Figure 6. Confocal laser-scanning micrographs of Rhizobium alamii wt and MSΔGT mutant.

mg.L⁻¹ of Cd²⁺ in the form of cadmium nitrate, 3 days (left lane) and 5 days (right lane), Bacteria were grown on polycarbonate membranes in a minimal M9 medium supplemented with 2 g.L⁻¹ of glucose, with 0 or 2 mg.L⁻¹ of cadmium following inoculation of the medium with 2.10⁵ cell.mL⁻¹. The bacterial cells were localized by constitutive expression of GFP (in green); EPS was labeled by binding with fluorescent Alexa660-Concanavalin A (in red) in observations performed at 3 days of growth. 5-day biofilms were unlabeled for EPS. A) R. alamii wt after 3 days of growth in the absence of cadmium nitrate. Projections of Z-sections (1 µm) through 5 µm. The scale bar corresponds to 20 µm. Few cells were attached to the membrane surface as a small core. The EPS can be seen in red, surrounding a single cell. B) After 5 days of growth in the absence of cadmium. Projections of Z-sections (1 µm) through 5 µm. The scale bar corresponds to 50 µm. Bacterial cells colonized the membrane, in the form of a loose spherical biofilm. The EPS can be seen in red in some areas of the biofilm (see picture insert projection of Z-sections through 15 µm, the scale bar corresponds to 20 µm). C) R. alamii wt after 3 days of growth in the presence of cadmium nitrate (Cd²⁺ 2 mg.L⁻¹). Projections of Z-sections (1 µm) through 5 µm. The scale bar corresponds to 20 µm. The cells grew as a sphere. The EPS was localized inside the biofilm. D) R. alamii wt after 5 days of growth in the presence of cadmium nitrate (Cd2+ 2 mg.L⁻¹). Projections of Z-sections (1 µm) through 15 µm. The scale bar corresponds to 20 µm. The cells were organized in the form of fused, expanded spheres. E) MSΔGT mutant cell development on a membrane surface after 3 days of growth, in the absence of cadmium nitrate. Projection of Z-sections (1 µm) through 6 µm. The scale corresponds to 20 µm. The cells adhered to the membrane surface, although as single cells. F) MSΔGT mutant after 5 days of growth in the absence of cadmium. Projections of Z-sections (1 µm) through 17 µm. The scale bar corresponds to 50 µm. The bacterial cells formed a flat, loose biofilm architecture. G) MSΔGT mutant after 3 days of growth in the presence of cadmium nitrate (Cd²⁺ 2 mg.L⁻¹). Projection of Z-sections (1 µm) through 5 µm. The scale bar corresponds to 50 µm. Biofilms developed in the yz plane. 2 h. MSΔGT mutant after 5 days of growth in the presence of cadmium nitrate (Cd²⁺ 2 mg.L⁻¹). Projection of Z-sections (1 µm) through 17 µm. The scale bar corresponds to 50 µm. The cells formed a distributed, dense biofilm.