Glycine Betaine Transport in the Obligate Halophilic Archaeon Methanohalophilus portucalensis

Abstract

Transport of the osmoprotectant glycine betaine was investigated using the glycine betaine-synthesizing microbe Methanohalophilus portucalensis (strain FDF1), since solute uptake for this class of obligate halophilic methanogenic Archaea has not been examined. Betaine uptake followed a Michaelis-Menten relationship, with an observed K_t of 23 μM and a V_max of 8 nmol per min per mg of protein. The transport system was highly specific for betaine: choline, proline, and dimethylglycine did not significantly compete for [¹⁴C]betaine uptake. The proton-conducting uncoupler 2,4-dinitrophenol and the ATPase inhibitor N,N-dicyclohexylcarbodiimide both inhibited glycine betaine uptake. Growth of cells in the presence of 500 μM betaine resulted in faster cell growth due to the suppression of the de novo synthesis of the other compatible solutes, α-glutamate, β-glutamine, and N^ɛ-acetyl-β-lysine. These investigations demonstrate that this model halophilic methanogen, M. portucalensis strain FDF1, possesses a high-affinity and highly specific betaine transport system that allows it to accumulate this osmoprotectant from the environment in lieu of synthesizing this or other osmoprotectants under high-salt growth conditions.

The Bacteria employ the adaptive strategy of accumulating a broad spectrum of osmotically active solutes, including potassium, proline, glutamic acid, glutamine, α-aminobutyric acid, ectosine, and betaine (4, 9–11, 15, 41); Eucarya accumulate glycerol, polyols, proline, betaine, and amino acids as compatible solutes (19, 24–26, 42). Of the Archaea examined thus far, the predominant compatible solute in the extremely halophilic Euryarchaeota, such as the Halobacterium and Halobium species, is potassium (16). The methanogenic Archaea also accumulate potassium but to a much lesser extent. In addition they can accumulate α-glutamate, betaine, and the β-amino acids N^ɛ-acetyl-β-lysine, β-glutamine, and β-glutamate as compatible solutes in response to high external NaCl levels (20, 21, 36, 39, 40). Ability to uptake these solutes has only been examined in one methanogen, Methanosarcina thermophila TM-1 (33). This nonhalophile possesses a single high-affinity betaine transporter whose velocity varies over eightfold depending on the osmotic strength of the medium and the availability of betaine in the environment. However, glutamate was not taken up. Little is known about solute uptake in any other methanogenic Archaea including those of the recently described obligate halophilic class of methanogens.

The moderate and extreme halophilic methanogens including Methanohalophilus portucalensis strain FDF1 and the Methanohalophilus strains SF1, SF2, SD1, Ret-1, Z7301, Z7401, SLP, Z7302, and Z7304 grow optimally within the salt range of 1.2 to 4.3 M NaCl (2, 27). In response to increasing external osmotic strength, they can accumulate β-glutamine, N^ɛ-acetyl-β-lysine, α-glutamate, and betaine as compatible solutes (21). In M. portucalensis strain FDF1, betaine is synthesized de novo from glycine by three successive methylation reactions where S-adenosyl-methionine is the methyl donor (22). Interestingly, neither sarcosine nor dimethylglycine can be used in place of betaine when the solute is provided exogenously.

Since ability to take up betaine has not been examined in any of the obligate hypersaline-loving methanogenic Archaea, M. portucalensis strain FDF1 was used as a model system to examine this process. This organism is shown to contain a specific high-affinity betaine transporter for accumulating this compatible solute in lieu of synthesizing it de novo.

Effect of exogenous betaine on cell growth.

The halophilic methanogen M. portucalensis can synthesize betaine and apparently accumulate it if betaine is present in the culture medium (21). However, little is known about how the cell acquires this solute or whether addition of betaine to cultures affects the rate of cell growth. To examine the latter question, M. portucalensis was grown in the presence and absence of betaine in a mineral medium containing 2.1 M NaCl where trimethylamine was the methanogenic substrate (Fig. 1). The specific cell growth rate was increased by 40% from 0.05 (cell doubling time of ca. 13 h) to 0.07 (cell doubling time of 10 h) by addition of either 0.5 or 1 mM betaine. However, betaine addition did not affect the final cell density of the culture. In prior betaine turnover studies performed with M. portucalensis by nuclear magnetic resonance methods, betaine was shown to have a relatively long half-life (ca. 32 h) inside the cell (35). Other studies showed that betaine was not used as a methanogenic substrate unlike mono-, di-, and trimethylamine (21). Betaine is apparently used solely as a compatible solute.

