Effect of NO production on cell growth and catalase activity in Δ<i>nirS</i> mutant, the wild-type PAO1 and its isogenic mutants.

<p><i>P. aeruginosa</i> Δ<i>nirS</i> mutant and Δ<i>nirS</i>(pHERD<i>nirS</i>) were cultured anaerobically in LBN for 24 hrs. Different concentration of arabinose (0–0.1%) were added to the cultures of strain Δ<i>nirS</i>(pHERD<i>nirS</i>) to induce expression of the <i>nirS</i> gene. Cells were sampled to measure NO production (A), catalase activity (B) and cell growth (OD₆₀₀) (C). NO levels were determined using the fluorescent indicator compound, DAF-2A. Overnight anaerobic cultures were normalized and incubated with 10 μM of DAF-2A for 30 min at 37°C. Catalase activity was measured spectrophotometrically by following the decomposition of 19 mM H₂O₂. See Materials and Methods for detail. All results were presented as a value of fold changed compared to the Δ<i>nirS</i> mutant that was assigned a value of 1. The black, light gray and white bars represented PAO1, Δ<i>katA</i> mutant and Δ<i>nirS</i> mutant, respectively. The gray, dotted, diagonal striped and vertical striped bars were referred to Δ<i>nirS</i>(pHERD<i>nirS</i>) exposed to 0, 0.005, 0.05 and 0.1% arabinose, respectively. D. Catalase activity of the Δ<i>nirS</i> mutant upon SNP treatment. Exponential phase aerobic culture of Δ<i>nirS</i> in LBN was sub-cultured anaerobically with varying concentration of the NO donor SNP for 24 hrs. Cells were harvested, disrupted by sonication and the cleared cell lysates were used to measure catalase activity. Representative data are shown. E. Aconitase activity assay. Cells of anaerobically-grown PAO1, Δ<i>katA</i>, Δ<i>nirS</i> and Δ<i>norCB</i>. The assay was performed three times as described previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091813#pone.0091813-Varghese1" target="_blank">[51]</a>, and the standard deviation was shown.