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Applied and Environmental Microbiology, May 2002, p. 2133-2139, Vol. 68, No. 5
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.5.2133-2139.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Identification of a Salt-Induced Primary Transporter for Glycine Betaine in the Methanogen Methanosarcina mazei Gö1

M. Roeßler,¹ K. Pflüger,¹ H. Flach,¹ T. Lienard,^2,3 G. Gottschalk,^2,3 and V. Müller^1*

Lehrstuhl für Mikrobiologie der LMU München, 80638 Munich,¹ Abteilung Allgemeine Mikrobiologie,² Göttingen Genomics Laboratory, Georg-August-Universität, 37077 Göttingen, Germany³

Received 1 November 2001/ Accepted 31 January 2002

Top Abstract Introduction Materials and Methods Results Discussion References

 
The salt adaptation of the methanogenic archaeon Methanosarcina mazei Gö1 was studied at the physiological and molecular levels. The freshwater organism M. mazei Gö1 was able to adapt to salt concentrations up to 1 M, and the addition of the compatible solute glycine betaine to the growth medium facilitated adaptation to higher salt concentrations. Transport studies with cell suspensions revealed a salt-induced glycine betaine uptake activity in M. mazei Gö1, and inhibitor studies argue for a primary transport device. Analysis of the genome of M. mazei Gö1 identified a homolog of known primary glycine betaine transporters. This gene cluster was designated Ota (osmoprotectant transporter A). Its sequence and gene organization are very similar to those of the glycine betaine transporter OpuA of Bacillus subtilis. Northern blot analysis of otaC revealed a salt-dependent transcription of this gene. Ota is the first identified salt-induced transporter for compatible solutes in Archaea.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Methanogenic archaea are a phylogenetically diverse group of organisms inhabiting ecosystems with very different physical parameters, such as temperature, pH, or salt concentration. Methanogens have been isolated from freshwater, marine, and even saline environments (11). Therefore, like other living cells, methanogens have derived mechanisms to cope with salt stress. Generally, the cytoplasmic membrane is freely permeable to water, and therefore, cells tend to loose water upon a hyperosmotic shock; however, this is prevented by protective mechanisms allowing the cells to adjust their turgor pressure. Two different mechanisms for turgor regulation have been realized in living cells. First, the intracellular salt concentration is adjusted to the extracellular one. This mechanism, which is found in aerobic, halophilic archaea (Halobacteriales) and in some anaerobic bacteria (Haloanaerobiales) (8, 35), requires far-reaching adaptations of the intracellular machineries to high salt and limits growth to a narrow range of external salt concentrations (23). The second mechanism relies on the accumulation of compatible solutes, which do not interfere with the metabolism and whose intracellular concentrations can be varied over a wide range (5). Therefore, growth can occur over a wide range of external salt concentrations. This mechanism is well conserved in all three lines of descent and was found in most bacteria and also in methanogenic archaea (26, 35).

In recent years, the physiology of osmoadaptation and its molecular basis have been worked out for bacteria and eukarya (12, 15, 24, 37), but little is known about the molecular basis of osmoadaptation in archaea. As the final response, methanogenic archaea accumulate a wide variety of compatible solutes, either by de novo synthesis or uptake from the environment. However, the molecular basis of salt adaptation in methanogenic archaea is completely unknown, and salt-induced genes or proteins have never been described (26).

As a first step towards the molecular basis of salt adaptation in methanoarchaea, we wanted to identify a salt-induced gene(s). As a model system we chose Methanosarcina mazei Gö1, because other Methanosarcina strains have been shown before to be halotolerant (4, 20, 31, 32). In addition, M. mazei Gö1 is a metabolically versatile, methylotrophic methanogen whose genomic sequence has been determined, which makes it an ideal candidate for the study of regulation of gene expression. As a model for a salt-induced gene, we aimed to identify the gene(s) encoding a transporter for the compatible solute glycine betaine, because methanogens have been shown before to accumulate glycine betaine from the exterior (17, 19) and it has been reported that this uptake is induced at high external salt concentrations (18, 25). To identify the transporter(s) we screened the genomic sequence for genes whose products might be involved in the uptake of glycine betaine or other compatible solutes and employed Northern blot analyses to identify salt-induced genes. This approach revealed a salt-induced gene, otaC, which is part of a cluster encoding a primary ABC-type transporter with high similarity to glycine betaine transporters from bacteria.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Organism, culture conditions, and growth experiments.
M. mazei Gö1 (DSMZ 3647) was obtained from the Deutsche Sammlung für Mikroorganismen und Zellkulturen and grown under strictly anaerobic conditions as described (9). Substrates and NaCl were supplemented as indicated. For growth experiments in minimal medium, the standard Methanosarcina medium (DSMZ 120) without Casitone and yeast extract was used. Growth experiments were done in 16-ml Hungate tubes containing 5 ml of medium. After inoculation (10%) from an appropriate preculture, cultures were incubated at 37°C, with gentle shaking for growth on H₂-CO₂. The optical density at 600 nm was determined in a photometer (type 1101 M; Eppendorf, Hamburg, Germany). All data points given reflect the means of duplicate tubes of one experiment, and diagrams display a representative growth curve of at least three independent replications.

