INTRODUCTION

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All microorganisms have to cope with fluctuations in the osmolarity of their environment. The mechanisms of osmoregulation are very similar in all living organisms (plants, animals, and bacteria). In response to the elevated osmolarity of their environment, they accumulate, at relatively high intracellular concentrations, inorganic ions and neosynthesized low-molecular-mass organic molecules called compatible solutes. Amino or imino acids (glutamate, proline, pipecolic acid [PIP], and ectoine), carbohydrates (trehalose, sucrose, and glycerol) and methylated onium compounds (glycine betaine [GB] and dimethylsulfoniopropionate [DMSP]) are the most frequently accumulated compatible solutes (6, 8, 10, 19, 37, 42). After a sudden decrease in osmolarity, accumulated compatible compounds can be liberated into the surrounding environment and subsequently used as osmoprotectants by the same or other organisms if they are under a hyperosmotic stress (25, 27). Such compounds that are able to improve growth of cells under inhibitory osmolarities are thus called osmoprotectants. Hence, in natural environments, the concept of an osmoprotectant supposes an ecological cycle in which the compatible solutes are shuttled from producers to consumers exposed to hyperosmotic constraint. Several osmoprotectants produced by plants are therefore of prime environmental interest for soil bacteria. Osmoprotection of the soil bacterium Sinorhizobium meliloti has been reported to be effective with a variety of compounds: GB; GB derivatives or analogues, such as proline betaine, -butyrobetaine, trigonelline, dimethylglycine, and DMSP (5, 11, 22, 34); ectoine (39); sucrose (16); and some disaccharides derived from plant polymers (15).

PIP is a nonprotein imino acid the occurrence or accumulation of which has already been reported in several organisms: animals (1, 2, 7, 21, 43); microbes like Neurospora crassa (31), Pseudomonas (3, 4, 9, 26, 32, 33), and Brevibacterium ammoniagenes (13); and plants (12, 35, 36, 38). L-PIP and other amino acids added to soil and rhizosphere soil of various plants are readily oxidized (17, 18). Recently, PIP was isolated from seeds of Cycas circinalis and Phaseolus vulgaris (23). Hence, under a hypoosmotic stress, after cell decay, or during the process of seed germination, PIP could be released by these organisms in the rhizosphere and could serve as a possible osmoprotectant for osmotically stressed cells of soil bacteria. The osmoprotective capacity of PIP has not been studied extensively in bacteria, and to our knowledge, it was examined only in Escherichia coli (14). Only the L isomer of PIP exerts a beneficial effect on growth of this bacterium under stressing conditions.

In S. meliloti, the mechanism of osmoprotection depends on the nature of the osmoprotectant present in its environment. While betaines and other methylated onium compounds are accumulated within stressed cells of S. meliloti (5, 34, 39), all of the other osmoprotectants supplied to this bacterium are catabolized even at elevated osmolarities. Specific attention has been paid to ectoine (39), sucrose (16), and other osmoprotective disaccharides (15) which are readily metabolized and hence are unable to counteract by themselves any decrease in intracellular water activity. The question raised by such a behavior was partially answered by the observations that increased intracellular osmolarity was provided by an enhancement of the intracellular levels of endogenously neosynthesized compatible solutes in S. meliloti (glutamate, the dipeptide N-acetylglutaminylglutamine amide [NAGGN], and trehalose [15, 16, 39, 40]). Hence, the osmoregulation strategy in S. meliloti appears to be atypical of those of other species in the bacterial kingdom.

To add further evidence to S. meliloti osmoregulating mechanisms, we have investigated the osmoprotective effect and the fate of PIP in osmotically stressed cells. The differential effect of D- and L enantiomers of the imino acid was analyzed.

	MATERIALS AND METHODS

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Bacterial strain and growth conditions. S. meliloti 102F34, a standard laboratory strain, was grown aerobically at 30°C overnight in mannitol-salts-yeast extract (MSY) medium until the late exponential phase of growth was reached (16). Bacteria were inoculated at a final concentration of 1% (vol/vol) into minimal LAS medium containing 10 mM sodium L-aspartate, 10 mM sodium DL-lactate, and the same mineral salts as in MSY medium. The osmolarity of the medium was increased by the addition of 0.5 M NaCl or an isoosmotic concentration of the nonelectrolyte mannitol (0.8 M). D-, L-, and DL-PIP, DL-lactate, and L-aspartate stock solutions (Sigma Chimie, Saint-Quentin Fallavier, France) were sterilized by filtration. Bacterial growth was monitored by optical density at 570 nm (OD₅₇₀) measurements. The protein contents of the cultures were determined by the method of Lowry et al. (24).

