This was previously demonstrated in S. meliloti by Gouffi et al [35]. On the other hand, mannosucrose

and glutamate were the main osmolytes in A. tumefaciens 10c2 grown at high salinity, whereas at low salt only mannitol was observed. Mannosucrose accumulation was found to be NaCl-dependent in A. tumefaciens 10c2 (this study), A tumefaciens strains C58 and NT1 [31] and in rhizobial isolates from Acacia nodules [36], supporting the hypothesis that this compatible solute participates in alleviating osmotic stress. However, isolation and analysis of osmosensitive mutants would be necessary to prove the latter statement, and additional mechanisms involved in A. tumefaciens 10c2 osmoadaptation cannot be ruled out. In the tested strains, mannitol was not CYT387 accumulated when glucose was used as a carbon source (Figure 4, and data not shown). On the other hand, cells this website grown SHP099 with [1/6-13C]mannitol as a carbon source accumulated [1/6-13C]mannitol, indicating that mannitol was not synthesized de novo but accumulated upon

transport from the external medium. Bacteria rarely synthesize mannitol as a compatible solute, but it is frequent to find it as an external osmoprotectant [4]. In general, uptake and accumulation of osmoprotectants is preferred over the synthesis of endogenous compatible solutes, as the latter is energetically more costly [37]. However, R. tropici CIAT 899 and A. tumefaciens 10c2 used mannitol both as carbon source and as an osmoprotectant solute at low salinity, but mannitol

was replaced by endogenous compatible solutes (i.e. trehalose or mannosucrose) when cells were exposed to hyperosmotic stress (see Figures 3 and 4). This finding may be explained by two, non-exclusive, reasons: (i) that trehalose and mannosucrose are better osmolytes than mannitol, and/or (ii) that energy-requiring systems, other than trehalose or mannosucrose synthesis, were operating at high salinity, and mannitol catabolism was enhanced many in detriment of its accumulation. The role of trehalose as a compatible solute involved in bacterial tolerance to osmotic stress has been widely demonstrated in the literature. Thus, E. coli [38], S. meliloti [12] and B. japonicum [13] mutants lacking the otsA gene for the synthesis of trehalose are osmosensitive. In another study, Alarico et al. [39] found a direct correlation between the presence of genes for trehalose synthesis (otsA/otsB) in Thermus thermophilus strains and their halotolerance. In this work, we found that trehalose synthesis in R. tropici CIAT 899 is osmoregulated (Figure 6), suggesting the involvement of trehalose in the osmotolerance of this strain. However, we could not find a direct correlation between the trehalose content of the rhizobial strains and their osmotolerance. On the contrary, trehalose levels in the less salt tolerant strains grown at 0.1 M NaCl were 10 fold-higher than those of the more salt-tolerant R. tropici CIAT 899 grown under the same conditions (Figure 6).