Figure 4.

Hypothetical model of the role of pyocyanin in anaerobic survival in biofilm and construction of the multilayered structure of the multispecies biofilm. (A) Pyocyanin is predicted to act as an electron acceptor for the anaerobic cells and shuttle electrons between anaerobic and aerobic niches. Pyocyanin stimulates excretion of pyruvate. The secreted pyruvate is expected to be utilized for the anaerobic pyruvate fermentation. PYO_red and PYO_ox indicate the reduced- and oxidized-forms of pyocyanin, respectively. pO₂ indicates partial oxygen pressure. (B,C) FISH images of three-species biofilms in vertical sections. A pyocyanin-overproducing P. aeruginosa strains P1 (P1) and its pyocyanin-non-producing derivative (ΔphzM) appear blue with a Cy3-labeled probe, A pyocyanin-resistant Raoultella strain (R1) and a pyocyanin-sensitive Brevibacillus strain (S1) appear green and red with the FITC- and Cy5-labeled probes, respectively. Strain P1 forms a multilayered biofilm with strains R1 and S1 (B). An intermingled biofilm is formed when strain ΔphzM is used (C). Small arrows indicate the bottom of biofilms. White bars indicate 50 μm.