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

Abstract

(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