Preparation of resting cell suspensions.
Fresh cell suspensions of M. mazei Gö1 were prepared for each experiment. Cells were grown with 100 mM methanol as the substrate at the given NaCl concentration and harvested by centrifugation (5,000 x g, 15 min, 4°C) in the exponential growth phase. The cells were washed in isosmotic assay buffer (containing, per liter, 0.348 g of K₂HPO₄, 0.227 g of KH₂PO₄, 0.5 g of NH₄Cl, 0.5 g of MgSO₄ · 7H₂O, 0.002 g of FeSO₄ · 7H₂O, and 0.25 g of CaCl₂ · 2H₂O), which contained titanium(III) citrate solution (3) (2 ml/liter), 5 mM dithioerythreitol, resazurin (1 mg/liter), and NaCl as indicated, and resuspended in 5 ml of the same buffer. The resulting cell suspension containing 12 to 18 mg of protein/ml was stored in a 16-ml Hungate tube on ice until use. The protein concentration was determined as described (30). All manipulations were done in an anaerobic glove box (Coy, Ann Arbor, Mich.) containing an atmosphere of N₂-H₂ (95:5).

Determination of glycine betaine uptake.
All experiments were carried out under strictly anaerobic conditions in airtight 58-ml bottles containing 9 ml of the assay buffer with the indicated NaCl concentration. After the bottles were flushed with oxygen-free nitrogen for 10 min, 1 ml of the concentrated cell suspension was added, resulting in a final protein concentration of 1.2 to 1.8 mg of protein/ml. Additions of all components were made from anaerobic stock solutions with a syringe. Methanol was added to a final concentration of 100 mM, and the suspension was preincubated for 10 min at 37°C. Methane was determined by gas chromatography as described previously (2). The reaction was started by addition of [¹⁴C]glycine betaine (American Radiolabeled, St. Louis, Mo.) to a final concentration of 100 µM (1 µCi; specific activity, 55 µCi/µmol). 4,5,4',5'-Tetrachlorosalicylanilide (4,5,4',5'-TCS) was added as indicated from an ethanolic stock solution to a final concentration of 10 µM. Ethanol itself had no effect on the uptake of glycine betaine. At the time points indicated, 500-µl samples were withdrawn from the cell suspension by a syringe and transferred to microcentrifuge tubes containing 200 µl of silicon oil that had been incubated for at least 12 h in an anaerobic glove box. The cells were separated from the assay buffer by centrifugation through silicon oil (28). At a density of 1.012, 1.023, or 1.048 mg/ml for 38.5, 400, and 800 mM NaCl, respectively, separation was optimal. Under these conditions the assay buffer remained anaerobic as judged from the redox indicator resazurin. After centrifugation, the tip of the microcentrifuge tube with the cell pellet was cut off and transferred into a scintillation vial. A 5-ml aliquot of Rotiszint ecoplus (Roth, Karlsruhe, Germany) was added, the vials were vigorously shaken, and radioactivity was determined in a liquid scintillation counter (type 2100 TR; Packard, Dreieich, Germany). Internal glycine betaine was calculated from the specific activity, which was determined for each experiment by measuring the radioactivity of an aliquot of the cell suspension.

Isolation of chromosomal DNA.
Chromosomal DNA of M. mazei Gö1 was essentially isolated as described (21). Cells were grown to the late logarithmic growth phase, sedimented by centrifugation, and lysed by an alkaline sodium dodecyl sulfate solution. The protein was precipitated with 6 M guanidinium thiocyanate and isobutanol; subsequently the DNA was bound to glass milk in the presence of NaI (90.8 g of NaI, 1.5 g of Na₂SO₃, and 100 ml of H₂O). After washing with wash buffer (50% ethanol, 0.1 M NaCl, 0.02 M Tris-HCl [pH 7.5], 1 mM EDTA), the DNA was eluted with Tris-EDTA buffer. The isolated chromosomal DNA was digested with BamHI or XhoI, respectively, and subjected to Southern hybridization as described (29).

Probe construction and labeling.
otaC was amplified from chromosomal DNA of M. mazei Gö1 by PCR using oligonucleotides otaC1 (5'-TGGAACTGGTTGTGTCAT-3') and otaC2 (5'-TTATCTGCTTCTCCGTAA-3'). mcrG was amplified using oligonucleotides mcrG1 (5'-TACGAATCACAGTATTAC-3') and mcrG2 (5'-GATCCTCTGTACCCATTC-3'). The amplified products were purified by agarose gel electrophoresis and the QIAEX II gel extraction kit (Qiagen, Hilden, Germany). The DNA fragments were radiolabeled with [-³²P]dATP (Hartmann Analytic GmbH, Braunschweig, Germany) using the Random Primed DNA Labeling System (Gibco BRL GmbH, Eggenstein, Germany) (7). Following ³²P labeling, probes were separated from unincorporated nucleotides by using the QIAquick Nucleotide removal kit (Qiagen). The specificity of the probes was confirmed by Southern blot analysis as described (29).

