TEXT

Top Abstract Text References

Microorganisms must be able to adapt to changes in the osmolarity of their environment. To adapt to these changes, bacteria accumulate some compounds, named compatible solutes, that confer protection against the deleterious effect of the low water activity (8). Moderately halophilic bacteria are defined as those which can grow optimally between 0.5 and 2.5 M salt (9). Among this heterogeneous group of microorganisms, Halomonas elongata has a very promising potential for use in biotechnology (13), and since it can grow at a very wide range of salinities (from 0.5 to 3 M NaCl in a minimal medium) (4), it is also a good model organism to study the molecular basis of prokaryotic osmoadaptation (14). To cope with changes in the salt concentration of the environment, H. elongata is able to synthesize ectoine and hydroxyectoine (5, 8) and to take up a variety of organic compounds which can serve as osmoprotectants when present externally (4). Accordingly, exogenous glycine betaine (called betaine hereafter), choline, and choline-O-sulfate have been demonstrated to play an osmoprotective role in H. elongata (4). Many organisms, including gram-positive (1, 15) and gram-negative (11) bacteria, as well as higher plants (12, 18), can generate betaine for osmoprotection by oxidation of choline. We have investigated the transport of choline and its conversion to the osmoprotectant compound betaine in the moderately halophilic bacterium H. elongata. The role of salinity and betaine in the regulation of this osmoprotective mechanism has also been investigated.

Effect of choline on the salinity growth range of H. elongata. H. elongata DSM 3043 (16) has been shown to be able to grow in minimal medium M63 in a wide range of salt concentrations in the absence of any added osmoprotectant (4). To test the effect of choline on the growth of H. elongata at different salinities, cells were inoculated in M63 (6) (with 20 mM glucose as sole carbon source) plus 0.5 to 4.0 M NaCl in the presence or absence of 1 mM choline, and the optical density at 600 nm was measured after 24 h of incubation at 37°C (Fig. 1). Choline stimulated the growth of H. elongata in minimal medium at salinities above the optimal concentration of 2 M NaCl, so that it extended the salinity range of growth to at least 3.5 M NaCl. It is noteworthy that the growth of H. elongata was also stimulated by choline at 0.5 M NaCl (Fig. 1). This result is consistent with our earlier observation that betaine was likewise stimulatory for this bacterium at all salinities, including the suboptimal concentration of 0.5 M NaCl (4). Thus, compared to a culture grown at 0.5 M NaCl, the growth rate of H. elongata can be increased by two seemingly contradictory additives: increased NaCl concentration up to 2 M NaCl or an osmoprotectant such as betaine or its precursor, choline. These observations present somewhat of a paradox, because these two additives have contradictory effects: increasing the NaCl concentration presumably increases osmotic stress, while the osmoprotectants are believed to alleviate the inhibitory effects of high osmolarity. Resolution of this apparent paradox may lie in the hypothesis that while H. elongata requires high concentrations of NaCl (up to 2 M) for some unknown biochemical reason(s), it is nevertheless under osmotic stress, even at 0.5 M NaCl, and addition of osmoprotectants relieves the inhibitory effects of high osmolarity.

View larger version (19K): [in this window] [in a new window]   FIG. 1.   Effect of choline on growth of H. elongata in M63-glucose-minimal medium with increasing salinity. Cultures were grown at 37°C in M63 containing the indicated concentrations of NaCl, and the optical density was determined after 24 h. Symbols: , M63 alone; , M63 with 1 mM choline. For every growth experiment, data are the averages of three different repeats which on no occasion varied by more than 5%. OD600, optical density at 600 nm.

Transport of choline and its intracellular conversion to betaine. To study the effect of salinity on the transport of choline, cells were incubated in M63 with 0.5 or 2.0 M NaCl and 10 µM [methyl-¹⁴C]choline. Samples (0.5 ml) were taken at different time intervals, and cells were collected after 2 min of centrifugation in a microcentrifuge. Cell pellets were frozen in liquid nitrogen. For each sample, 0.3 ml of supernatant was used for scintillation counting to measure the choline remaining in the culture medium. At 2.0 M NaCl, almost the entire exogenous choline was taken up within 5 min, while at 0.5 M NaCl, the cells required 10 min to transport 85% of the external choline, and transport was completed only after 20 min of incubation (data not shown).