FIG. 1.

Effect of exogenous betaine on M. portucalensis strain FDF1 cell growth. Cells were grown at 37°C with 2.05 M NaCl and 20 mM trimethylamine. Symbols: squares, 1.0 mM betaine; circles, 0.5 mM betaine; triangles, no added betaine. Absorbance was measured at 540 nm. M. portucalensis strain FDF1 was obtained (OCM59) from R. Mah (2, 27). The mineral medium defined by Lai (21) was used for cell growth. Trimethylamine (20 mM) was the sole carbon and energy source. Sealed serum bottles were inoculated with a 0.5% volume of late-exponential-phase culture using a N₂-flushed syringe. Cells were grown at 37°C as previously described (21). Cell growth rates were monitored by removing 1 ml of the culture with a N₂-flushed syringe, placing it into a 1-cm cuvette, and measuring the optical density of the culture at 540 nm.

Glycine betaine transport.

To determine if M. portucalensis possesses an active transport system for betaine, [¹⁴C]betaine accumulation was followed using a standardized anaerobic betaine uptake assay (Fig. 2). Uptake was linear during the first 10 min of the experiment except when very low levels of [¹⁴C]betaine were used (data not shown). The kinetic parameters of betaine transport by M. portucalensis were determined with cells grown at 2.1 M NaCl using substrate concentrations from 5 μM to 1 mM. The accumulation was saturable. Initial rates were calculated, and double-reciprocal Lineweaver-Burk plots gave a straight line, thus indicating that betaine uptake follows kinetics characteristic of the presence of a single transporter. The analysis of the Lineweaver-Burk plot revealed a K_t value of 23 μM (Fig. 2). A maximum initial rate of betaine uptake (V_max) of 8.0 nmol min⁻¹ mg of protein⁻¹ was achieved.

FIG. 2.

Kinetics of glycine betaine transport by M. portucalensis strain FDF1. Cells were grown in minimal medium with 2.05 M NaCl at 37°C. Transport rates were measured with the indicated concentration of [¹⁴C]betaine. The points shown are the mean values of triplicate assays from three different experiments. The insert shows the Lineweaver-Burk plot of the initial rates of glycine betaine transport. For the assays, mid-exponential growth-phase cells (optical density at 540 nm [OD₅₄₀] of 0.33) were harvested anaerobically by introducing the serum bottles into a Coy Environmental Chamber (Coy Laboratory, Ann Arbor, Mich.). The cell culture (25 ml) was transferred into Oak-Ridge centrifuge bottles which were then sealed and removed from the chamber for centrifugation at 10,000 × g for 10 min at room temperature. Each bottle was reintroduced into the Coy chamber and opened, and the cell pellet was resuspended with 4.95 ml anaerobic HP buffer containing the indicated concentration of NaCl. The resulting cell suspension was typically 0.5 mg of protein/ml. HP buffer was identical in composition to the mineral medium used for cell growth except that NaCl and trimethyl amine were omitted (21): NaCl and trimethyl amine were added at the concentrations indicated for each experiment. Individual [¹⁴C]betaine transport experiments were initiated inside the Coy chamber by adding 50 μl of a [¹⁴C]-labeled betaine solution at the indicated concentration (from 5 μM to 1 mM) to 10-ml serum vials that contained 4.95 ml of cell suspension (ca. 2.5 mg of protein). The serum vial was sealed with a butyl rubber stopper, removed from the Coy chamber, and incubated in a 37°C water bath. At each indicated time point, a 0.5-ml sample was removed with a N₂-flushed syringe and filtered using a 13-mm-diameter, 0.45-μm-pore-size filter (type HA; Millipore Corp., Bedford, Mass.). The filter was then quickly washed at room temperature with 1 ml of a solution containing NaCl at the same concentration as in the HP buffer. Filters were solubilized in scintillation vials containing 3 ml of Fluoransafe 2 scintillation fluid (Merck Co., Rahway, N.J.) for 36 h, and the radioactivity was determined using a Wallac 1410 LSC liquid scintillation spectrometer (Pharmacia Co., Piscataway, N.J.). The first aliquot was generally taken 4 min after the addition of [¹⁴C]betaine and was used as the initial time point. Since betaine uptake remained linear in most cases for an additional 4 to 10 min, the transport rates were easily determined for the various conditions. Methyl [methyl-¹⁴C]-labeled glycine betaine was prepared from choline (specific activity, 54 Ci/mol) obtained from Du Pont NEN (Wilmington, Del.) as described by Ikuta et al. (14). Glycine, glycine betaine, sarcosine, choline, N,N-dimethylglycine, DNP, and DCCD were purchased from Sigma Chemical Co.