Transcript analysis.
Cells were grown in medium with the substrate and NaCl concentration as indicated until the late logarithmic growth phase. Nine milliliters of the culture 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 using the RNeasy system (Qiagen) according to the instructions of the manual. Residual DNA was removed by a DNase I treatment (Boehringer, Mannheim, Germany). The resulting RNA preparations had an RNA content of 210 to 1,200 µg/ml as determined by absorption at 260 nm. Denaturing agarose gel electrophoresis of RNA in the presence of formaldehyde, transfer to nylon membranes (Amersham Buchler GmbH, Braunschweig, Germany), and Northern blot hybridization were essentially performed as described (29). Finally, the blots were visualized by autoradiography.

Cloning and sequencing of the ota gene.
The complete genomic sequence of M. mazei Gö1 was determined by a whole-genome shotgun approach. More than 18,000 clones carrying inserts of approximately 2.5 kb in length from small insert libraries representative of the whole genome were sequenced from both ends using LICOR IL 4200 and ABI PRISM 377 DNA sequencers. The generated sequence readings were assembled into contigs with the Prap software implemented in the STADEN software package.

Nucleotide sequence accession number.
The ota gene sequences have been deposited in GenBank under the accession number AF475089.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Salt range of growth of M. mazei Gö1.
M. mazei Gö1 was isolated from a municipal sewage plant, indicating that it is a freshwater organism. Although other Methanosarcina strains have been shown to be halotolerant (4, 20, 31, 32), it was important to confirm this for M. mazei Gö1 and to determine the kinetics of growth after a hyperosmotic shock. M. mazei Gö1 was pregrown on methanol at the standard NaCl concentration of 38.5 mM and then transferred to complex media having NaCl concentrations from 38.5 to 1,500 mM. At NaCl concentrations up to 200 mM growth was indistinguishable from that at 38.5 mM NaCl. NaCl concentrations above 200 mM led to a lag phase, whose length increased with increasing NaCl concentrations. Growth occurred up to 1,000 mM NaCl; higher concentrations were inhibitory. After cells adapted to increased NaCl concentrations, growth resumed with identical growth rates of 0.053 h^-1 up to 400 mM, while growth was reduced by 48% to 0.028 h^-1 at NaCl concentrations of 800 and 1,000 mM. On the other hand, final optical densities were only marginally affected by the external NaCl concentration (Fig. 1). This experiment shows that M. mazei Gö1 is a halotolerant archaeon with the ability to adapt to NaCl concentrations up to 1 M. However, the results obtained here differ slightly from those in other studies (see Discussion). When cells preadapted to 400, 600, or 800 mM NaCl were transferred to fresh iso-osmotic medium, there was no longer a lag phase, and growth rates as well as final optical densities were identical to those under standard growth conditions.

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FIG. 1. Salt adaptation of M. mazei Gö1. Growth was determined in Methanosarcina medium with 100 mM methanol containing 38.5 (), 200 (), 400 (), 800 (), 1,000 (), or 1,500 (•) mM NaCl as a substrate. Cultures were inoculated with a 10% inoculum from a preculture grown on methanol at 38.5 mM NaCl. OD600, optical density at 600 nm.

The carbon sources used by methanogens differ significantly with respect to their energy content, with acetate being at the far lower end of the scale, allowing the synthesis of only 0.5 mol of ATP per mol of acetate under cellular conditions (22). Furthermore, a compound like trimethylamine allows the synthesis of 2 mol of ATP/mol of trimethylamine, and in principle, it could be used as an osmolyte in addition. Therefore, it was of interest to study the possible dependence of the salt adaptation on the carbon source used. Cells were first adapted to the carbon source (acetate, H₂-CO₂ or trimethylamine) at 38.5 mM NaCl and then, after three transfers, shifted to higher NaCl concentrations. Lag phases and growth rates were affected by NaCl in a way observed before with methanol-grown cells. However, the acetate cultures did not show reliable growth at NaCl concentrations of 0.8 to 1.0 M NaCl (data not shown).

Stimulation of growth at elevated salt concentrations by glycine betaine.
Various methanogens are known to accumulate the compatible solute glycine betaine upon an increase of external salt concentration (17, 18, 19, 25, 33). A first hint for the presence of a transport system for compatible solutes can be obtained from the analysis of growth parameters in cultures grown in minimal media at high salt concentrations in the absence or presence of compatible solutes. When M. mazei Gö1 was grown in minimal medium at a high salt concentration, growth occurred in all cases but was stimulated significantly by the addition of 1 mM glycine betaine (Fig. 2). While the addition of glycine betaine had no effect on growth at 38.5 mM NaCl, growth rates at 800 mm NaCl were increased three- to fourfold and final optical densities approximately doubled in the presence of glycine betaine. These experiments are in accordance with the presence of a transport system for glycine betaine.

