Pseudomonas aeruginosa Dnr-regulated denitrification in microoxic conditions

Abstract

Pseudomonas aeruginosa causes acute and chronic infections, such as those that occur in the lungs of people with cystic fibrosis (CF). In infection environments, oxygen (O₂) concentrations are often low. The transcription factor Anr (anaerobic regulation of arginine deiminase and nitrate reduction) responds to low O₂ by upregulating genes necessary for P. aeruginosa fitness in microoxic and anoxic conditions. Anr regulates Dnr (dissimilative nitrate respiration regulator), a gene encoding a transcriptional regulator that promotes the expression of genes required for using nitrate as an alternative electron acceptor during denitrification. In CF sputum, transcripts involved in denitrification are highly expressed. While Dnr is necessary for the anoxic growth of P. aeruginosa in CF sputum and artificial sputum media (ASMi), the contribution of denitrification to P. aeruginosa fitness in oxic conditions has not been well described. Here, we show that P. aeruginosa requires dnr for fitness in ASMi, and the requirement for dnr is abolished when nitrate is excluded from the media. Additionally, we show that P. aeruginosa consumes nitrate in lysogeny broth (LB) under microoxic conditions. Furthermore, strains without a functioning quorum sensing regulator LasR, which leads to elevated Anr activity, consume nitrate in LB even in normoxia. There was no growth advantage for P. aeruginosa when nitrate was present at concentrations from 100 to 1,600 µM. However, P. aeruginosa consumption of nitrate in oxic conditions created a requirement for Dnr and Dnr-regulated NorCB, likely due to the need to detoxify nitric oxide. These studies suggest that Anr- and Dnr-regulated processes may impact P. aeruginosa physiology in many common culture conditions.IMPORTANCEPseudomonas aeruginosa is an opportunistic pathogen commonly isolated from low-oxygen environments such as the lungs of people with cystic fibrosis. While the importance of P. aeruginosa energy generation by denitrification is clear in anoxic environments, the effects of denitrification in oxic cultures are not well understood. Here, we show that nitrate is consumed in microoxic environments and, in some strains, in normoxic environments. While nitrate does not appear to stimulate microoxic growth rate or yield, it does impact physiology. We show that the regulators Anr (anaerobic regulation of arginine deiminase and nitrate reduction) and Dnr (dissimilative nitrate respiration regulator), which are best known for their roles in anoxic conditions, contribute to P. aeruginosa fitness in common laboratory media in the presence of oxygen.

Keywords: Anr; Dnr; Pseudomonas aeruginosa; denitrification; microoxia; nitrate; nitric oxide.

Fig 1

Contribution of Anr and Dnr to P. aeruginosa growth in media ± nitrate. (A) P. aeruginosa strain culture density of PA14 wild type (WT), the ∆anr mutant, and the ∆anr+anr strain after 16 h in LB at 21% and 1% O₂ in a 96-well plate. (B) Levels of NO₃⁻ in LB, TB, a 0.5% yeast extract solution, and a 0.5% sodium chloride (NaCl) solution. The dotted line indicates the lower limit of detection (LOD). (C) WT, ∆anr, and ∆anr+anr culture density after 16 h of growth in TB (solid) or TB + 200 µM KNO₃ (vertical stripes) at 1% O₂. Each point represents an average of replicates from 1 day, and lines connect data from the same experiment. (D) The % decrease in growth of the ∆anr strain relative to WT in TB versus TB + 200 µM KNO₃ in 96-well plates at 1% O₂ for 16 h with shaking. (E) The levels of NO₃⁻ in LB before and after WT growth for 16 h at 21% and 1% O₂. NO₃⁻ levels were calculated using a standard curve of KNO₃ in water and normalized to OD₆₀₀. The levels of nitrate in LB are the same as in panel B. (F) WT, the ∆dnr mutant, and the ∆dnr+dnr strain culture density after growth in LB (diagonal stripes) at 1% O₂ for 16 h in a 96-well plate with shaking. P-values were calculated using a paired one-way analysis of variance (ANOVA) with multiple comparisons (A, C, and F) and an unpaired t-test (B, D, and E).

