Fig. 4.

Analyses of antibiotic sensitivity and gene expression indicate that diverse redox-balancing pathways are functioning in PA14 biofilms. a Overview of the redox-balancing pathways investigated. NADH can be re-oxidized by pyruvate fermentation via LdhA or by the electron transport chain via terminal oxidases, such as the cbb₃-type Cco complexes implicated in phenazine reduction. b Ciprofloxacin (100 µg/mL) tolerance observed for cells from biofilms formed by cco mutants. Data for the parent strains (WT and ∆phz) are shown in gray. p values are based on an unpaired two-sided t-test between strain pairs as indicated (n.s., not significant; **p ≤ 0.01). Data for growth without antibiotics does not show significant differences between strains (Supplementary Figure 3). The center line of the boxplot shows the median, the lower and upper hinges correspond to the first and third quartiles, and the whiskers extend to the most extreme points, limited to 1.5 times the interquartile range. c Expression analyses of WT and ∆phz colony paraffin sections show lactate production (which activates expression of the lldPDE operon) in biofilms grown on defined medium with glucose as the sole carbon source. One representative biological replicate is shown (data for all replicates (N = 3) is shown in Supplementary Figure 9e). Scale bar is 25 µm. d Model depicting the metabolisms that could support redox balancing in oxic (activity 1) and hypoxic (activity 2) biofilm subzones, contributing to activities detected by isotope labeling/SRS imaging and to antibiotic tolerance