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FIG. 2. Stimulation of growth of M. mazei Gö1 at high salt concentrations by external glycine betaine. Growth was determined in minimal medium with 100 mM methanol containing 38.5 (, ) or 800 (, •) mM NaCl as a substrate. Cultures were inoculated with a 10% inoculum from a preculture grown on methanol at 38.5 mM NaCl in minimal medium. Glycine betaine was added to a final concentration of 1 mM (closed symbols).OD600, optical density at 600 nm.

Glycine betaine transport is salt induced and, most likely, catalyzed by a primary transporter
Salt-induced glycine betaine transporters were described before in several methanogenic archaea (18, 25). As a next step toward the identification of a glycine betaine transporter in M. mazei Gö1, transport studies were performed to determine glycine betaine transport activity directly and to characterize the transporter involved. Of special interest was the salt dependence of transport activity. Transport of glycine betaine was studied in cell suspensions derived from cells grown on methanol. Cells were grown at different salt concentrations, harvested in the exponential growth phase, washed, and resuspended in buffer at conditions iso-osmotic with the growth media. Transport of glycine betaine was studied using [¹⁴C]glycine betaine and a silicon oil centrifugation assay. Upon addition of methanol, methane formation started in every case (data not shown). However, the rate of [¹⁴C]glycine betaine transport was a function of the salt concentration of the growth medium (Fig. 3). Cells grown at 38.5 mM NaCl did not exhibit measurable glycine betaine transport activity. On the other hand, cells grown at 400 mM NaCl did accumulate glycine betaine, at a rate of 0.057 nmol/min·mg of protein, and transport activity was further increased by a factor of 4.6 in cells grown at 800 mM NaCl. These cells transported glycine betaine at a rate of 0.26 nmol/min·mg of protein. Assuming an intracellular volume of 3 µl/mg of protein (3), one can calculate that after 40 min of incubation at 400 and 800 mM NaCl, glycine betaine was enriched 7- and 28-fold, respectively. A plateau (160-fold enrichment) was reached after 120 min (data not shown). This experiment gives clear evidence for a salt-induced glycine betaine transporter(s) in M. mazei Gö1.

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FIG. 3. Salt dependence of glycine betaine uptake in M. mazei Gö1. Cells were grown in Methanosarcina medium containing 38.5 (), 400 (), or 800 () mM NaCl and harvested, and uptake experiments were performed in cell suspensions with [14C]glycine betaine in assay buffer under isosmotic conditions. The protein concentration was 1.2 mg/ml. The experiments were performed at least three times, and the data presented reflect a typical result.

Uptake of [¹⁴C]glycine betaine required the presence of a substrate, reflecting the energy requirement for the uptake process (data not shown). 4,5,4',5'-TCS is a very potent protonophore for methanogens and known to dissipate the membrane potential as well as the cellular ATP content (3). Upon addition of 4,5,4',5'-TCS in the course of methanogenesis and [¹⁴C]glycine betaine transport, methanogenesis and, at the same time, [¹⁴C]glycine betaine transport ceased (Fig. 4). However, the intracellular glycine betaine concentration did not decrease but stayed at a level 17-fold higher than the extracellular concentration. Since glycine betaine is not known to be further metabolized by M. mazei Gö1 and since secondary, ion-coupled transporters, but not ATP-driven transporters, are mostly reversible machines mediating equilibration of the substrate in the absence of a driving force, the lack of observed export of glycine betaine along its chemical gradient after the addition of the protonophore is in accordance with a primary transport system. However, a secondary transport mechanism cannot be excluded by these experiments.

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FIG. 4. Energy dependence of glycine betaine uptake in M. mazei Gö1. Cells were grown in Methanosarcina medium containing 800 mM NaCl and harvested, and uptake experiments with [14C]glycine betaine were performed in assay buffer under iso-osmotic conditions. At the time point indicated by the arrow, 4,5,4',5'-TCS was added to a final concentration of 10 µM (open symbols). The protein concentration was 1.2 mg/ml. The experiments were performed at least three times, and the data presented reflect a typical result.

Identification of genes putatively encoding transporters for compatible solutes.
To identify genes encoding transporters for compatible solutes, we did BLAST searches of the genome of M. mazei Gö1. Because our experiments argued for the presence of a primary glycine betaine transporter, we looked for potential glycine betaine transporter genes by using the substrate binding protein of a known ABC-type glycine betaine transporter from Bacillus subtilis, OpuAC, as a query sequence. Using this approach we could identify a homolog of OpuA in M. mazei Gö1, which was designated Ota (osmoprotectant transporter A). This gene cluster encodes a substrate binding protein (OtaC), a transmembrane protein (OtaB), and an ATP binding protein (OtaA) (see below).

otaC of M. mazei Gö1 is induced at high salt concentrations.
To test the salt-dependent expression of ota, part of the substrate binding protein-encoding gene (otaC) was amplified by PCR and used as a probe. The specificity of the probe was verified in Southern blots (data not shown).