Extraction of cellular solutes. The pellet of freshly harvested and washed cells was extracted twice with 80% (vol/vol) ethanol under vigorous magnetic stirring at room temperature for 30 min. After centrifugation, the supernatant (ethanol-soluble fraction or ESF) was evaporated to dryness at 40°C and redissolved in distilled water or deuterated water for ¹³C nuclear magnetic resonance (NMR) analysis as described previously (16). The pellet (ethanol-insoluble fraction or EIF) contained the intracellular macromolecular components and cell envelopes.

Production of L-[¹⁴C]PIP. L-[U-¹⁴C]PIP was synthesized by B. ammoniagenes ATCC 6872 from L-[U-¹⁴C]lysine monochloride (11.1 GBq/mmol; Amersham, Les Ullis, France). The labeled lysine and 1 M NaCl were added to a cell suspension growing in M63 medium. The mixture was incubated for 2 h, and L-[¹⁴C]PIP was purified as described previously (13). The specific activity (1.83 GBq/mmol) was determined after quantification by high-performance liquid chromatography.

Radiolabeling assays. Cells of S. meliloti were grown in Erlenmeyer's flasks containing 10 ml of LAS medium with 0.5 M NaCl and 1 mM L-[¹⁴C]PIP or a mixture of unlabeled D-PIP and L-[¹⁴C]PIP (0.5 mM each isomer). Respired CO₂ was trapped on a strip of filter paper (3 by 1 cm) moistened with 30 µl of 5 M KOH. Samples were removed periodically, washed twice in 1 ml of isoosmotic LAS medium without PIP, and submitted to an ethanolic extraction as described above. The radioactivity of the ESF and EIF was measured by liquid scintillation counting. An aliquot of the soluble fraction was analyzed by paper chromatography as described below.

Chromatographic analysis and purification of glutamate and NAGGN. Identical numbers of cells were extracted at each stage of growth in LAS medium-0.5 M NaCl in the presence of 1 mM L-[¹⁴C]PIP or D-PIP-L-[¹⁴C]PIP. The ESF was analyzed by two-dimensional paper chromatography (Whatman no. 1) run in two different solvents: aqueous phenol (80%)-ethanol (1/1 vol/vol) and n-butanol-acetic acid-water (12:3:5 vol/vol). The amino acids and PIP were detected by spraying ninhydrin onto the chromatogram and heating at 80°C; the radiolabeled spots were localized by electronic autoradiography and counted by liquid scintillation spectroscopy. Spots comigrating with radiolabeled NAGGN and glutamate were purified after a preparative chromatography on Whatman 3MM paper by using the solvent mixture n-butanol-acetic acid-water (12:3:5 vol/vol) and analyzed by ¹³C NMR spectroscopy.

Transport assays. Cells were grown in LAS medium and LAS medium with 0.5 M NaCl. D-, L-, or DL-PIP (1 mM [ratio of 1:1]) was added to a culture in mid-exponential growth. After 4 h of growth under these conditions, cells were centrifuged (5,000 × g for 5 min), washed twice with an isotonic minimal medium deprived of PIP, and concentrated in the same medium to an OD₅₇₀ of 10 U. L-[¹⁴C]PIP uptake assays were performed as described previously (16). L-[¹⁴C]PIP (1.83 GBq/mmol) was used at a final concentration of 0.5 mM in 500 µl of transport assay medium containing 1 OD₅₇₀ unit of bacterial suspension, with or without 0.5 mM D-PIP. Initial rates of uptake were determined over a 2- to 5-min period. Transport assays were carried out three times each, and the errors were less than 10%.

	RESULTS

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Osmoprotection of S. meliloti is effective by DL-PIP but not by the L or D isomer provided separately. Cells were cultivated in defined LAS medium with NaCl or mannitol added. PIP was supplied as either 1 mM DL-PIP, 1 mM D- or L-PIP, or 1 mM D- and L-PIP (0.5 mM each isomer). GB (1 mM), the most potent osmoprotectant known for S. meliloti so far (5), was used in this experiment as a positive control (Table 1). Addition of 1 mM L-, D-, or DL-PIP to the growth medium had no significant effect on unstressed cells. In sharp contrast, DL-PIP enhanced both the growth yields and the growth rates of NaCl- and mannitol-stressed cells about twofold and was as effective as GB (Table 1). Similar results were obtained when a mixture of D and L isomers of PIP was supplied to osmotically stressed cells. However, provided separately, neither L- nor D-PIP was able to improve the growth of cells under hyperosmotic conditions (Table 1).

