Accumulation of compatible solutes, by uptake or de novo synthesis, enables bacteria to reduce the difference between osmotic potentials of the cell cytoplasm and the extracellular environment. To examine this process in the halophilic and halotolerant methanogenic archaebacteria, 14 strains were tested for the accumulation of compatible solutes in response to growth in various extracellular concentrations of NaCl. In external NaCl concentrations of 0.7 to 3.4 M, the halophilic methanogens accumulated K+ ion and low-molecular-weight organic compounds. j-Glutamate was detected in two halotolerant strains that grew below 1.5 M NaCl. Two unusual ,8-amino acids, N.-acetyl-13-lysine and 1-glutamine (3-aminoglutaramic acid), as well as L-a-glutamate were compatible solutes among all of these strains. De novo synthesis of glycine betaine was also detected in several strains of moderately and extremely halophilic methanogens. The zwitterionic compounds (j3-glutamine, N,-acetyl-I3-lysine, and glycine betaine) and potassium were the predominant compatible solutes among the moderately and extremely halophilic methanogens. This is the first report of j3-glutamine as a compatible solute and de novo biosynthesis of glycine betaine in the methanogenic archaebacteria.Prokaryotic and eukaryotic microorganisms have evolved mechanisms to adapt to osmotic stress ranging from low solute concentrations in spring water to saturated solutes in salt brines (38). In environments where the extracellular solute concentration exceeds that of the cell cytoplasm, microorganisms accumulate low-molecular-weight organic compounds, known as compatible solutes, that enable them to minimize water loss and maintain cell turgor pressure (42). These compounds function by reducing the difference between the osmotic potentials of the cell cytoplasm and the extracellular environment, thus maintaining a constant turgor pressure, and by protecting enzymes from the low water activity caused by solute accumulation (4, 9, 16, 37). Compatible solutes in eukaryotic organisms include proline and betaine in plants (15,35), glycerol in lower fungi (22), and polyols, proline, and amino acids in marine algae (39). Eubacteria also contain a broad spectrum of osmotically active solutes, including potassium, proline, glutamic acid, glutamine, -y-aminobutyric acid, alanine, and glycine betaine (9, 40).Several types of compatible solute have been identified in archaebacteria. The predominant compatible solute in extremely halophilic archaebacteria such as Halobacterium and Halobium species is potassium (11); organic compatible solutes do not accumulate to a large extent in these organisms. Methanogenic archaebacteria, however, accumulate 1-amino acids as compatible solutes in response to external NaCl concentrations. ,B-Glutamate accumulates in thermophilic strains of methanococci as well as the mesophile Methanogenium cariaci (26, 28), and the 13-amino acid derivative N8-acetyl-13-lysine accumulates in Methanosarcina thermophila and M. cariaci (30). Glycine betaine also...

Methanosarcina thermophila, a nonmarine methanogenic archaebacterium, can grow in a range of saline concentrations. At less than 0.4 M NaCi, Ms. thermophila accumulated glutamate in response to increasing osmotic stress. At greater than 0.4 M NaCI, this organism synthesized a modified (3-amino acid that was identified as N6-acetyl-.8-lysine by NMR spectroscopy and ion-exchange HPLC.This (-amino acid derivative accumulated to high intracellular concentrations (up to 0.6 M) in Ms. thermophila and in another methanogen examined-Methanogenium cariaci, a marine species. The compound has features that are characteristic of a compatible solute: it is neutrally charged at physiological pH and it is highly soluble. When the cells were grown in the presence of exogenous glycine betaine, a physiological compatible solute, N6-acetyl--lysine synthesis was repressed and glycine betaine was accumulated. N8-Acetyl-(3-lysine was synthesized by species from three phylogenetic families when grown in high solute concentrations, suggesting that it may be ubiquitous among the methanogens. The ability to control the biosynthesis of N'-acetyl-(3-lysine in response to extracellular solute concentration indicates that the methanogenic archaebacteria have a unique (3amino acid biosynthetic pathway that is osmotically regulated.Microorganisms can proliferate in a diverse range of saline concentrations from low saline environments such as freshwater lakes to saturated brines found in solar salterns (1, 2). Cell size and the intracellular water activity must remain relatively constant to maintain physiological processes. Eukaryotic and eubacterial microorganisms have evolved mechanisms that enable them to minimize water loss when the extracellular solute concentration exceeds that of the cell cytoplasm (1-3). The mechanism of this adaptation involves the uptake or synthesis of low molecular weight organic compounds known as physiological compatible solutes (4, 5). These compounds reduce the osmotic potential between the extracellular milieu and the cytoplasm and protect enzymes from the low water activity that results from solute accumulation.Methanogenic archaebacteria have been isolated from environments with NaCl concentrations ranging from <0.05 M for many nonmarine species to >4 M for halophilic species (6). Individual species can also adapt to a range of saline concentrations. Methanosarcina thermophila, which was isolated from a thermophilic sludge digestor, grows in medium containing 0.05-1.2 M NaCl (7-9), and the marine species Methanogenium cariaci (10) grows in medium containing 0.17-1.4 M NaCl (D.N. and M.F.R., unpublished results). Compatible solutes have been detected in methanogenic bacteria (11-13). Several species of marine methanogens synthesize ,-glutamate in response to external NaCl (11, 13). Methanogens have also been reported to accumulate glycine betaine if it is provided in the medium (12). However, the mechanism for adaptation to high osmotic stress has not been investigated. Here we report the structure of ...