The possible salt dependence of expression of otaC was analyzed in Northern blots. Therefore, cells of M. mazei Gö1 were grown on methanol at either 38.5, 400, or 800 mM NaCl and harvested at the end of the logarithmic growth phase, and RNA was isolated and blotted. The blots were hybridized against otaC or a probe derived from the methyl coenzyme M reductase gene (mcrG). The product of the latter is involved in methanogenesis, and its expression is not expected to be salt dependent. As can be seen from Fig. 5, mcr expression only slightly increased at high salt concentrations of the growth medium. The transcript size of 6.0 kb corresponds well to the predicted size of 4,893 bp. The slight difference may result from flanking noncoding regions. Additionally, the determined transcript size varied depending on the RNA standard used. On the other hand, expression of otaC at the standard NaCl concentration of 38.5 mM was very low but increased drastically with increasing salt concentrations in the growth medium. The very strong signal at 1.1 kb corresponds to otaC (921 bp) only. This fragment could arise from independent transcription of otaC or from processing of a large fragment (the smear from 3 to 4 kb). These results correlate nicely with the transport studies which revealed maximal induction of the transport activity at 800 mM NaCl. The salt dependence of expression of otaC was also observed in cells grown on trimethylamine, H₂-CO₂, or acetate (data not shown). Again, this is in agreement with the growth experiments described above.

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FIG. 5. Expression of ota is salt dependent. RNA of M. mazei Gö1 grown at the indicated NaCl concentrations was isolated, and 5 µg of RNA was subjected to a denaturing agarose gel electrophoresis. After a transfer to nylon membranes, RNA was probed with the mcr probe (A) or the ota probe (B).

Nucleotide sequence and structure of the ota gene cluster from M. mazei Gö1.
The ota gene cluster contains three genes arranged in the order otaA, otaB, and otaC. otaA and otaB overlap by 1 bp, and otaB and otaC are separated by 228 bp. Immediately downstream of otaC is a potential rho-independent transcriptional terminator. Downstream (313 bp) of the stop codon of otaC is the stop codon of a divergently transcribed gene whose product is similar to S-layer proteins. otaA, otaB, and otaC are 1,353, 834, and 921 bp, respectively. Each gene is preceded by a well-conserved and well-placed Shine-Dalgarno sequence. Sequence data suggest that each gene contains an unusual start codon (GTG for otaA, otaB, or otaC).

Properties of the gene products and similarities to other proteins.
The properties of the ota gene products are summarized in Table 1. Database searches, primary sequence alignments, secondary structure predictions, and comparisons of the molecular masses and isoelectric points were used to identify homologs in other organisms. OtaA is a hydrophilic protein with an M_r of 49,510. It contains a Walker motif, indicating its capability of binding ATP; it is very similar to the ATP hydrolyzing subunit of ABC-type glycine betaine transporters. OtaB has an M_r of 30,230. Hydrophobicity plots indicate that OtaB is a transmembrane protein with six predicted transmembrane helices. Again, it is very similar to the membrane-bound translocator subunit of ABC-type glycine betaine transporters. OtaC is a hydrophilic protein with an M_r of 36,340; it is very similar to substrate binding proteins from ABC-type glycine betaine transporters from bacteria.

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TABLE 1. Properties of subunits of the putative primary glycine betaine transporter Ota of M. mazei Gö1g

Top Abstract Introduction Materials and Methods Results Discussion References

 
Microorganisms sense and respond to changing salt concentrations. However, the signal sensed as well as the signal transduction chain leading to the expression of transporters for compatible solutes is unknown in archaea. These questions could not even be addressed in archaea before now since transporters had not been identified on a molecular level. OtaC is the first identified subunit of a transporter for compatible solutes in archaea which is clearly induced at high salt concentrations and will, therefore, be a useful tool to study salt-dependent gene regulation in the domain Archaea.

The results presented here with M. mazei Gö1 are in line with the previous observations that various Methanosarcina strains are slightly halotolerant (4, 20, 31, 32). However, previous studies have been performed with preadapted cultures, and information on the response of Methanosarcina to a hyperosmotic shock is scarce. Here, we have demonstrated that cells respond to a hyperosmotic shock with a lag phase in which the metabolism has to be reprogrammed; i.e., enzymes have to be synthesized and compatible solutes have to be accumulated in order to cope with the salt stress. The more turgor that has to be created, the more time is required. Therefore, the time required for this shift is a function of the external salt concentration. The response is rather slow (up to 4 days), but it corresponds to the low growth rates (doubling time = 13 h). In light of this experiment it would be interesting to determine the rates of protein synthesis and solute accumulation in M. mazei Gö1, which could be rather low and would explain the rather long time required for the adaptation.

After the lag phase, growth started and the growth rates obtained at 38.5, 200, and 400 mM NaCl were similar. A further increase in the external salt concentration decreased the growth rate slightly. The same dependence of the growth rate on the external salt concentration was observed with preadapted cells of various Methanosarcina strains (20). In contrast, with preadapted cells, Sowers and Gunsalus (32) observed an increase in the growth rate parallel to the increase in the external salt concentration since a plateau was reached at 0.7 osmol/kg; a further increase in the external salt concentration led to a decrease in the growth rate (32). These differences could be due to the use of different media, different strains of the same species, and/or time of preadaptation.