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View larger version (14K): [in this window] [in a new window]   FIG. 1.   Growth of S. meliloti 102F34 in the presence of D-, L-, and DL-PIP as carbon sources. Cells were cultivated in aspartate-S medium containing 10 mM DL-PIP (), L-PIP (), or D-PIP ().

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Fate of PIP in stressed growing cells of S. meliloti. To investigate the intracellular fate of DL-PIP (osmoprotective form of PIP) in stressed cells of S. meliloti, an analysis of cell extracts by natural abundance ¹³C NMR spectroscopy was undertaken. Spectra of ethanolic extracts (Fig. 2) from cells cultivated in LAS medium containing 0.5 M NaCl and 1 mM DL-PIP showed both the characteristic peaks of PIP and those of the three major endogenous osmolytes: glutamate, NAGGN, and trehalose. These three compounds were also observed in the absence of exogenous osmoprotectant (data not shown). The signals of PIP appeared together with those of glutamate and NAGGN in the early and mid-exponential phases of growth (Fig. 2A and B) and with those of glutamate, NAGGN, and trehalose in the late exponential phase (Fig. 2C). The resonances of PIP were absent thereafter in spectra from cells in the stationary phase (Fig. 2D). Extracts obtained from stationary-phase cells exhibited a spectrum similar to that of cells grown under osmotic stress without osmoprotectant and harvested at stationary phase.

View larger version (22K): [in this window] [in a new window]   FIG. 2.   Representative 13C NMR spectra from cultures of S. meliloti 102F34 grown in LAS medium containing 0.5 M NaCl and 1 mM DL-PIP. Cells were harvested at the early (A), mid- (B), late exponential (C), and stationary (D) phases of growth. Resonances from endogenously synthesized glutamate (g), the dipeptide NAGGN (d), and trehalose (t), as well as exogenously supplied PIP (p), are indicated when these compounds were accumulated as cytosolic osmolytes.

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View larger version (59K): [in this window] [in a new window]   FIG. 3.   Fate of L-[14C]PIP in the presence of D-PIP in stressed cells of S. meliloti. Cells were cultivated in LAS medium-0.5 M NaCl containing L-[14C]PIP (1 mM, 5 GBq/mmol) with (A and B) or without (C and D) D isomer. , growth expressed as OD570. Other symbols express radioactivity in the medium (), 14CO2 (), EIF (), and ESF (). Histograms B and D represent the percentages of ESF radiolabeling: , glutamate; and , NAGGN.

Stressed cells of S. meliloti immediately catabolize both the L and D isomers of PIP supplied separately. The fate of L-PIP was observed over the entire bacterial growth cycle in LAS medium-0.5 M NaCl containing 1 mM L-[¹⁴C]PIP. In contrast to the situation observed with DL-PIP (Fig. 3A), bacterial cells incorporated labeled PIP at a lower velocity (Fig. 3C), corresponding to about 19 nmol min¹ mg of protein¹. Consequently, 48% of the supplied L-[¹⁴C]PIP still remained in the growth medium after 30 h of culture. During the lag phase of growth, the intracellular radiocarbon was recovered in the ESF, CO₂, and EIF at 63, 16, and 20% of the incorporated radioactivity, respectively. As observed with DL-PIP, the radioactivity increased in CO₂ as soon as the cells started to grow (i.e., after 10 h of incubation). The labeling of the EIF increased until 30 h of culture and then remained constant over the entire growth cycle (about 25 to 30% of the incorporated radioactivity). That of ¹⁴CO₂ reached a maximal value of 67% when cells entered the late exponential phase of growth (Fig. 3C).

DL-PIP, the only osmoprotective form of PIP, increases endogenous osmolyte concentrations in stressed cells of S. meliloti. The amounts of intracellular glutamate, NAGGN, and trehalose were determined during the growth cycle of S. meliloti cultivated in LAS medium containing 0.5 M NaCl without and with 1 mM D-PIP, L-PIP, or DL-PIP (1/1 D-/L-PIP ratio).

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	DISCUSSION

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In this study, we have demonstrated for the first time the atypical and the differential behavior of D and L enantiomers of PIP in bacterial osmoprotection. Osmoprotection of S. meliloti cells was effective only when the two isomers of PIP were present together in the stressing medium of culture. Such a response differs from that of E. coli cells, where exogenously supplied L-PIP, but not D-PIP, exerts a protective effect under an inhibitory osmolarity (14). To understand this uncommon behavior, we have focused our attention on the uptake activity and the fate of L-PIP in hyperosmotically stressed cells of S. meliloti grown in the presence and the absence of D-PIP. The most important observations were that (i) NaCl was able to stimulate L-PIP influx within cells of S. meliloti as described previously for osmoprotective compounds such as betaines (5, 11) and DMSP (34), in both S. meliloti and other bacteria; and (ii) the uptake of L-PIP was stimulated by the D isomer regardless of the osmolarity of the growth medium.

