Then, absorption of samples was measured at 562 nm in a Perkin Elmer Lambda 25 UV/Vis spectrophotometer and compared to protein standards containing bovine serum albumin in a concentration range of 0-600 μg ml-1. Extraction selleck kinase inhibitor and determination of intracellular trehalose content Trehalose determination was performed basically as described by Blázquez et al. [52] by the following procedure. Cell pellets from 15 ml of early stationary phase cultures in their optimal minimal medium were washed with isotonic carbon-free medium and resuspended in 1 ml of the same medium. Cells were lysed by 30 min selleck chemical incubation at 95°C and, after

centrifugation, trehalose was assayed in a 200 μl total volume reaction containing 100 μl of the supernatant,

90 μl of 25 mM sodium acetate buffer (pH 5.6) and 0.02 U of commercial trehalase (Sigma). For each culture sample, endogenous glucose content was monitored by performing a parallel reaction in which trehalase was substituted by water. After overnight incubation at 37°C, glucose released by trehalose hydrolysis was determined on 150 μl of the previous reaction by SIS3 mouse addition of 150 μl of a glucose oxidase/peroxidase mixture (0.66 mg ml-1) Aspergillus niger glucose oxidase and 0.25 mg ml-1 horseradish peroxidase in 0.5 M phosphate buffer, pH 6.0 (Sigma) and 50 μl of 2.33 mg ml-1 o-toluidine. After 30 min incubation at 37°C, 1.5 ml of water was added to the samples and absorption was measured at 420 nm in a Perkin Elmer Lambda 25 UV/Vis spectrophotometer and compared to glucose standards in a concentration range of 0-300 μg ml-1. Finally, trehalose content was inferred from the glucose content by performing a standard curve with commercial trehalose (Sigma) ranging from 1 to 5 mM. Trehalose concentration was expressed as μmol mg Lenvatinib research buy protein-1. Isolation of the otsA and 16S rRNA genes Total DNA was isolated by using the CTAB method [53]. Amplification of about 1-kb of the otsA gene from R. gallicum bv. phaseoli 8a3, R. leguminosarum bv. phaseoli 31c3, and R. etli 12a3 was performed by

using the primers OTA1: 5′-ATC TGG ATG GGA TGG TCG GGA-3′ and OTA2: 5′-GAC ATA TTC CTT GGC AAC GAG GTT-3′. For strain CIAT 899, otsA was amplified by using the degenerated primers: OTAS1: 5′-CAT CTG GAT GGG (CT)TG GTC GG-3′ and OTAS2: 5′-GGC GAC ATA TTC CTT GGC (GC)AC (GC)AG GTT-3′. The amplification protocol consisted of the following steps: initial denaturation at 94°C for 5 min followed by 30 cycles of denaturation (45 seconds at 94°C), annealing (45 seconds at 58°C), extension (1 min at 72°C), and a final extension step at 72°C for 10 min. Sequencing of the otsA genes was performed by the company Newbiotechnics (NBT, Seville, Spain). PCR amplifications of the complete 16S rRNA genes were carried out as previously described [54].