0.6, supplemented with 100 μM of [14C]-glucose. After different times of incubation at 37°C, the glucose remaining in the supernatant (S) and cytoplasmatic see more solutes synthesized from ectoine,

present in the ethanol insoluble (EIF) and soluble (ESF) fractions, respectively, were determined as described in Methods. The data are the averages of three different replicates ± SD (standard deviation). Mutant CHR95 possesses a deregulated ectoine uptake As mutant CHR95, but not the wild type strain, could use ectoines as nutrients at low salinities, we investigated the transport and metabolism of ectoine in both strains in response to increasing osmolarity. As previously reported by Vargas et al [25], the wild type strain showed its maximal ectoine transport rate at the optimal salinity for growth (1.5 M NaCl), which was 3- and 1.5-fold higher than those observed at 0.75 and 2.5 M NaCl, respectively (Figure 3). Notably, the ectoine transport rates of strain CHR95 were 8-, 2.3-, and 2.5-fold higher at 0.75, 1.5, and 2.5 M NaCl, respectively, than those of the wild type grown at the same salt concentrations (Figure 3). Figure 3 C. salexigens CHR95 shows a deregulated ectoine uptake. The wild-type strain and the mutant

CHR95 (ΔacseupRmntR::Tn1732) were grown in glucose M63 minimal medium containing the Epacadostat indicated concentration of NaCl. The measurement of 40 [14C]-ectoine uptake rates (vi, expressed as nmol min-1 OD-1 units) was performed as described in Methods. Experiments were repeated twice, and the data correspond to mean values. To test if the metabolism of ectoine was affected in CHR95, the fate of radioactive ectoine was analysed in the presence

or absence of 20 mM glucose as described in Methods, and compared to that of the wild type strain. According to previous studies [25], CO2 production due to ectoine catabolism in the wild type strain was lower (40-fold) in the presence of glucose, suggesting that ectoine utilization is partially repressed by glucose. No significant differences were found between CO2 production from ectoine by CHR95 and the wild type strain, neither with nor without glucose addition (Figure 4a). In both strains, most of the carbon backbone of ectoine (ca. 70% of the total radioactivity added) was found in the ethanol soluble fraction (ESF), whereas only about 3.82% of the total Dipeptidyl peptidase radioactivity added was found in the ethanol insoluble fraction (EIF). No significative differences were found in the radioactivity present in the ESF and EIF MDV3100 in vitro fractions of the wild type and mutant strain. Glucose did not influence the biosynthesis of molecules from ectoine in any of these fractions (Figure 4b). These results suggested that whereas ectoine transport is deregulated in mutant CHR95 at any salinity, ectoine metabolism is not affected in this strain. Figure 4 C. salexigens CHR95 is not affected in the metabolism of ectoine. Cells grown in M63 with 1.