Activation of fro promoter transcription by flow and during infection of human tissue.

(A) RNA-seq heatmap of P. aeruginosa genes that are upregulated by at least 3-fold by flow and during infection in human wounds. Datasets are from [3,18]. (B) Schematic of a P. aeruginosa fluorescent reporter strain that contains a transcriptional fusion of yfp to the fro promoter and expresses mCherry under a constitutive promoter. (C) Representative phase contrast, fluorescence, and merged images of the P. aeruginosa reporter strain co-incubated with neutrophils, macrophages, or host cell-free medium. Dashed lines indicate boundaries of the host cells and white arrows indicate P. aeruginosa with high YFP/mCherry ratios. Images are representative of three independent experiments. Scale bars represent 5 μm.

Conditioned medium from stimulated neutrophils induces fro expression.

(A) Representative phase-contrast and fluorescence images and (B) fro expression as determined by ratios of YFP to mCherry fluorescence, of P. aeruginosa that were cultured in medium only, medium with PMA, conditioned medium from PMA-stimulated neutrophils, or conditioned medium from unstimulated neutrophils. Scale bars represent 5 μm. Data points indicate an average from at least one hundred individual P. aeruginosa. Gray columns indicate the mean of at least three independent experiments and error bars represent the standard error of the mean (SEM). P-values were obtained using two-tailed t-tests with unequal variances and values of p>0.05 are denoted as ns (nonsignificant). (C) Schematic depicting that PMA-stimulated neutrophils produce respiratory burst products that could activate fro expression.

NaOCl but not H2O2 or HNO3 induces fro expression.

(A) Growth profiles as measured by optical density (OD600) of P. aeruginosa treated with NaOCl, H2O2, HNO3, or no treatment. Data points indicate a mean of at least two independent experiments and error bars indicate SEM. (B) fro expression as determined by ratios of YFP to mCherry fluorescence in P. aeruginosa treated with NaOCl, H2O2, or HNO3, or no treatment (UTR). Horizontal bars indicate the mean of at least three independent experiments and error bars indicate SEM. The dashed line indicates the average YFP/mCherry ratio from the untreated condition. (C) Abundance of froA transcripts in P. aeruginosa following treatment with 1 µM NaOCl, 4 µM H2O2, or no treatment (UTR), as measured by RT-qPCR. Measurements were normalized to 5S ribosomal RNA and the logarithm (base 2) of the fold-change in transcription was computed relative to the untreated condition. Horizontal bars indicate the average of four independent experiments and error bars indicate SEM. (D) fro expression, measured by YFP to mCherry ratio, in P. aeruginosa after treatment with 1 µM NaOCl, 1 µM NaCl, 200 µM HCl, 6 mM NaOH, or no treatment (UTR). Error bars indicate SEM. In panels B and D, data points indicate the average from at least one hundred individual P. aeruginosa. P-values were obtained using two-tailed t-tests with unequal variances, with values of p>0.05 denoted as ns.

The expression of fro is activated during corneal infection and inhibited by methionine and antioxidants.

(A) Abundance of froA transcripts in P. aeruginosa 0, 2, or 20 hours after being inoculated on a mouse corneal abrasion, as measured by RT-qPCR. Measurements were normalized to 5S ribosomal RNA and the logarithm (base 2) of the fold-change in transcription was computed relative to the initial inoculum. Data points represent individual experiments. Horizontal bars indicate the average of at least six independent experiments and error bars represent SEM. (B) Heatmap showing P. aeruginosa gene transcripts that were upregulated by at least 4-fold in wild type compared to the ΔfroR mutant after treatment with 1 µM NaOCl and that had p-values less than 0.05 (n≥3), sorted by genomic locus. Raw values are in Table S1 in the SI. Log2(FC) indicates the log2 of the fold-change. (C) fro expression, determined by ratios of YFP to mCherry, in P. aeruginosa with no treatment (UTR), or treated with 1 µM NaOCl, or 1 µM NaOCl with methionine (Met), cysteine (Cys), or β-mercaptoethanol (βME). (D) fro expression, determined by ratios of YFP to mCherry, in P. aeruginosa after incubation with conditioned medium only (replotted from Fig. 2B), conditioned medium from stimulated neutrophils, or in conditioned medium from stimulated neutrophils with 100 µM methionine. Data points indicate an average from at least one hundred individual P. aeruginosa. Gray columns indicate the mean of at least three independent experiments and error bars represent SEM. P-values were obtained using two-tailed t-tests with unequal variances and values of p>0.05 are denoted as ns.

FroR improves P. aeruginosa growth against HOCl stress and regulates transcription of genes involved in antioxidant defense.

(A) Growth profiles of wild-type P. aeruginosa (WT) and ΔfroR mutants treated with 16 µM NaOCl or untreated. Data points represent an average of at least three independent experiments and error bars indicate SEM. (B-C) Statistical significance as a function of fold-change in transcript abundance in (B) WT and (C) ΔfroR P. aeruginosa after treatment with 1 µM NaOCl (n≥3). Genes in red (upregulated) and blue (downregulated) were altered by at least four-fold (raw values in Table S2-S3 in the SI). The unlabeled genes have not been previously annotated. P-values were determined using the Wald test. Greater values along the vertical axis indicate greater statistical significance and the dashed horizontal line indicates a p-value of 0.05. (D) Schematic that depicts a model in which reactive chlorine species (RCS) reacting with methionine (Met) produces oxidized methionine, which upregulates fro and in-turn increases the production of methionine sulfoxide reductases.