Each of the mutated gene/s was introduced into the genome of R. leguminosarum by homologous recombination. The flaA/B/C/D mutants have deletions in the following: flaA 3′ end; flaB; flaC; and flaD 5′ end. Epoxomicin Southern hybridization and/or PCR were performed for each gene to confirm replacement of the wild-type gene with the mutated gene/s. Construction of gene fusions and ß-glucuronidase (gusA) reporter gene assays The promoter BLZ945 mouse region

of flaB was cloned upstream of a promoterless gusA gene in pFus1 [33]. The resulting construct was introduced into VF39SM and 3841 by biparental mating. VF39SM and 3841 strains containing the flaB-gusA fusion were grown in TY broth for 48 hours at 30°C [33]. β-glucuronidase activity was measured as described by Jefferson et al. [37] and modified AC220 in vitro by Yost et al. [38]. The data given are the means of triplicate experiments. Swimming motility test The strains were grown in TY broth for 24 hours. Swimming motility was determined

by inoculating the strains into a motility medium (YES) containing the following: 0.3% agar, 0.01% yeast extract, and 1 mM MgSO4 [39]. The optical densities (OD600) of the cultures were standardized and equal amounts of inoculum were inoculated into the swimming agar using a fine-point pipette tip. The swimming diameter was measured 3-4 days after inoculation. Swarming Motility Test The swarm assay was performed following the method described by Tambalo et al. [29]. Briefly, R. leguminosarum wildtype and fla mutant strains were grown in TY broth for 24 hours. Equal amounts of inoculum from the TY culture was used to inoculate

swarm plates. The plates were incubated at 22°C for two to three weeks and the swarming motility of the fla mutants was compared with the wildtype. Flagellar filament RVX-208 isolation Flagellin proteins were isolated from R. leguminosarum based on the procedure described by Maruyama et al. [40]. Cells were grown in 100 ml of TY broth for 48 hours with slow agitation (50 rpm). The bacterial cells were collected by centrifugation at 12,000 × g for 10 minutes. The pellet was resuspended in 40 mM phosphate buffer. The bacterial cells were vigorously agitated using a vortex to detach the flagella from the cells. The mixture was centrifuged at 12,000 × g for 10 minutes using a Sorval centrifuge. The supernatant was removed and centrifuged again at the same speed and time. The supernatant containing the detached flagella was centrifuged in an ultracentrifuge at 50,000 × g for 2 hours. The pellet was resuspended in 200 μL of 40 mM phosphate buffer. Immunoblot The flagellar protein samples were denatured at 100°C for 5 minutes and then separated on 12% acrylamide SDS-PAGE gel at 200V for 45 minutes. Molecular size markers from Bio-Rad and Fermentas were used.