Fig 2

Contribution of Dnr to P. aeruginosa growth in concentrations of O₂ that lead to nitrate consumption. (A) The levels of NO₃⁻ in LB before and after ∆lasR growth for 16 h at 21% and 1% O₂. NO₃⁻ levels were normalized to OD₆₀₀. The levels of nitrate in LB are the same as in Fig. 1B. (B) ∆lasR, the ∆lasR∆dnr mutant, and the ∆lasR∆dnr+dnr strain culture density after growth in LB (diagonal stripes) at 1% O₂ for 16 h in a 96-well plate with shaking. (C) The % decrease in growth of the ∆dnr and ∆lasR∆dnr mutants relative to WT and ∆lasR parental strains in LB after 16 h of growth in 96-well plates in 21% or 1% O₂. Data points represent an average of technical replicates, with lines showing comparisons of averages of data from the same day. P-values were calculated using paired one-way ANOVA with multiple comparisons (B) and a paired t-test (C).

Fig 3

Dnr contribution to microoxic growth and overall yield of P. aeruginosa in media ± nitrate. (A) WT, the ∆dnr mutant, and the ∆dnr+dnr strain culture density after growth in TB (solid) and TB + KNO₃ (horizontal stripes) at 1% O₂ for 16 h in a 96-well plate with shaking. (B) Culture densities of ∆lasR, a ∆lasR∆dnr mutant, and the ∆lasR∆dnr+dnr strain after 16 h of growth in TB (solid) and TB + 200 µM KNO₃ (horizontal stripes) in 96-well plates at 1% O₂. Each data point represents an average of replicates from 1 day, with lines connecting data from the same experiment. (C) Comparison of growth of WT and ∆lasR in TB (solid) and TB + 200 µM KNO₃ (horizontal stripes) from panels A and B. (D) Comparison of OD₆₀₀ of WT, ∆dnr, and ∆dnr+dnr in TB with 0, 100, 200, 400, and 1,600 µM KNO₃ added after 16 h of growth in 1% O₂. The area under the curve was calculated and used for dose-response analysis. P-values were calculated using a paired one-way ANOVA (A and B), a paired t-test (C), and a t-test comparison of area under the curve (D).

Fig 4

Dnr contribution to microoxic growth of P. aeruginosa in ASMi ± nitrate. (A) WT, the ∆dnr mutant, and the ∆dnr+dnr strain culture density after growth in ASMi − KNO₃ (dots) and ASMi (vertical stripes) at 1% O₂ for 16 h in a 96-well plate at 1% O₂ with shaking. (B) The ∆lasR, ∆lasR∆dnr, and the ∆lasR∆dnr+dnr strain culture densities after growth in ASMi − KNO₃ (dots) and ASMi (vertical stripes) for 16 h in a 96-well plate at 1% O₂ with shaking. (C) Comparison of WT and ∆lasR culture densities after growth in ASMi without and with KNO₃ for 16 h at 1% O₂ from panels A and B. Each data point represents an average of replicates from 1 day, with lines connecting data from the same experiment. P-values were calculated using a paired one-way ANOVA with multiple comparisons (A and B), or a paired t-test (C).

Fig 5

Nitrate consumption of dnr and anr mutants, and growth comparison of P. aeruginosa WT and transposon mutants defective in denitrification at different nitrate concentrations. (A) The levels of NO₃⁻ in LB before and after WT, ∆dnr, ∆anr, and ∆anr∆dnr growth for 16 h at 1% O₂. NO₃⁻ levels were normalized to OD₆₀₀. Nitrate levels in LB are the same as in Fig. 1B, and nitrate levels in WT supernatants at 1% O₂ are the same as in Fig. 1E. (B) Growth of WT, a ∆dnr mutant, and a ∆dnr+dnr strain in TB with indicated concentrations of KNO₃ relative to growth in TB alone. Data in each cell represents the average of nine experiments. For statistical analyses, the relative growth of ∆dnr was compared to WT and ∆dnr+dnr at each concentration of KNO₃ added. P-values were calculated using a paired one-way ANOVA with multiple comparisons. Only P-values of ∆dnr compared to WT are shown. (C) A visual representation of the Anr- and Dnr-regulated identification pathway. (D) Relative growth of WT and confirmed PA14 TnM mutants with insertions in specified genes, the ∆norC mutant, and the ∆norC+norC complemented strain. Colors represent growth at the specified [KNO₃] divided by growth in TB without added KNO₃. Cultures were grown in 96-well plates at 1% O₂ for 16 h on a shaker. Data in each cell represents the average of 3–5 experiments. For statistical analyses, the relative growth of mutants was compared to WT at each concentration of KNO₃ added. P-values were calculated using an ordinary one-way ANOVA with multiple comparisons. Only P-values <0.05 are shown.