Glycine betaine is known as a compatible solute in methanogens and has been found in members of the genera Methanococcus, Methanogenium, Methanohalophilus, and Methanosarcina (17, 27, 32). From the results presented here one can conclude that glycine betaine is also used in M. mazei Gö1 as a compatible solute. However, like other organisms, methanogens do not rely on glycine betaine only, and even one organism can have a cocktail of different compatible solutes. For example, at an external salt concentration of 1.77 M NaCl, Methanohalophilus portucalensis FDF1 synthesizes glycine betaine at a concentration of only 0.3 M, which is far too low to counterbalance the external osmolality. However, in addition, the cells accumulated -glutamate, N^--acetyl-ß-lysine, ß-glutamine, and potassium ions in concentrations of 0.2, 0.47, 0.20, and 0.61 M, respectively. Furthermore, it was shown that Methanosarcina thermophila accumulates -glutamate and other -amino acids and N-acetyl-ß-lysine, as well as glycine betaine, when grown at elevated salinities (32). A cocktail of different solutes can also be expected in M. mazei Gö1 since we measured only 16 mM internal glycine betaine at 1,000 mM NaCl. The determination of the composition and dynamics of the internal solute pool in M. mazei Gö1 is a challenge for future studies.

Whether the salt-induced glycine betaine uptake activity is solely due to the activity of Ota or is also due to additional, different transport systems remains to be elucidated. M. thermophila and Methanohalophilus portucalensis were shown by inhibitor studies in cell suspensions to have secondary, ion-coupled glycine betaine transporters (18, 25). Our experiments could be interpreted to indicate a primary transport mechanism. In any case, these conclusions are based on inhibitor studies and therefore have to be considered with caution. Anyway, the molecular data revealed a potential, salt-induced primary transporter for glycine betaine. However, one or more additional secondary transporters are not excluded, and future studies are aimed at identifying other transporters for compatible solutes in M. mazei Gö1.

Ota is a typical member of the ABC-type transporters which catalyze a primary, ATP-dependent transport. Because highest similarities were found to glycine betaine transporters of various bacteria (Table 1), it is most likely that Ota of M. mazei Gö1 also catalyzes glycine betaine transport. The overall genetic organizations and the properties of the deduced subunits of glycine betaine transporters in bacteria (14, 34) and of Ota of M. mazei Gö1 are very similar, indicating a common ancestor of these transporters. Like gram-positive bacteria, methanogenic archaea have no periplasm, and, therefore, the "periplasmic" substrate binding protein has to be anchored to the cell membrane. Currently, two ways are envisaged for anchoring substrate binding proteins in archaea. One is by the hydrophobic N terminus of the mature protein, and another is by lipoylation of the N-terminal cysteine, thus producing a lipoprotein which is anchored in the membrane via its lipid anchor (1, 6, 36). Both features are present in Ota of M. mazei Gö1 (Fig. 6). The conserved cysteine residue preceded by a hydrophobic stretch in OtaC of M. mazei Gö1, PhoX and DppA of Archaeoglobus fulgidus (16), and MalE of Thermococcus litoralis (10) as well as those in hypothetical substrate binding proteins of ABC-type transporters from A. fulgidus and Pyrococcus horikoshii (13, 16) favors a membrane anchoring via a lipid anchor, but this assumption has to be verified by biochemical analyses.

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FIG. 6. N-terminal sequence alignments of putative substrate binding proteins of Archaea. The stretch of hydrophobic amino acids is underlined; the conserved cysteine, target for lipoylation, is given in boldface type. Numbers denote gene designations given in the databases. Abbreviations: MM, M. mazei Gö1; AF, A. fulgidus; PH, P. horikoshii (shinkaj) OT3; TL, T. litoralis.

Future studies using Ota as a model system will help in understanding the molecular basis of salt adaptation and regulation of stress genes in methanoarchaea.

 
This work was supported by grants from the Deutsche Forschungsgemeinschaft and the Ministry of Science and Culture of the state of Lower Saxony.

We are grateful to M. Hube for experimental assistance.

 
* Corresponding author. Mailing address: Lehrstuhl für Mikrobiologie der LMU München, Maria-Ward-Str. 1a, 80638 Munich, Germany. Phone: (49) 89 2180 6126. Fax: (49) 89 2180 6127. E-mail: v.mueller{at}lrz.uni-muenchen.de.