View larger version (21K): [in this window] [in a new window]   FIG. 2.   Analysis of the conversion of choline to betaine in response to salinity. Choline and betaine in cell extracts were separated by thin-layer chromatography and visualized by autoradiography. Signals corresponding to choline (closed symbols) and betaine (open symbols) in the autoradiogram were analyzed by computing densitometry. Cells were grown at 0.5 (circles) or 2.0 (squares) M NaCl. Experiments (at both salt concentrations) are means of duplicates which on no occasion varied by more than 5%.

Effect of betaine in the intracellular oxidation of choline. The uptake and conversion of choline were assayed in the presence of betaine. Cells were incubated in M63 plus 0.5 or 2.0 M NaCl with 10 µM [methyl-¹⁴C]choline and 10 µM betaine, and the disappearance of choline from the medium and the rate of intracellular oxidation of choline to betaine were determined as described above. Although the initial uptake of choline was slightly delayed at both salt concentrations, choline was completely taken up by 10 min at 2.0 M NaCl or 20 min at 0.5 M NaCl (data not shown). After 10 min of incubation at 2.0 M NaCl, 34% of choline remained in the cytoplasm in the presence of betaine (Fig. 3), whereas no choline could be detected at the same time in the absence of betaine (Fig. 2). After 10 min of incubation at 0.5 M NaCl, 60% of nonoxidized choline remained in the cytoplasm in cells grown with betaine (Fig. 3), whereas at the same time less than 20% of the choline remained in the cytoplasm of cells grown without betaine (Fig. 2). Thus, whereas betaine exerted a slight inhibition of the uptake of choline in H. elongata (maybe due to a competition for the choline transporter), its major negative regulatory effect occurred at the oxidation of choline to betaine. This inhibition seems to be stronger at high salinity, where the almost instantaneous conversion of choline to betaine was delayed by betaine. However, the fact that both the transport and the oxidation of choline were faster at 2 M NaCl than at 0.5 M NaCl, regardless of the presence or absence of betaine, indicates that the salinity of the medium is the main factor that regulates the choline-betaine pathway of H. elongata under the experimental conditions used.

View larger version (21K): [in this window] [in a new window]   FIG. 3.   Effect of betaine on the intracellular oxidation of choline. H. elongata was grown in M63 containing 0.5 (circles) or 2.0 (squares) M NaCl and [methyl-14C]choline (closed symbols) and betaine (open symbols). Oxidation of choline was determined as described in the legend to Fig. 2. Experiments (at both salt concentrations) were repeated twice, and the data correspond to means which on no occasion varied by more than 5%.

Accumulation of betaine from choline in response to salt stress. The intracellular betaine level derived from choline was determined from cells grown in M63 containing 0.75 to 3 M NaCl, 10 mM [methyl-¹⁴C]choline, and 20 mM [³H]glucose as described previously (4) (Fig. 4). Because choline was converted rapidly to betaine (see above), the radioactivity contained in the cells was due to betaine. The accumulation of betaine by synthesis from choline was salt dependent and increased as the NaCl concentration was raised up to 2.5 M NaCl. At 3 M NaCl, the level of betaine accumulated was slightly lower than at 2.5 M NaCl, suggesting that the transport of choline, its transformation to betaine, or its retention at this high salinity was affected by an uncharacterized mechanism. Studies of the accumulation of betaine by a high-affinity transport system (4) are in good agreement with the data reported here. In both cases (with choline or betaine added externally), the levels of betaine accumulated in the cytoplasm at different salt concentrations were nearly identical, suggesting a common regulatory mechanism for the two systems.

View larger version (18K): [in this window] [in a new window]   FIG. 4.   Accumulation of betaine from choline by H. elongata in M63 at increasing osmolarities. Cells were grown overnight in the presence of [methyl-14C]choline and [3H]glucose. Samples were centrifuged and washed, and the accumulated radioactivity was measured by scintillation counting. Experiments (at both salt concentrations) were repeated twice, and the data correspond to means which on no occasion varied by more than 5%. conc, concentration.