Figure 1.

STEM Imaging and Tomography Revealed Distinct Bacterial Phenotype Distribution in PAO1 and PAO1ΔwspF Biofilms

(A) STEM images of the in vitro PAO1 biofilm and PAO1ΔwspF biofilm showed a distinct spatial distribution of two bacterial phenotypes named as bacteria_white and bacteria_gray. Unlike that in PAO1 biofilm where the bacteria_white and bacteria_gray were homogenously distributed throughout the biofilm, bacteria_white and bacteria_gray are segregated in the PAO1ΔwspF biofilm. Bacteria_white was observed from lower-middle to apical area, and bacteria_gray was observed from basal to lower-middle area. The pseudo-colored rendering derived from 3D STEM tomographic structure of the PAO1 biofilm and PAO1ΔwspF biofilm. Scale bar, 1 μm.

(B) STEM images showed the successful separation of bacteria_white and bacteria_gray in PAO1 biofilm and PAO1ΔwspF biofilm. The bac_light fraction and bac_heavy fraction were obtained from the density gradient centrifugation of biofilm following DNaseI treatment. Scale bar, 1 μm.

(C) Real-time changes in oxygen consumption rate (OCR, in picomoles of molecular oxygen per minute) measured on a Seahorse XFe Extracellular Flux Analyzer in bac_light fraction and bac_heavy fraction of PAO1 and PAO1ΔwspF biofilm. (n = 10). Data are mean ± SD.

(D) Live dead staining of PAO1 and PAO1ΔwspF biofilm using SYTO Green and PI at 48 h. Scale bar, 20 μm.

See also Figures S1 and S2.