The first stage consisted of an attenuated exponential phase of 20 h (if compared with that of the wild type) followed by 30 h of arrested growth with OD600 values of around 0.5 units. In the second one, growth was restarted, showing a second exponential phase during 40 h, followed by a second stationary phase with absorbance values comparable to those of the wild type strain (Figure

5A). As Berzosertib clinical trial in otsAch cells collected at the beginning of the first stationary phase (see Figure 4B), trehalose was absent from extracts prepared from samples harvested at the entrance of this second stationary phase. Instead, they contained large amounts of glutamate (Figure 4C). However, when glucose and trehalose were used as the sole carbon source, this biphasic 10058-F4 pattern of growth was

not observed. Growth of the otsAch strain with both carbon sources was delayed with respect to the wild-type strain, even in the absence of osmotic stress (see Additional file 3: Figure S2). At 35°C, R. etli wild-type strain was able to grow well in B- medium with NaCl concentrations ranging from 0 to 0.15 M. As described above (see Figure 1), growth of the wild type was impaired at 35°C with 0.2 M NaCl, showing absorbance values not exceeding 1.0 unit of OD600(Figure 5B). At this temperature, growth of the otsA mutant was severely affected, regardless of the salinity of the culture medium, with cultures showing OD600 around 0.5 OD units. The above data suggest that trehalose is essential for growth of R. etli at high temperature. Osmotically induced trehalose synthesis improves desiccation tolerance in R. etli Involvement

of trehalose in desiccation tolerance in rhizobia has been firmly established in R. leguminosarum bv. trifolii[7]. On the other hand, in S. meliloti[55] or rhizobia nodulating Acacia[56], Urease desiccation tolerance was stimulated by osmotic and/or temperature pre-treatment. To check the influence of trehalose on desiccation tolerance of R. etli, wild type and otsAch PF-6463922 datasheet strains were grown at 28°C in minimal medium B-alone or additioned with 0.2 M NaCl, and harvested at early stationary phase. For cell drying, we used two variants of the protocol described by Manzanera et al. for E. coli[39], a drying process (induced by vacuum at 30°C) or a drying + high temperature process (including a second step with a controlled increase of temperature from 20 to 30°C under vacuum). In the absence of osmotic stress, both wild type and otsAch strains showed survival levels under 0.01%, regardless of the drying protocol (data not shown). In contrast, wild type cells osmotically pre-conditioned by the presence of 0.2 M NaCl showed ca. 35% survival levels after drying, although viability after 4 days storage dropped down to 1.4% (Figure 6). Compared to the drying treatment, the drying + high temperature protocol did not enhance wild type cell survival (Figure 6).