The experiment was repeated twice. Assays for sensitivity to antibiotics, detergents, and osmotic stress The sensitivity of R. leguminosarum bv. trifolii strains to sodium deoxycholate (DOC), sodium dodecyl sulfate (SDS), and ethanol was studied, and minimal inhibitory concentration of particular agents was determined. Bacteria were collected from TY agar medium into sterile water to an OD600 of 0.3 and 10 μl of each suspension was plated on TY containing a defined concentration of DOC (0.005-1% w/v), SDS (0.005-1% w/v) or ethanol (0.25-6% v/v).

After 3 days, the growth of strains on individual media was determined. Three independent experiments were done for each strain. To assess the effect of osmolarity on growth of the R. leguminosarum bv. trifolii Rt24.2 and the rosR mutants, see more the strains were grown in TY medium supplemented with a defined concentration of NaCl (0-510 mM). Cultures were incubated at 28°C for 48 h, and then the OD600 was measured. Tolerance to hypo-osmotic stress was determined using low-osmolarity selleck compound glutamate-yeast extract-mannitol (GYM) medium

[35]. Antibiotic sensitivity assays were performed using commercially available filter disks with the appropriate antibiotic: ampicillin (10 μg), erythromycin (15 μg), chloramphenicol (30 μg), gentamicin (10 μg), bacitracin (10 μg), augmentin (30 μg), streptomycin (10 μg), polymyxin B (10 μg), carbenicillin (20 μg), penicillin G (10 U), and tetracycline (30 μg) (Mast Diagnostics, Merseyside, UK). Filter disks were placed on the surface of 79CA medium, where 100 μl of R. leguminosarum cultures were previously spread. The diameter of the growth inhibition zone was measured after 3 days of incubation. Isolation and analysis of extracellular and membrane proteins For analysis of extracellular and membrane proteins, the Rt2472 and Rt24.2 strains were grown at 28°C for 2 days to an OD600 of 0.6 in 200 ml TY medium. To study the influence of clover root exudates on membrane protein profiles, these strains were grown at 28°C for 3 days in 400 ml M1 medium supplemented Branched chain aminotransferase with Dilworth’s

vitamins and with or without 5 μM exudates. Cells were removed by twice centrifugation at 5,000 × g for 20 min at 4°C, and supernatants were used for purification of extracellular proteins. The proteins were concentrated by precipitation with 10% trichloroacetic acid according to the procedure by Russo et al. [14]. Membrane proteins from cell pellets were isolated according to the method described by Kucharczyk et al. [70]. The cells were washed in 200 ml 50 mM Tris-HCl (pH 7.4), and centrifuged at 5,000 × g for 20 min at 4°C. Cell pellet was resuspended in 1.6 ml 200 mM Tris-HCl (pH 8.0), and then 1.6 ml 1 M sucrose in 200 mM Tris-HCl (pH 8.0), 16 μl lysozyme (12 mg/ml in 100 mM EDTA, pH 8.0) and 3.2 ml ice cold water were added. Next, 25.6 μl saturated ethanol-phenylmethylsulfonylfluoride (PMSF) solution and 12.