Interdependency of Respiratory Metabolism and Phenazine-Associated Physiology in Pseudomonas aeruginosa PA14

ETC composition influences tetrazolium dye reduction. (A) Depiction of the terminal oxidase complexes present in the WT (center) and the strains PaCco (left) and PaCio (right). Electrons enter the ETC via primary dehydrogenases (not shown) and are transferred to the quinone pool (Q). Reduced Q can act as a substrate for the quinol oxidases (Cyo and Cio) or the cytochrome bc₁ complex (bc₁). A cytochrome c protein mediates electron transfer between bc₁ and the terminal oxidases Cco1, Cco2, and Cox. (B) The 32 carbon sources that support PA14 growth, grouped based on tetrazolium dye reduction patterns of each phenazine-producing and phenazine-null strain. Groups were designated based on data from three experiments. Carbon sources highlighted in colored boxes are those on which dye reduction patterns changed relative to the strains with the full complement of terminal oxidases (i.e., WT and Δphz, the center column). Carbon source descriptors are as provided by the manufacturer.

Electron donor and phenazines affect terminal oxidase expression in liquid culture. Mean fluorescence (RFU) of reporter strains engineered to express GFP under the control of the cox, cio, cco1, or cco2 promoter during liquid culture growth on the indicated carbon sources is shown. Mean fluorescence values were corrected for cell density (OD at 500 nm), and the fluorescence values of a strain expressing GFP without a promoter (the MCS control) were subtracted from each data point. Onset of stationary phase (time = 0 h) was determined for each strain from the respective growth curves (see Fig. S4 in the supplemental material), and fluorescent reporter expression data are plotted from 5 h prior to onset of stationary phase to 10 h into stationary phase. Gray boxes indicate the time points for which P values were determined using unpaired, two-tailed t tests. Data represent the mean from three biological replicates; error bars denote standard deviation and are not drawn in instances where they would be obscured by point markers.

Electron donor and phenazines affect terminal oxidase expression in colony biofilms. Representative images show expression of GFP under the control of the cox, cio, cco1, or cco2 promoter in thin sections prepared from biofilms grown for 3 days on the indicated carbon sources. Reporter fluorescence is shown in green and is overlain on the DIC image of each colony biofilm. Dotted lines indicate the air-biofilm (top) or agar-biofilm (bottom) interfaces. Graphs show fluorescence values (RFU) relative to biofilm depth of the corresponding images. Fluorescence values of the MCS control (a strain expressing GFP without a promoter) in either the WT or Δphz background have been subtracted from each plot. The y axis for each graph provides scale for the respective image, which is representative of at least six biological replicates.

Phenazine production is affected by carbon source. (A) The precursor phenazine phenazine-1-carboxylic acid (PCA) can be modified by PhzH to produce phenazine-1-carboxamide (PCN) and/or by PhzM to produce 5-methyl-PCA (5-Me-PCA). 5-Me-PCA can be further modified by PhzS to yield pyocyanin (PYO). The redox potential of each phenazine is indicated (E_1/2 versus a normal hydrogen electrode at pH 7) (39, 44). (B) Phenazines produced by WT PA14 in liquid cultures (top) and biofilms (bottom) grown on the indicated carbon sources. The area of each pie represents the combined concentration of all measured phenazines, and each “slice” represents the mean concentration in micromolar (listed for each phenazine produced alongside the respective “slice”) of PCA, PCN, or PYO. Mean values are representative of data from at least six biological replicates. For numerical data, including error and total n, see Fig. S6 in the supplemental material.