PMC

Biofilm assay of MR-1 and aggA mutant. (A) Pellicle formation of MR-1, ΔaggA, ΔaggA* (aggA in-frame deletion mutant containing pBBR-AGGA). (B) SSA Biofilm was assessed for the strains indicated after 16 and 24 h, respectively. Cultures were prepared as described in Methods. The averaged OD readings of four independent culture tubes were given with images of representative CV-stained tubes.

EPS analysis. (A) Effects of proteinase K on pellicle formation and developed pellicles. Upper-panel, pellicle formation of the WT in static LB, in which the proteinase K was added at inoculation to 100 mg/ml (final concentration). Lower panel, developed pellicles of the WT (48 h after inoculation) were treated with 100 mg/ml (final concentration). (B) TLC analysis of monosaccharide in pellicles and supernatants. P and S represent pellicle and supernatant, respectively. Man, gal, and glu represent mannose, galactose, and glucose, respectively. Supernatants of the aggA mutant culture were included in the analysis.

The ΔflgA mutant displayed slow pellicle formation. (A) Swimming and swarming motility assays of the ΔflgA mutant. In both panels, the ΔflgA mutant (Upper) was compared to the WT (Lower). The ΔflgA* strain refers to the ΔflgA mutant containing pBBR-FLGA. (B) Electron micrographs of WT and the ΔflgA mutant. No flagellum was observed on the mutant. (C) Left panel, pellicle formation of the ΔflgA mutant. Right panel, the cell densities of cells in pellicles of the WT and the ΔflgA mutant. The WT, dark red; the ΔflgA mutant, light blue. E represents the time at which the cell density of ΔflgA mutant catches up (10 days after inoculation in the experiment). Presented are averages of four replicates with the standard deviation indicated by error bars.

Treatment of S. oneidensis pellicles with EDTA and divalent cations. (A) Pellicle formation of the WT after 48 h in static LB in the presence of 0.3 mM EDTA and certain divalent cation (0.3 mM) under aerobic conditions. (B) Cells in pellicles formed in the presence of 0 (light blue), 0.1 (dark red), 0.2 (light yellow), and 0.3 mM (dark blue) EDTA at the different time points. Presented are averages of four replicates with the standard deviation indicated by error bars. (C) Effects of divalent cations on the inhibition of pellicle formation by EDTA. Pellicle formation of the WT after 48 h in static LB in the presence of 0.3 mM EDTA and one of indicated divalent cations (0.3 mM) under aerobic conditions was shown. The WT in static LB without EDTA was used as the control. The relative pellicle formation ((EDTA and indicated cation)/EDTA-absence control) was presented in the figure. EDTA only ('No cation' was used as the negative control. Presented are averages of four replicates with the standard deviation indicated by error bars.

Pellicle formation of S. oneidensis in LB under aerobic conditions. (A) Growth of S. oneidensis in static liquid LB under aerobic conditions. Cell density of all cells (planktonic and pellicle cells combined) (brown square), pellicle cells (yellow triangle), planktonic cells (blue circle), and the ΔflgA mutant (green cross) was shown. Growth of agitated cultures (black diamond) is included for comparison. Presented are averages of four replicates with the standard deviation indicated by error bars. (B) Pellicle formation of MR-1 in static liquid LB under aerobic conditions. The pellicles started to form about 12 h after inoculation based on the altered growth rate of planktonic cells at the room temperature. (C) Dissolved oxygen concentrations at 1 cm below the surface in the static MR-1 cultures.