Top Abstract Introduction Materials and Methods Results Discussion References

 

1
1. Albers, S. V., W. N. Konings, and A. J. Driessen. 1999. A unique short signal sequence in membrane-anchored proteins of Archaea. Mol. Microbiol. 31:1595-1596.[CrossRef][Medline]
2
2. Blaut, M., and G. Gottschalk. 1982. Effect of trimethylamine on acetate utilization by Methanosarcina barkeri. Arch. Microbiol. 133:230-235.[CrossRef]
3
3. Blaut, M., and G. Gottschalk. 1984. Coupling of ATP synthesis and methane formation from methanol and molecular hydrogen in Methanosarcina barkeri. Eur. J. Biochem. 141:217-222.[Medline]
4
4. Boone, D. R., and R. A. Mah. 1989. Methanosarcinaceae, p. 2198-2205. In J. G. Holt (ed.), Bergey's manual of systematic bacteriology. Williams & Wilkins, Baltimore, Md.
5
5. Brown, A. D. 1976. Microbial water stress. Bacteriol. Rev. 40:803-846.[Free Full Text]
6
6. Diez, J., K. Diederichs, G. Greller, R. Horlacher, W. Boos, and W. Welte. 2001. The crystal structure of a liganded trehalose/maltose-binding protein from the hyperthermophilic archaeon Thermococcus litoralis at 1.85 Å. J. Mol. Biol. 305:905-915.[CrossRef][Medline]
7
7. Feinberg, A. P., and B. Vogelstein. 1983. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 132:6-13.[CrossRef][Medline]
8
8. Galinski, E. A., and H. G. Trüper. 1994. Microbial behaviour in salt-stressed ecosystems. FEMS Microbiol. Rev. 15:95-108.
9
9. Hippe, H., D. Caspari, K. Fiebig, and G. Gottschalk. 1979. Utilization of trimethylamine and other methyl compounds and methane formation by Methanosarcina barkeri. Proc. Natl. Acad. Sci. USA 76:494-498.[Abstract/Free Full Text]
10
10. Horlacher, R., K. B. Xavier, H. Santos, J. DiRuggiero, M. Kossmann, and W. Boos. 1998. Archaeal binding protein-dependent ABC transporter: molecular and biochemical analysis of the trehalose/maltose transport system of the hyperthermophilic archaeon Thermococcus litoralis. J. Bacteriol. 180:680-689.[Abstract/Free Full Text]
11
11. Jones, W. J., D. P. Nagle, and W. B. Whitman. 1987. Methanogens and diversity of archaebacteria. Microbiol. Rev. 51:135-177.[Free Full Text]
12
12. Jung, K., B. Tjaden, and K. Altendorf. 1997. Purification, reconstitution, and characterization of KdpD, the turgor sensor of Escherichia coli. J. Biol. Chem. 272:10847-10852.[Abstract/Free Full Text]
13
13. Kawarabayasi, Y., M. Sawada, H. Horikawa, Y. Haikawa, Y. Hino, S. Yamamoto, M. Sekine, S. Baba, H. Kosugi, A. Hosoyama, Y. Nagai, M. Sakai, K. Ogura, R. Otsuka, H. Nakazawa, M. Takamiya, Y. Ohfuku, T. Funahashi, T. Tanaka, Y. Kudoh, J. Yamazaki, N. Kushida, A. Oguchi, K. Aoki, and H. Kikuchi. 1998. Complete sequence and gene organization of the genome of a hyper-thermophilic archaebacterium, Pyrococcus horikoshii OT3 (supplement). DNA Res. 5:147-155.[CrossRef][Medline]
14
14. Kempf, B., and E. Bremer. 1995. OpuA, an osmotically regulated binding protein-dependent transport system for the osmoprotectant glycine betaine in Bacillus subtilis. J. Biol. Chem. 270:16701-16713.[Abstract/Free Full Text]
15
15. Kempf, B., and E. Bremer. 1998. Uptake and synthesis of compatible solutes as microbial stress responses to high-osmolality environments. Arch. Microbiol. 170:319-330.[CrossRef][Medline]
16
16. Klenk, H.-P., R. A. Clayton, J.-F. Tomb, O. White, K. E. Nelson, K. A. Ketchum, R. J. Dodsen, M. Gwinn, E. K. Hickey, J. D. Peterson, D. L. Richardson, A. R. Kerlavage, D. E. Graham, N. C. Kyrpides, R. D. Fleischmann, J. Quackenbush, N. H. Lee, G. G. Sutton, S. Gill, E. F. Kirkness, B. A. Dougherty, K. McKenny, M. D. Adams, B. Loftus, S. Peterson, C. I. Reich, L. K. McNeil, J. H. Badger, A. Glodek, L. Zhou, R. Overbeek, J. D. Gocayne, J. F. Weidman, L. McDonald, T. Utterback, M. D. Cotton, T. Spriggs, P. Artiach, B. P. Kaine, S. M. Sykes, P. W. Sadow, K. P. D'Andrea, C. Bowman, C. Fujii, S. A. Garland, T. M. Mason, G. J. Olsen, C. M. Fraser, H. O. Smith, C. R. Woese, and J. C. Venter. 1997. The complete genome sequence of the hyperthermophilic, sulphate-reducing archaeon Archaeoglobus fulgidus. Nature 390:364-370.[CrossRef][Medline]
17
