maripaludis, and Methanosarcina barkeri (Fig. (Fig.2).2). Methanococcus jannaschii contains a gene encoding a potential lysine-2,3-aminomutase (MJ0634), but the gene downstream (MJ0635) has no apparent similarities to acetyltransferases.

FIG. 2.
Comparison of the genomic organizations of the lysine-2,3-aminomutase (ablA) and acetyltransferase (ablB) genes of the methanogens M. mazei Gö1 (6), M. acetivorans (MA3979) (11), M. maripaludis LL/S2 (J. Leigh, personal communication), M. barkeri ...

Properties of the gene products and similarities to other proteins.

It was shown that the C. subterminale lysine-2,3-aminomutase (Kam) (EC has a hexameric quaternary structure composed of identical subunits with a molecular mass of 48 kDa (4, 45), which corresponds nicely to the deduced molecular mass (47.7 kDa) of AblA. Kam contains three [4Fe-4S] clusters, which are bound by conserved cysteine residues; these are also present in AblA (Fig. (Fig.3).3). The conversion of lysine to β-lysine requires S-adenosylmethionine (SAM). Crystal structure analyses of SAM-dependent enzymes revealed a glycine-rich region forming a flexible loop that is involved in SAM binding. This region is found in Kam (37) and AblA. Zinc is essential for the activity of Kam, and another group of three cysteines near the C terminus of Kam is suggested to present the zinc binding site (37). These three cysteines are conserved in AblA as well. Kam contains six molecules of pyridoxal-5′-phosphate per hexamer (29, 45), but a pyridoxal-5′-phosphate binding domain could not be identified by sequence analyses. The same is true for AblA of M. mazei Gö1. Taken together, these data indicate that ablA of M. mazei Gö1 codes for an aminomutase very similar to Kam of C. subterminale. Therefore, the mechanism for the conversion of α-lysine to β-lysine is suggested to be identical to that of Kam of C. subterminale (10) and other aminomutases (37).

FIG. 3.FIG. 3.
Alignment of the lysine-2,3-aminomutase of M. mazei Gö1 with lysine-2,3-aminomutases of M. acetivorans (MA3979) (11), M. maripaludis LL/S2 (J. Leigh, personal communication), C. subterminale (37), Bacillus halodurans (BH2255) (49), Mesorhizobium ...

The acetyltransferase is a member of the GNAT (GCN-5-related N-acetyltransferase) superfamily of enzymes. This superfamily spans all kingdoms of life and is characterized by the common acetyl donor acetyl coenzyme A (7). The acetylation of β-lysine by AblB is proposed to function in a similar way as in Escherichia coli strains that are resistant to nourseothricin, where the antibiotic is detoxified upon the acetylation of its β-lysine moiety (33, 41). The acetyltransferases of the GNAT superfamily were shown to have four poorly (in primary structure) conserved motifs (A to D), arranged in the order C-D-A-B (1). Motif A is the best conserved one, as it contributes many of the most critical contacts to the acetyl coenzyme A molecule (7). Therefore, the best-conserved residues across the GNAT superfamily, notably the motif Q/R-x-H/K-G-x-G/A-K/R, are present in this region (7, 50). In AblB of M. mazei Gö1, this R-G-K-G-H-M-K motif is also found.

Transcription of ablA and ablB is induced at high salt concentrations.

Because the products of the two genes ablA and ablB might be involved in the synthesis of Nepsilon-acetyl-β-lysine, the question arose whether their transcription is salt regulated. To address this question, Northern blot analyses were employed. Parts of ablA or ablB were amplified by PCR and used as probes. Cells of M. mazei Gö1 were grown in minimal medium with 38.5, 400, or 800 mM NaCl and harvested at the end of the exponential growth phase, and RNA was isolated and blotted. The blots were hybridized against the ablA or ablB probes or against a probe derived from the coenzyme M reductase gene (mcr). The product of mcr is involved in methanogenesis, and as its expression was shown to be only little affected by the salt concentration (36), it was used as an internal control.

As can be seen from Fig. Fig.4,4, mcr expression increased only slightly at high salt concentrations. The transcript size of about 5,500 bases corresponds well to the predicted size of 4,893 bases. On the other hand, expression of ablA and ablB at the standard NaCl concentration of 38.5 mM was not detectable, but it increased drastically with increasing salt concentrations in the growth medium. The ablA and ablB probes resulted in identical hybridization patterns, which indicated that the two genes are in fact transcribed as one operon, which was termed the abl operon. The determined transcript size of about 2,600 bases corresponds well to the predicted size of the abl operon of 2,448 bases. The level of transcription of the abl operon was apparently identical in cells grown with 400 or 800 mM NaCl.

