Redox Reactions of Phenazine Antibiotics with Ferric (Hydr)oxides and Molecular Oxygen

Phenazines are small redox-active molecules produced by a variety of bacteria. Beyond merely serving as antibiotics, recent studies suggest that phenazines play important physiological roles, including one in iron acquisition. Here we characterize the ability of four electrochemically reduced natural phenazines—pyocyanin (PYO), phenazine-1-carboxylate (PCA), phenazine-1-carboxamide, and 1-hydroxyphenazine (1-OHPHZ)—to reductively dissolve ferrihydrite and hematite in the pH range 5–8. Generally, the reaction rate is higher for a phenazine with a lower reduction potential, with the reaction between PYO and ferrihydrite at pH 5 being an exception; the rate decreases as the pH increases; the rate is higher for poorly crystalline ferrihydrite than for highly crystalline hematite. Ferric (hydr)oxide reduction by reduced phenazines can potentially be inhibited by oxygen, where O2 competes with Fe(III) as the final oxidant. The reactivity of reduced phenazines with O2 decreases in the order: PYO > 1-OHPHZ > PCA. Strikingly, reduced PYO, which is the least reactive phenazine with ferrihydrite and hematite at pH 7, is the most reactive phenazine with O2. These results imply that different phenazines may perform different functions in environments with gradients of iron and O2

Ligand-Enhanced Abiotic Iron Oxidation and the Effects of Chemical versus Biological Iron Cycling in Anoxic Environments

This study introduces a newly isolated, genetically tractable bacterium (Pseudogulbenkiania sp. strain MAI-1) and explores the extent to which its nitrate-dependent iron-oxidation activity is directly biologically catalyzed. Specifically, we focused on the role of iron chelating ligands in promoting chemical oxidation of Fe­(II) by nitrite under anoxic conditions. Strong organic ligands such as nitrilotriacetate and citrate can substantially enhance chemical oxidation of Fe­(II) by nitrite at circumneutral pH. We show that strain MAI-1 exhibits unambiguous biological Fe­(II) oxidation despite a significant contribution (∼30–35%) from ligand-enhanced chemical oxidation. Our work with the model denitrifying strain Paracoccus denitrificans further shows that ligand-enhanced chemical oxidation of Fe­(II) by microbially produced nitrite can be an important general side effect of biological denitrification. Our assessment of reaction rates derived from literature reports of anaerobic Fe­(II) oxidation, both chemical and biological, highlights the potential competition and likely co-occurrence of chemical Fe­(II) oxidation (mediated by microbial production of nitrite) and truly biological Fe­(II) oxidation

Engineering the Soil Bacterium Pseudomonas synxantha 2–79 into a Ratiometric Bioreporter for Phosphorus Limitation

Microbial bioreporters hold promise for addressing challenges in medical and environmental applications. However, the difficulty in ensuring their stable persistence and function within the target environment remains a challenge. One strategy is to integrate information about the host strain and target environment into the design-build-test cycle of the bioreporter itself. Here, we present a case study for such an environmentally motivated design process by engineering the wheat commensal bacterium Pseudomonas synxantha 2–79 into a ratiometric bioreporter for phosphorus limitation. Comparative analysis showed that an exogenous P-responsive promoter outperformed its native counterparts. This reporter can selectively sense and report phosphorus limitation at plant-relevant concentrations of 25–100 μM without cross-activation from carbon or nitrogen limitation or high cell densities. Its performance is robust over a field-relevant pH range (5.8–8), and it responds only to inorganic phosphorus, even in the presence of common soil organic P. Finally, we used fluorescein-calibrated flow cytometry to assess whether the reporter’s performance in shaken liquid culture predicts its performance in soil, finding that although the reporter is still functional at the bulk level, its variability in performance increases when grown in a soil slurry as compared to planktonic culture, with a fraction of the population not expressing the reporter proteins. Together, our environmentally aware design process provides an example of how laboratory bioengineering efforts can generate microbes with a greater promise to function reliably in their applied contexts

Glucose tolerance and two-year pulmonary exacerbation history.

<p>CFRD subjects had approximately twice as many pulmonary exacerbations as subjects with impaired (IGT) or normal glucose tolerance (NGT, left panel). The average length of admission was not significantly different between these groups (right panel). Diamonds are centered on each category’s mean and the vertical distance represents the 95% confidence interval.</p

R² and p-values from correlations between SG and clinical parameters.

<p>R² and p-values from correlations between SG and clinical parameters.</p

Physiological consequences of long-term PCA oxidation for C. portucalensis MBL.

