Onofre, Personal Communication  pHP45Ω pBR322 derivative carrying the Ω cassette; AprSmrSpr [36]  pRK600 Helper plasmid; Cmr tra [37]  pJQ200-SK Suicide vector; Gmr mobsac [38]  pMotsA1

4.2-kb blunt fragment from R. etli CE3 genome (containing frk, otsAch, pgi) cloned into pUC19301 in EcoRV; Apr This study  pMotsA4 4,1-kb BglII-XbaI fragment from pMotsA1 cloned into pSK in BamHI-XbaI; Apr This study  pMotsA5 pMotsA4 derivative containing an BglII recognition site within otsAch; Apr This study  pMotsA6 pMotsA5 derivative with Ω casete within otsAch; AprSmrSpcr This study  pMotsA7 6.1-kb ApaI-XbaI fragment from pMotsA6 (containing frk,otsAch, pgi) cloned into pJQ200-SK; GmrSmrSpcr This study Tolerance to desiccation Aliquot volumes (1 ml) of B- medium cultures in early stationary phase were harvested by Opaganib in vivo centrifugation. Cell pellets were washed with the same medium without any carbon source, centrifuged for 5 min at 13000 rpm and, after removing the CH5424802 research buy supernatant, vacuum dried. Two variations of the protocol described by Manzanera

et al. [39] were used. In a first step, two replicates of all samples were dried by vacuum in a Memmert V0200 vacuum oven at 20°C and 313 mbar for 20 h. After that, for each sample, one replica was taken out from the oven, sealed and stored at 28°C, and the other was subjected to a further step under vacuum consisting on a temperature ramping of 2°C/min with a 15-min pause after every increase of 2°C, up to a maximum temperature of 30°C, followed by storage at 28°C. For assessment of viability, after variable time periods, dried samples were resuspended in 1 ml of TY complex medium, and serial dilutions were plated PJ34 HCl on TY plates, incubated at 28°C, and counted to determine CFU. Viability was measured before (taken as 100% survival) and just after drying, and at 4 days, 1, 2, and 3 weeks storage, and

expressed as percentage of viable cells. Extraction and determination of intracellular solutes by 13C-NMR spectroscopy R. etli wild-type and otsA mutant strain (CMS310) were grown in B-medium with 0.2 M NaCl at 28°C until early-stationary phase. Cells were collected by centrifugation and washed with the same medium without any carbon source. Cell pellet was resuspended in 10 ml of extraction mixture (methanol:chloroform:water; 10:5:4) and extracted by gently shaking for 30 min at 37°C. Cell debris was removed by centrifugation, and supernatants were extracted once with chloroform:water (1:1) and freeze-dried. The solids were dissolved in D2O (0.6 ml). 13C-NMR spectra were recorded at 25°C on a Brucker AV500 spectrometer at 125 MHz. The chemical shifts are reported in ppm on the δ scale relative to tetramethylsilane. Signals were assigned by comparison with previously published chemical shift values [6] and compared with 13C-NMR of pure compounds.