Specificity of the betaine uptake system.

Betaine transport was also investigated in the presence of potential substrates of the uptake system. A 20-fold molar excess of each unlabeled compound was added to the assay mixture containing [¹⁴C]betaine as a competitor of betaine uptake. Whereas unlabeled betaine inhibited [¹⁴C]betaine transport by 94%, proline, a common bacterial compatible solute, did not (Table 1). Choline, a precursor of betaine in Escherichia coli, Rhizobium meliloti, and other Bacteria (8, 23), was also unable to compete for betaine uptake. In separate experiments we also tested whether [¹⁴C]choline could be taken up by M. portucalensis: it was not (data not shown). Both glycine and sarcosine inhibited the [¹⁴C]betaine transport by 26 and 29%, respectively (Table 1). These data are in contrast to studies with E. coli (30) and Ectothiorhodospira halochloris (31) where neither glycine nor sarcosine (or N,N-dimethylglycine) inhibited betaine transport. The de novo biosynthetic pathway of betaine in M. portucalensis occurs by successive methylation reactions of glycine to give sarcosine, N,N-dimethylglycine, and then betaine (22, 34). N,N-dimethylglycine did not inhibit [¹⁴C]betaine transport (Table 1). The fact that neither sarcosine nor N,N-dimethylglycine added to M. portucalensis cell cultures could be used as a source of betaine (22) suggests that these molecules are not recognized by the betaine uptake system.

TABLE 1.

Effect of potential competitors and metabolic inhibitors on methyl [methyl-¹⁴C]-glycine betaine transport by M. portucalensis strain FDF1

Competitor or inhibitor (concn)	% Inhibitiona
Competitors
None	0
Proline (2 mM)	3
Choline (2 mM)	−1
Dimethylglycine (2 mM)	1
Sarcosine (2 mM)	26
Glycine (2 mM)	29
Inhibitors
DCCD (200 mM)	86
DNP (10 mM)	97
Sodium azide (1 mM)	−6

^aThe data are expressed in percent reduction from the uninhibited betaine uptake rate. Values are the averages of the results of two different experiments where the variation was less than 5%. Uptake is expressed in nanomoles per milligram of cell protein. Cells were preincubated for 30 min at 37°C in the presence of the indicated inhibitor before the addition of [¹⁴C]betaine (33). Cells were pregrown at 37°C in a medium containing 2.05 M NaCl. For the competitor experiments, a mixture of labeled betaine (100 μM) and unlabeled competitor was added to the suspended cells (e.g., 20-fold molar excess). The substrate concentration was four times the K_t value so that weak competition may not have been observed. For the inhibitor studies, the indicated compound was added 30 min prior to the initiation of the assay. For some assays, the initial rate of glycine betaine uptake was calculated from the amount of radioactive glycine betaine accumulated by the cells in 4 to 10 min. During this period, uptake was linear with time. Protein was determined by the Biuret method (13) using bovine serum albumin (Sigma Chemical Co., St. Louis, Mo.) as the standard. 

Energy dependency of betaine transport.