(A) Diagram of the bioelectrochemical reactor. The C. portucalensis MBL culture was incubated in the main chamber with a working electrode poised to -500 mV that continuously reduced any available PCA. The reference electrode communicated with the potentiostat to retain a constant voltage. Cells oxidized PCA when nitrate was present. In a sidearm, separated by a dense glass frit, the counter-electrode completed the circuit. Reduced PCA is green and oxidized PCA is colorless. (B) Current traces for biological replicates with and without provided PCA and an initial concentration of 10 mM nitrate. Samples were taken from these replicates at the times displayed in (C)-(E), where each data point corresponds to an independent biological replicate. (C) Time point measurements of survival in the culture, as determined by colony forming units (CFUs). The asterisks represent a Bonferroni-corrected statistically significant difference between the two conditions at the final time point (p = 0.0025). (D) The nitrite that was produced (from nitrate reduction) by the cultures over the experiment. At the third and fourth time point there were Bonferroni-corrected statistically significant differences between the two conditions: p = 0.0029 and p = 0.0019, respectively. (E) The normalized ATP content per CFU over the time course. There were no statistically significant differences. The Bonferroni corrected threshold was p < 0.0083 given hypothesis testing across the six time points.</p

Comparison of the roles of the three terminal nitrate reductases in PCA oxidation.

(A-C) Comparison of homologous recombination knockouts versus translational knockouts for each individual terminal nitrate reductase. Each graph shows the oxidation of PCA over time (measured by the decay of PCAred, which is fluorescent) with the abiotic control (nitrate plus reduced PCA in reaction medium without cells) in grey. (A) NarZ comparison: the deletion (Δ) and translational knockout (tlKO) strains all have a PCA oxidation deficit relative to the wildtype (WT, blue). The most severe phenotype is in the ΔnarUZYWV strain (green), and the ΔnarZYWV and narZ-tlKO (yellow and orange, respectively) phenotypes are indistinguishable. (B) NarG comparison: the deletion (green) and translational knockout (orange) strains have the same slight PCA oxidation deficit relative to the wildtype control (WT, blue). (C) NapA comparisons: the translational knockout (orange) has a more severe PCA oxidation deficit that the deletion strain (green), relative to the wildtype control (blue). Each thick line corresponds to the mean of three biological replicates plotted in semitransparent circles. (D-E) Comparisons of the maximum observed PCA oxidation rate for each combinatorial nitrate reductase mutant (and abiotic control). (D) The phenotypes after shaking (oxic) pregrowth. (E) The phenotypes after standing (hypoxic) pregrowth. (F-G) Comparisons of the time until half of the PCA was oxidized for each combinatorial nitrate reductase mutant (and abiotic control). (F) The phenotypes after oxic pregrowth. (G) The phenotypes after hypoxic pregrowth. N.D. stands for “not detected,” corresponding to strains that did not reach the 50% PCA oxidized threshold over the 48-hour assay. Squares represent the means of technical triplicates and circles are independent biological replicates with no technical replicates. Double asterisks represent statistical significance after Bonferroni correction, and single asterisks represent p S1 Text), and the complete matrices of pairwise comparisons are presented in S2 Fig. The conversion of PCA oxidation curves to rate and time metrics is described in S3 Fig.</p

Re-analysis of HPLC peaks previously assigned as phenazines from original data collected on an older HPLC.

UV–vis chromatograms are shown on the left, and the full UV–vis spectra of the peaks indicated with an arrow are shown on the right. Where indicated on the y-axis, the data have been normalized to bring the data into the same scale for clarity. (A) Analysis of the PCA peak, comparing a pure PCA standard (chromatogram at 364 nm, orange) to a sputum sample (chromatogram at 398 nm, black). (B) Analysis of the pyocyanin peak, comparing pyocyanin in P. aeruginosa-free sputum (blue) to a P. aeruginosa-positive sputum sample (black) (both chromatograms at 387 nm).</p

Correlation between ferriprotoporphyrin IX concentration and disease status as measured by percent predicted FEV1 (Spearman’s ρ = −0.47, <i>p</i> = 3.6 × 10⁻⁵).

Each data point represents a single sputum sample from a unique patient. The dashed line illustrates a linear regression through the data.</p

Comparisons of HbA_1c and SG by glucose tolerance.

<p>CFRD subjects had significantly higher HbA_1c than subjects with impaired (IGT) or normal glucose tolerance (NGT, left panel), while NGT subjects had significantly higher average sputum glucose than IGT or CFRD subjects (right panel). Diamonds are centered on each category’s mean and the vertical distance represents the 95% confidence interval.</p