Monosaccharides in the form of alditol acetates and methyl PLX3397 order glycosides of trimethylsilyl ethers were analysed by GC-MS on the Hewlett-Packard (5890) gas chromatograph interfaced to the 5971 mass selective detector using the 30 m HP-5MS capillary column (temperature program 150°C for 5 min, raised to 310°C at 5°C/min). NMR spectroscopy – 1H experiments were recorded with the Varian Unity plus 500 instrument in D2O solutions at 70°C with acetone as an internal standard

(d 2.225 ppm) using standard Varian software. Motility assay R. leguminosarum motility assay was conducted in 0.3% M1 agar medium. 5 μl culture grown in liquid TY medium at 28°C for 24 h to an OD600 of 0.4 was stabbed into plates with M1 medium. P005091 To eliminate

the flocculation of the rosR mutants, cell clumps were wiped and broken up on the inner surface of a glass tube using a sterile wooden stick. Then, the tube was left standing for 15 min so that the remaining clumps sunk to the bottom. The suspended cells from the top were taken carefully and, if needed, diluted down into TY to get the desired cell density (OD600 of 0.4). The plates were incubated at 28°C for 3 days, and bacterial growth from the point of inoculation was measured. Motility assay was done twice in triplicate. Biofilm formation assay – microtiter plate method The biofilm formation assay CAL-101 mw was done according to method described by Rinaudi and Gonzalez [15]. Briefly, R. leguminosarum strains were grown in M1 medium supplemented with Dilworth’s vitamins at 28°C for 48 h. The cultures were diluted to an OD600 of 0.4, inoculated into the polystyrene microplate wells in 100 μl aliquots, and incubated with agitation (100 rpm) at 28°C for 48 h. After this time, bacterial growth was assessed by measuring the

OD600. The contents of wells were removed and each well was washed three times with 150 μl of 0.85% NaCl, stained for 15 min with 150 μl of 0.1% crystal violet, and then rinsed three times with water. Biofilm formation was quantified by the addition of 150 μl of L-NAME HCl 95% ethanol and measurement of the absorbance at 560 nm in a microplate reader. The experiment was performed in triplicate, repeated three times, and averaged. Confocal laser scanning microscopy To visualize different stages of R. leguminosarum biofilm formation in a 4-day time-course experiment in polystyrene microplate wells, the inverted microscope Axiovert 200M equipped with LSM 5 Pascal head (with magnification 200x) was used. To obtain images of biofilm formation, bacterial cultures were stained with either Calcofluor (Sigma) or Bacterial Viability kit LIVE/DEAD BacLight™ (Invitrogen).