Figure 2

The pIG50 plasmid enhances MG1655 adherence to polystyrene. The GFP-tagged MG1655 strain (S29) and its engineered version were transformed with pIG50 (S61) and incubated in 96-well plates for 24 h at 30°C in M63G medium supplemented with various concentrations of metal ranging from 0.05 to 5 μM as indicated in the figure. The efficiency of biofilm formation was estimated based on microscopic observation or the fluorescence of the attached cells as described in the Methods section. A) Fluorescence microscopy. Biofilms formed by the S29 and S61 strains in the presence or absence of 1 μM Ni and 1 μM Co were observed. A sharp increase of adherence resulting from transformation with the pIG50 plasmid was observed. This phenomenon is quantified in Figure 2C. B) Confocal laser scanning microscopy. The thickness of the biofilm formed by the S61 strain in the presence or absence of cobalt and nickel was estimated using confocal microscopy to illustrate the slight increase in adherence conferred by the presence of metal. Five fields were observed per strain, and three measurements of biofilm thickness were performed per field. The biofilms of the engineered strains were significantly thicker in the presence of metal (p < 0.005, Dunnett’s test). C) Spectrofluorimetry. The effect of metal concentration on S61 biofilm formation was estimated based on the fluorescence intensity of the attached cells. The fluorescence intensity of the S61 biofilms was converted to the corresponding number of cells/mm² based on a standard curve as described in the Methods section section and compared to the reference values obtained from biofilms formed under the same conditions by the parental S29 strain. Data represent the mean of 3 replicates (=3 wells), and error bars represent standard deviations. Significant differences are indicated using lowercase letters, and different letters indicate significant differences (Tukey’s test, p < 0.05).