), we have investigated the involvement of this molecule in the osmotic adaptation of Rhizobium meliloti. Ectoine appeared almost as effective as glycine betaine in improving the growth of R. meliboti under adverse osmotic conditions (0.5 M NaCI). Moreover, improvement of growth of rhizobial strains insensitive to glycine betaine was also observed. Ectoine transport proved inducible, periplasmic protein dependent, and, as shown by competition experiments, distinct from the transport of glycine betaine. Medium osmolarity had little effect on the uptake characteristics, since the rate of influx increased from 12 to only 20 nmol min-' mg of proteinwhen NaCI concentrations were raised from 0 to 0.3 or 0.5 M, with a constant of transport of 80 FM. Natural-abundance "C-nuclear magnetic resonance and radiolabelling assays showed that ectoine, unlike glycine betaine, is not intracellularly accumulated and, as a consequence, does not repress the synthesis of endogenous compatible solutes (glutamate, N-acetylglutaminylglutamine amide, and trehalose). Furthermore, the strong rise in glutamate content in cells osmotically stressed in the presence of ectoine suggests that, instead of being involved in osmotic balance restoration, ectoine should play a key role in triggering the synthesis of endogenous osmolytes. Hence, we believe that there are at least two distinct classes of osmoprotectants: those such as glycine betaine or glutamate, which act as genuine osmolytes, and those such as ectoine, which act as chemical mediators.Rhizobia are soil bacteria which display symbiotic interactions with specific legume hosts. Most of these bacteria are very sensitive to a soil water deficit, which adversely affects their dinitrogen fixation capacity and hence the productivity of the whole legume plant (1,8,19,38). Much attention has been focused during the last decade on the mechanisms of osmotic adaptation in several rhizobium species (18,41,42). Rhizobium meliloti, the microsymbiont in alfalfa root nodules, has been extensively investigated (5,6,22,36), and the mechanisms governing turgor restoration of bacterial cells growing under hyperosmotic conditions have been partially elucidated. Specifically, concomitant accumulations of potassium and glutamate ions were pointed out as the primary response in R meliloti (6), and the same mechanism has been found in most of the bacterial species investigated thus far (7, 27). Trehalose and the dipeptide N-acetylglutaminylglutamine amide (NAGGN) (37) are also accumulated, probably to help the bacterial cells in balancing the high osmotic pressure of the medium. Among a series of exogenously supplied osmoprotectants, glycine betaine and proline betaine proved the most effective (3, 14).Their effect is quite similar to that found in members of the family Enterobacteriaceae (23) except that lowering medium osmolality triggers catabolism of betaines in R meliloti (3, 14, 36). A transport system involving a periplasmic glycine betainebinding protein was identified previously (24, 40). Glycine beta...