FIG. 4.
Expression of ablA and ablB is salt dependent. RNA of M. mazei Gö1 grown at the NaCl concentration indicated was isolated and subjected to denaturing agarose gel electrophoresis. After transfer to nylon membranes, the RNA was hybridized with a ...

Construction of abl deletion mutants of M. maripaludis JJ.

The experiments described so far are compatible with the hypothesis that the products of the abl operon are key players in the biosynthesis of Nepsilon-acetyl-β-lysine. To determine whether the abl operon is involved in Nepsilon-acetyl-β-lysine formation, Δabl mutants were generated and analyzed with respect to Nepsilon-acetyl-β-lysine formation and their salt tolerance. Because there is not yet a genetic system for M. mazei Gö1 and the sequence of M. acetivorans had not been published at the time that we performed these experiments, we used the genetically tractable M. maripaludis JJ for the inactivation of the abl operon (13). The ablA and ablB genes of M. maripaludis LL/S2 are tandemly organized as in M. mazei Gö1 and are very similar to those of M. mazei Gö1 (Fig. (Fig.2).2). To construct a deletion in the abl operon, the integrative shuttle plasmid pKP1 was constructed; 587 bp of the 5′ end of ablA was cloned upstream of the pac cassette, and 597 bp of ablB was cloned downstream of the pac cassette. This plasmid was linearized and transformed into M. maripaludis JJ. Two isolates, named M. maripaludis JJ Δabl1 and M. maripaludis JJ Δabl2, were obtained. The correct integration of the construct into the abl operon of M. maripaludis JJ was confirmed by Southern blotting and PCR analysis.

Formation of Nepsilon-acetyl-β-lysine is impaired in the Δabl mutants.

M. maripaludis JJ grew optimally at an NaCl concentration equivalent to that in seawater (376.5 mM) but tolerated NaCl concentrations up to 1 M (Fig. (Fig.5).5). NMR analysis (Table (Table1)1) showed that at an NaCl concentration of 376.5 mM the wild type accumulated 0.41 μmol of glutamate/mg of protein but only 0.14 μmol of Nepsilon-acetyl-β-lysine/mg of protein. However, the intracellular Nepsilon-acetyl-β-lysine concentration increased 3.7-fold to 0.7 μmol/mg of protein when cells were grown with 800 mM NaCl and increased 7.8-fold when cells were grown in the presence of 1 M NaCl. In contrast, increasing amounts of NaCl did not have any effect on the intracellular glutamate concentration. These data demonstrate that M. maripaludis JJ induces the synthesis of Nepsilon-acetyl-β-lysine as the predominant compatible solute at high external salt concentrations. Of interest next was the Nepsilon-acetyl-β-lysine concentration in the Δabl mutants. Neither of the two mutants was able to synthesize Nepsilon-acetyl-β-lysine at 376.5 or 800 mM NaCl, proving that ablA and ablB in fact code for enzymes involved in the biosynthesis of Nepsilon-acetyl-β-lysine.

FIG. 5.
Growth of M. maripaludis Δabl mutants is impaired at elevated salt concentrations. Cells of M. maripaludis JJ ([filled lozenge]), M. maripaludis JJ Δabl1 ([open triangle]), and M. maripaludis JJ Δabl2 (○) were grown in minimal medium ...
Compatible solutes of wild-type M. maripaludis JJ and Δabl mutants

Growth of the Δabl mutants is impaired at 1 M NaCl.

M. maripaludis JJ grew at 376.5 mM NaCl at a rate of 0.306 h−1 to a final optical density of 1.45. When the salt concentration was increased to 800 mM, there was neither a significant lag phase nor a reduction in the growth rate or the final optical density. At 1 M NaCl, there was only a slight reduction in the growth rate. Salt concentrations above 1 M did not support reliable growth. Growth of the Δabl mutants was indistinguishable from that of the wild type at 376.5 mM NaCl, indicating that Nepsilon-acetyl-β-lysine is not an essential compatible solute at this salt concentration. In contrast, growth was severely inhibited at 800 mM NaCl and was almost completely impaired at 1 M NaCl. These experiments clearly demonstrate that Nepsilon-acetyl-β-lysine formation is essential for growth of M. maripaludis JJ at high salt concentrations.