Salt-stressed cells of S. meliloti were able to catabolize L-[¹⁴C]PIP, in both the presence and the absence of D-PIP, into glutamate and the dipeptide NAGGN, the two main endogenous osmolytes neosynthesized by this bacterium. Because the radioactivity of glutamate decreased during the exponential growth phase and that of NAGGN increased in parallel, a close metabolic relationship might exist between these two cytosolic solutes.

Analysis of cellular soluble extracts by ¹³C NMR and chromatography revealed that (i) in stressed cells grown in the presence of DL-PIP, D-PIP accumulated until the late exponential phase of growth and then disappeared as L-PIP was completely consumed; and (ii) both D- and L-PIP were immediately catabolized when they were provided separately to osmotically stressed cultures of S. meliloti. Thus, in cells grown under hyperosmotic conditions in the presence of DL-PIP, the catabolism of L-PIP might inhibit that of D-PIP, leading to the accumulation of the D isomer. The complete consumption of L-PIP from the medium triggers the catabolism of D-PIP. Consequently, the L isomer is the main (if not the sole) precursor of the endogenous osmolytes neosynthesized by stressed cells of S. meliloti. Because no intermediate between PIP and glutamate was detected in our chromatographic analyses, we can only speculate on the nature of the catabolic pathway of PIP. As already shown for Pseudomonas putida P2, which catabolizes PIP to glutamate (33), L-¹-piperideine-6-carboxylate and L--aminoadipate could be the products of oxidation of the imino acid. -Hydroxyglutarate could be an intermediate leading to -ketoglutarate and glutamate. Since PIP oxidase, the first enzyme involved in the catabolism of PIP, was shown to be highly specific for the L isomer (3, 29), one can expect that catabolism of the D isomer should proceed through a racemization as a first step. PIP racemase might therefore be considered as a key enzyme in the osmoprotection of S. meliloti cells in the presence of DL-PIP. Its activity would be negatively regulated by the L isomer under hyperosmotic conditions.

Several studies have reported the preferential utilization of one enantiomer of a molecule over the other as a carbon source by many microorganisms. This was observed, for example, in P. putida, which degrades only the L-carnitine when it grows in the presence of DL-carnitine (20, 28). In Pseudomonas sp. strain AK 1, grown on DL-carnitine, the L isomer is degraded first (30). Acinetobacter calcoaceticus is able to metabolize L-carnitine as a carbon source, but no growth was observed with D-carnitine (20); nevertheless, A. calcoaceticus is able to catabolize D-carnitine in the presence of L-carnitine (20).

Surprisingly, the transient intracellular accumulation of the D isomer when L-PIP is present in the growth medium does not inhibit the synthesis of endogenous osmolytes (glutamate and NAGGN), because it was usually observed in the presence of the other accumulated osmoprotectants GB and DMSP (5, 34, 40). Similarly to ectoine, sucrose, and a few other disaccharides (15, 16, 39), the imported DL-PIP leads to a significant increase in intracellular levels of glutamate and NAGGN in stressed cells of S. meliloti. The actual concentration of these osmolytes can be calculated according to the cell volumes determined elsewhere (39) for the same bacterium under identical conditions of culture. That leads to maximal concentrations of 320 mM for glutamate and 180 mM for NAGGN in the presence of 0.5 M NaCl and 1 mM DL-PIP. Since glutamate occurs mainly as potassium glutamate in osmotically stressed cells (8) and the dipeptide behaves as a neutral solute, the osmotic pressure developed by the cytosolic solution of these two osmolytes might account for almost 80% of that of the external stressing medium. Moreover, it should be mentioned that D-PIP itself, as long as it remains accumulated, might contribute with the endogenous osmolytes to the recovery of cell turgor.

In all, our data bring a novel insight into the versatile osmoadaptation processes in S. meliloti. That two isomers of a given molecule can exert together a beneficial effect under stressing conditions may confer to the bacterium a significant advantage over other organisms requiring osmoprotectants in the same habitat. The mechanism of action of the two isomers remains still unclear. Further work will pay attention to the regulation of both D-PIP catabolism and the level of endogenous osmolytes.