17. Lai, M. C., and R. P. Gunsalus. 1992. Glycine betaine and potassium ion are the major compatible solutes in the extremely halophilic methanogen Methanohalophilus strain Z7302. J. Bacteriol. 174:7474-7477.[Abstract/Free Full Text]
18
18. Lai, M. C., T. Y. Hong, and R. P. Gunsalus. 2000. Glycine betaine transport in the obligate halophilic archaeon Methanohalophilus portucalensis. J. Bacteriol. 182:5020-5024.[Abstract/Free Full Text]
19
19. Lai, M. C., K. R. Sowers, D. E. Robertson, M. F. Roberts, and R. P. Gunsalus. 1991. Distribution of compatible solutes in the halophilic methanogenic archaebacteria. J. Bacteriol. 173:5352-5358.[Abstract/Free Full Text]
20
20. Maestrojuan, G. M., J. E. Boone, R. A. Mah, J. A. G. F. Menaia, M. S. Sachs, and D. R. Boone. 1992. Taxonomy and halotolerance of mesophilic Methanosarcina strains, assignment of strains to species, and synonymy of Methanosarcina mazei and Methanosarcina frisia. Int. J. Syst. Bacteriol. 42:561-567.[Abstract/Free Full Text]
21
21. Marmur, J. 1961. A procedure for the isolation of deoxyribonucleic acid from microorganisms. J. Mol. Biol. 3:208-218.
22
22. Müller, V., M. Blaut, and G. Gottschalk. 1993. Bioenergetics of methanogenesis, p. 360-406. In J. G. Ferry (ed.), Methanogenesis. Chapman & Hall, New York, N.Y.
23
23. Oren, A. 1999. Bioenergetic aspects of halophilism. Microbiol. Mol. Biol. Rev. 63:334-348.[Abstract/Free Full Text]
24
24. Poolman, B., and E. Glaasker. 1998. Regulation of compatible solute accumulation in bacteria. Mol. Microbiol. 29:397-407.[CrossRef][Medline]
25
25. Proctor, L. M., R. Lai, and R. P. Gunsalus. 1997. The methanogenic archaeon Methanosarcina thermophila TM-1 possesses a high-affinity glycine betaine transporter involved in osmotic adaptation. Appl. Environ. Microbiol. 63:2252-2257.[Abstract]
26
26. Roberts, M. F. 2000. Osmoadaptation and osmoregulation in archaea. Front. Biosci. 5:D796-D812.[Medline]
27
27. Robertson, D. E., D. Noll, M. F. Roberts, J. A. G. F. Menaia, and D. R. Boone. 1990. Detection of the osmoregulator betaine in methanogens. Appl. Environ. Microbiol. 56:563-565.[Abstract/Free Full Text]
28
28. Rottenberg, H. 1979. The measurement of membrane potential and pH in cells, organelles and vesicles. Methods Enzymol. 55:547-569.[Medline]
29
29. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
30
30. Schmidt, K., S. Liaanen-Jensen, and H. G. Schlegel. 1963. Die Carotinoide der Thiorodaceae. Arch. Mikrobiol. 46:117-126.
31
31. Sowers, K. R., and R. P. Gunsalus. 1988. Adaptation for growth at various saline concentrations by the archaebacterium Methanosarcina thermophila. J. Bacteriol. 170:998-1002.[Abstract/Free Full Text]
32
32. Sowers, K. R., and R. P. Gunsalus. 1995. Halotolerance in Methanosarcina spp.: role of N-acetyl-ß-lysine, -glutamate, glycine betaine, and K⁺ as compatible solutes for osmotic adaptation. Appl. Environ. Microbiol. 61:4382-4388.[Abstract]
33
33. Sowers, K. R., D. E. Robertson, D. Noll, R. P. Gunsalus, and M. F. Roberts. 1990. N-acetyl-ß-lysine: an osmolyte synthesized by methanogenic archaebacteria. Proc. Natl. Acad. Sci. USA 87:9083-9087.[Abstract/Free Full Text]
34
34. van der Heide, T., and B. Poolman. 2000. Osmoregulated ABC-transport system of Lactococcus lactis senses water stress via changes in the physical state of the membrane. Proc. Natl. Acad. Sci. USA 97:7102-7106.[Abstract/Free Full Text]
35
35. Ventosa, A., J. J. Nieto, and A. Oren. 1998. Biology of moderately halophilic aerobic bacteria. Microbiol. Mol. Biol. Rev. 62:504-544.[Abstract/Free Full Text]
36
36. von Heijne, G. 1986. The distribution of positively charged residues in bacterial inner membrane proteins correlates with the membrane topology. EMBO J. 5:3021-3027.[Medline]
37
37. Wood, J. M. 1999. Osmosensing by bacteria: signals and membrane-based sensors. Microbiol. Mol. Biol. Rev. 63:230-262.[Abstract/Free Full Text]

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Applied and Environmental Microbiology, May 2002, p. 2133-2139, Vol. 68, No. 5
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.5.2133-2139.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

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