Microorganisms sense and respond to changing salt concentrations (12, 54). However, the signal sensed as well as the signal transduction chain leading to the expression of enzymes involved in the biosynthesis of compatible solutes is unknown for archaea (23, 30). A prerequisite for the elucidation of the signal transduction chain is the identification of genes involved in the accumulation of compatible solutes. Here, we identified the genes involved in the biosynthesis of Nepsilon-acetyl-β-lysine from α-lysine in methanogens.

Nepsilon-Acetyl-β-lysine has been found in many methanogenic archaea, such as Methanogenium cariaci, Methanosarcina spp., Methanohalophilus spp., and Methanococcus spp., (20, 31, 33, 46, 47). From the data presented here, it is evident that M. maripaludis also produces Nepsilon-acetyl-β-lysine at high external salt concentrations. The same holds true for M. mazei Gö1: whereas Nepsilon-acetyl-β-lysine was not detected in cells grown in the presence of 38.5 mM NaCl, the amount of intracellular Nepsilon-acetyl-β-lysine increased with increasing NaCl concentrations, from 0.14 μmol/mg protein at 400 mM NaCl to 0.82 μmol/mg at 800 mM NaCl (data not shown). It is important to note that the organisms known to have the well-conserved abl operon also produce Nepsilon-acetyl-β-lysine. Therefore, it seems safe to assume that the abl operon is involved in Nepsilon-acetyl-β-lysine formation in methanogens in general, although this has been shown here by mutational inactivation only for M. maripaludis. On the other hand, we could not find homologs of ablA and ablB in Methanothermobacter thermautotrophicus and Methanopyrus kandleri (43, 44), indicating that these organisms either do not produce or have a different mechanism for the production of Nepsilon-acetyl-β-lysine.

The Δabl mutants of M. maripaludis are completely impaired in the formation of Nepsilon-acetyl-β-lysine, which is evidence that the abl operon is essential for Nepsilon-acetyl-β-lysine formation. However, at 376.5 mM NaCl, growth of the mutants was indistinguishable from that of the wild type, and at 800 mM NaCl, although growth was largely impaired, the mutants still reached an optical density of 0.5 to 0.6. Like other organisms (17), methanogens do not rely on one compatible solute only, and even one organism can possess a cocktail of different compatible solutes (19, 20, 32). Compatible solutes found in methanogens are glycine betaine, α-glutamate, Nepsilon-acetyl-β-lysine, cyclic-2,3-bisphosphoglycerate, β-glutamine, and potassium ions, and the composition of the pool of compatible solutes in a given cell changes with increasing salt concentrations. At 400 mM NaCl, the proportion of Nepsilon-acetyl-β-lysine to the pool is negligible, and therefore growth inhibition is not expected in the mutants. Because the NMR analyses did not reveal an alternative organic solute at 800 mM NaCl, K+ might be used under these conditions. However, it can substitute for Nepsilon-acetyl-β-lysine to only a small extent.

NMR analyses suggested biosynthesis of Nepsilon-acetyl-β-lysine from α-lysine in a two-step process via β-lysine followed by subsequent acetylation of the β-lysine (31), and a lysine-2,3-aminomutase activity was detected in cell extracts (24). This pathway is supported by our finding that the abl operon comprises only two genes, ablA and ablB. AblA and AblB are similar to lysine-2,3-aminomutase and acetyltransferase and therefore are suggested to catalyze Nepsilon-acetyl-β-lysine from α-lysine. However, biochemical data are not yet available and have to await the purification of the enzymes either from methanogens or after heterologous overproduction. The latter research is in progress in our laboratory.

Since apparently only one route for α-lysine production is present in the methanogens examined, it was speculated that the formation of Nepsilon-acetyl-β-lysine is regulated via the activity of the aminomutase and/or the acetyltransferase (24, 31). This was corroborated by the finding of an eightfold induction of lysine-2,3-aminomutase activity in cells of Methanococcus thermolithotrophicus shifted from 0.68 to 1.4 M NaCl (24). Here, we demonstrate high expression of the abl operon in M. mazei Gö1 at 400 and 800 mM NaCl, while expression is apparently impaired at 38.5 mM NaCl. These data clearly show that the regulation of Nepsilon-acetyl-β-lysine is via the expression of the abl operon. Future studies are aimed at identifying the molecular basis of this regulation and the components of the signal transduction chain leading to expression of the abl operon. This system will be a useful tool to study salt-dependent gene regulation in the domain Archaea.


This work was supported by grants from the Deutsche Forschungsgemeinschaft and the Ministry of Science and Culture of the state of Lower Saxony. W.L. was supported by a grant from the U.S. Department of Energy to W. B. Whitman.

We acknowledge the helpful comments of W. B. Whitman. We thank Ana Mingote for solute extraction and NMR quantification.


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