Fig 1.
Presence of eDNA at the early stages of biofilm formation in Xcc.
Representative light (crystal violet, CV staining) and fluorescence (SYTO-9 staining) images of 72 h static cultures on LB or XVM2 media. Fibers were observed after both staining for strains Xcc 306 and Xcc 12879 Aw at the early stages of biofilm formation. Fibers interconnected cells at different stages of aggregation, from one to several cells are shown. In XVM2 medium, fibers were thicker and more uniform after staining with CV and SYTO-9. eDNA in XVM2 medium appeared to cover the surface like a sheet in contrast to individual fibers produced in LB medium.
Fig 2.
Presence of eDNA in preformed biofilms of Xcc.
Representative light (CV staining) and fluorescence (SYTO-9 staining) images of mature biofilms on LB or XVM2 media. Both Xcc 306 and Xcc 12879 Aw strains were more aggregated in XVM2 than LB. A high level of aggregation for strain Xcc 12879 Aw in XVM2 made it difficult to observe eDNA fibers in aggregates. In XVM2, strain Xcc 306 was less aggregated and CV and SYTO-9 staining revealed eDNA surrounding the cells. In LB both staining revealed long fibers interconnecting aggregates.
Fig 3.
eDNA in exponential growth phase of Xcc.
Representative fluorescence microscopy images of Xcc 306 and Xcc 12879 Aw in LB broth at exponential growth phase stained with SYTO-9. A high level of eDNA fibers (marked with white arrows) were produced by both strains. Bacillary shape bacteria are distinguishable from the fibers that appeared to connect cells over a long distance. Xcc Aw 12879 fibers were well developed while strain Xcc 306 fibers were more diffuse.
Fig 4.
Presence of eDNA in Xcc twitching motility.
Twitching halos from Xcc 306, 12879 Aw and Iran2 A* and light and fluorescence microscopy of twitching halos stained with CV or SYTO-9. Twitching motility was observed between the plate surface and the medium. CV and SYTO-9 staining of strains revealed similar twitching structures. Fibers observed after CV staining appeared to have a high eDNA content. Twitching fibres were longer for Xcc 12879 Aw and Iran2 A* than for Xcc 306. Some fibers are marked with white arrows to highlight diference from bacillary shape bacteria associated.
Fig 5.
Effect of DNAse in the extracellular matrix of Xcc.
Transmission Electron Microscopy of Xcc 306 and 12879 Aw strains in exponential growth phase, after DNAse and no-DNAse (Buffer) treatments. DNAse treated bacteria showed less extracellular structures than de buffer control. The remaining structures observed after treatment could be associated to extracellular proteins. Bacterial cell division is shown under DNAse treatment.
Fig 6.
Effect of DNAse I during biofilm formation in Xanthomonas.
Biofilm formation after treatment with DNAse I at different times after bacterial seeded with different Xanthomonas species and strains (see Table 1). A, DNAse added at 0 h post seeding (hps); B, DNAse added at 24 hps; C, DNAse added at 48 hps; and D, DNAse added at 72 hps. The absorbance values were normalized to the control XVM2 plus buffer in order to compare the response for different strains. Error bars represent the standard deviation. Graphs are a representative assay of at least three assays with three replicates per assay. Statistical analyses were performed using STATGRAPHICS Plus, version 5.1 (Copyright Manugistics Inc.).
Fig 7.
Xanthomonas biofilm rupture by DNAse I.
A, DNAse treatment of 1 h duration. B, DNAse treatment for an overnight period. The absorbance values were normalized to the control XVM2 plus buffer in order to compare the response for different strains. Error bars represents the standard deviation. Graphs are a representative assay of at least three assays with three replicates per assay
Table 1.
Strains of Xanthomonas spp. used in the study.