The high intracellular betaine concentration (22) suggests that betaine uptake must occur against an intracellular concentration gradient. When transport was examined without an added substrate (i.e., 20 mM trimethylamine) present, the betaine uptake rate was reduced about twofold (data not shown). The energy substrate for betaine uptake was clearly not depleted in the cells, suggesting that the cells contained sufficient amounts of trimethylamine to fuel the uptake process. The metabolic inhibitors N,N-dicyclohexylcarbodiimide (DCCD) and 2,4-dinitrophenol (DNP) each severely impaired betaine uptake, whereas sodium azide did not (Table 1). DCCD, an ATP synthase inhibitor, can decrease the rate of electron transport (6, 12, 37). When tested at a concentration of 200 μM, it inhibited betaine uptake by 86%. Since azide did not, there may be other cellular targets for DCCD. DNP, which may collapse the proton motive force, almost completely abolished betaine uptake (ca. 97%).

Comparison of betaine uptake in M. portucalensis with that in other Archaea and Bacteria.

Although a number of moderately to extremely halophilic methanogenic Archaea have been isolated from hypersaline environments, little was known about their ability to take up osmoprotectants. The present studies demonstrate the existence of a high-affinity transport system for betaine accumulation by M. portucalensis which can grow optimally in the salt range of 1.2 to 2.9 M NaCl. The betaine uptake system of M. portucalensis, with a K_t of 23 μM, is similar to the high-affinity transport system of Methanosarcina thermophila TM-1 (10 μM) (33) and is comparable in affinity to the betaine transport systems in Bacteria such as E. coli, Bacillus subtilis, Staphylococcus aureus, Lactococcus lactis, Listeria monocytogenes, Rhizobium meliloti and Corynebacterium glutamicam (1, 3, 7, 17, 18, 28–30, 32, 38). Interestingly, the betaine transporter of M. portucalensis exhibits a velocity eightfold higher than that of M. thermophila, while the K_t is about twofold weaker than that observed with the Methanosarcina strain (33).

Like betaine transport systems in the Bacteria that are energy dependent, betaine uptake in the Euryarchaeota member M. portucalensis also requires a driving force (presumably membrane ion gradients). For E. coli, betaine transport by ProP is impaired by DNP but is only slightly effected by DCCD: this demonstrates that an electrochemical proton gradient, generated by respiration, is the main driving force for betaine transport. ATP, rather than an ion gradient, is used as an energy source for the E. coli ProU uptake system (5). Related studies with E. halochloris showed that transport of glycine betaine might be driven by the electrochemical proton gradient generated by anaerobic photosynthesis (31). Our inhibitor tests showed that betaine transport was reduced 86% by DCCD and 97% by DNP. A Na⁺-stimulated ATPase activity has been detected in membrane preparations of the halotolerant methanogen M. halophilus (37). The process of methane formation is obligatorily coupled to the generation of primary proton and primary sodium ion gradients. Moreover, both proton gradient-driven ATP synthesis with A₁A_o-type ATPase and sodium-driven ATP synthesis by an analogous ATPase have been demonstrated in methanogens (6). The ATPase is specifically inhibited by sodium azide (6). Betaine transport in M. portucalensis was not affected by the inhibitor sodium azide (Table 1), which suggests that sodium-driven ATP synthesis may not be involved in this transport system. Recent studies with M. thermophila strain TM-1 suggest that betaine transport is H⁺- and/or Na⁺-driven, since transport was inhibited by several types of protonophores and sodium ionophores (33).

It is proposed that betaine accumulation in the moderately and extremely obligate halophilic methanogenic Euryarchaeota occurs by betaine transporters similar to that of M. portucalensis. In this microbe, osmolyte choice occurs in a hierarchical manner whereby betaine is taken up in preference to de novo synthesis of betaine, α-glutamate, β-glutamine, and N^ɛ-acetyl-β-lysine (21). This study also demonstrates that this class of obligate halophilic methanogens differs significantly from other obligate halophilic Archaea (i.e., the Halobacteriales) that use high internal salt concentrations (i.e., high molar amounts of potassium) to overcome osmotic stress.