The unusual compound j-aminoglutaric acid ("-glutamate) has been identified by 13C nuclear magnetic resonance spectroscopy in soluble extracts of marine methanogenic bacteria. We examined several methanogen species representing nine genera and found that a-glutamate occurred in methanococci and two methanogenium strains (Methanogenium cariaci JR1 and "Methanogenium anulus" AN9). The presence of this compound in the methanococci examined was further restricted to thermophilic members of the genus Methanococcus, including Methanococcus thermolithotrophicus strains, Methanococcus jannaschii, and "Methanococcus igneus." The two Methanogenium strains examined were mesophiles. Studies using Methanococcus thermolithotrophicus showed that levels of 1-glutamate in cells of that species were not affected by variation in growth temperature (40 to 65°C), N114+ (2 to 80 mM), Mg2e (10 to 50 mM), or K+ (2 to 10 mM) in the medium. In contrast, soluble pools of ,8-glutamate and L-a-glutamate (the other major free amino acid in all the methanococci) were proportional to NaCl levels in the growth medium. This dependence of 1-glutamate and L-a-glutamate concentrations on salt levels in the medium suggests that they function as osmolytes in these cells. P-Amino acids are relatively rare in nature (6, 12). One that has been detected at significant levels in marine sediments is P-aminoglutaric acid (,-glutamate).

Methanohalophilus strain FDF1, a member of the halophilic genus of methanogens, can grow over a range of external NaCl concentrations from 1.2 to 2.9 M and utilize methanol, trimethylamine, and dimethyl sulfide as substrates for methanogenesis. It produces the osmolytes glycine betaine, 13-glutamine, and NE-acetyl-13lysine with increasing external NaCl, but the relative ratio of these zwitterions depends primarily on the methanogenic substrate and less on the external osmolarity. When the cells are grown on methanol in defined medium, accumulation of glycine betaine predominates over the other zwitterionic solutes. The cells also synthesized a carbohydrate which was not detected in cells grown on trimethylamine. This negatively charged compound, identified as a-glucosylglycerate from the 13C and 'H chemical shifts, does not act as an osmoregulatory solute in the salt range 1.4 to 2.7 M in this methanogen as evidenced by its invariant intracellular concentration. 13CH30H-pulse/'2CH30H-chase experiments were used to determine half-lifes for these organic solute pools in the cells. L-ao-Glutamate showed a rapid loss of heavy isotope, indicating that L-aL-glutamate functions as a biosynthetic intermediate in these cells. Measurable turnover rates for both 13-glutamine, which acts as an osmolyte, and a-glucosylglycerate suggest that they function as metabolic intermediates as well. Molecules which function solely as osmolytes (glycine betaine and N-acetyl-13-lysine) showed a slower turnover consistent with their roles as osmotic solutes in Methanohalophilus strain FDF1.