To investigate surfactant production by R. leguminosarum swarm cells, a drop-collapsing test was conducted following the method described by Jain et al. (1991). Briefly, swarm cells were grown in the swarm medium and then a suspension of cells from the edge of a swarming population was prepared isocitrate dehydrogenase inhibitor 7 days and 3 weeks after inoculation. A 10 μL cell suspension (OD600 nmc. 2.0) was spotted on the surface of the hydrophobic lid of a plastic Petri dish. The cell suspension drop was observed for
spreading, which would indicate the presence of surfactants. Distilled water and 0.2% sodium dodecyl sulfate were used as negative and positive controls, respectively. Transmission electron microscopy was Talazoparib order performed by slightly modifying the procedure used by Miller et al. (2007). The R. leguminosarum strains were grown on solid (1.3% agar) TY plates (for vegetative cells) and on swarm plates (for swarmer
cells). A suspension of the bacteria from the plate cultures was prepared using sterile double-distilled water. For the swarm plates, cultures were taken from the tip of the swarm front and from the center of the plate. A formvar carbon-coated grid was placed on top of a cell suspension drop for 3 min and excess liquid was removed. To determine the arrangement of the swarmer cells, the grid was placed directly on top of the swarm plate, at the tip of the swarm front. Staining was performed using 1% uranyl acetate for 30 s. Samples were observed using a Hitachi-7650 transmission electron microscope and images were taken using an AMT Image Capture Engine. The expression of flagellar genes in R. leguminosarum VF39SM swarmer cells was compared with the expression in R. leguminosarum vegetative cells. We used pre-existing gusA fusions to flagellin (flaA) and flagellar PD184352 (CI-1040) regulatory genes (visN, and rem) (Tambalo et al., 2010). Vegetative cells were grown on a solid swarm medium (1.3% Bacto agar) for 8 days at room temperature and in swarm broth medium for 48 h. Swarmer cells were grown in swarm
plates for 2 weeks at 22 °C. Broth cultures were directly used for a β-glucuronidase (gusA) assay, whereas for plate cultures, cells were taken from the edge of a swarming population and vegetative cell population and were suspended in swarm broth. The gusA activity of the fusions was measured as described by Jefferson et al. (1986) and modified by Yost et al. (2004). All data given are the means of triplicate experiments. The antibiotic resistance patterns of vegetative and swarmer cells of R. leguminosarum were determined by growing the cells in swarm medium using 1.3% (solid plate) and 0.7% (swarm plate) Bacto agar. Antibiotic solutions were added onto sterile paper discs and then dried for 20 min. The antibiotics used were cephalexin (50 μg), nalidixic acid (50 μg), rifampicin (20 μg), and chloramphenicol (30 μg).