Characterization and binding of purified FinR to the <i>finR-fprA</i> promoter.

<p>(A) Nucleotide sequence showing the <i>finR</i>-<i>fprA</i> promoter structure. +1 indicates the transcriptional start site, and the bold sequences are the putative -35 and -10 promoter motifs. <b><i>CAT</i></b> and <b><i>ATG</i></b> are the translational start codons of FinR and FprA, respectively. The box shaded gray represents the proposed FinR binding site. (B), (C), and (D) Electrophoretic mobility shift assay using purified FinR. A ³²P –labeled DNA fragment (B), mutagenized MU1 and MU2 fragments (C), or the promoter fragments (EBI61-62), with (EBI 61–70) and without (EBI 62–69) proposed FinR binding site (D) spanning the <i>finR-fprA</i> promoter was incubated with increasing amounts of FinR. BSA represents an unrelated protein (2.5 μg BSA). CP and UD signify the cold probe (100 ng unlabeled promoter fragment) and unrelated DNA (1 μg of pUC18 plasmid), respectively, that were added to the binding reaction mixture containing 100 nM FinR. F and B indicate free and bound probes, respectively.</p

Determination of paraquat resistance levels in <i>P</i>. <i>aeruginosa</i> strains.

<p>(A) Paraquat resistance levels in PAO1 containing the mnin-Tn7 vector control (PAO1::Tn7T, red) and Δ<i>finR</i> mutants containing Tn7T (dotted green), Tn7T-<i>finR</i> (purple), Tn7T-<i>fprA</i> (dotted blue), Tn7T-<i>fprB</i> (yellow), or Tn7T-<i>fdx1</i> (dotted black) were determined using plate sensitivity assays. (B) Paraquat (150 μM) resistance levels of <i>P</i>. <i>aeruginosa</i> strains were determined using LB with and without 1% (w/v) KNO₃ supplementation and incubated under aerobic and anaerobic atmospheres. The survival is expressed as a percentage of the CFU on LB plates containing paraquat over the CFU on plates without paraquat. Data shown are means ± SD from three independent experiments.</p

Expression analysis <i>finR</i> and <i>fprA</i> in response to various stresses.

<p>The expression levels of <i>finR</i> (A) and <i>fprA</i> (B) were determined using real-time RT-PCR. Exponential-phase cultures of <i>P</i>. <i>aeruginosa</i> PAO1 were subjected to various stress conditions, including 1 mM H₂O₂, superoxide anion-generating agents (0.5 mM plumbagin [PB], 0.5 mM menadione [MD] and 0.5 paraquat [PQ]), organic hydroperoxides (1 mM cumene hydroperoxide [CHP] and 1 mM <i>t</i>-butyl hydroperoxide [tBH]), 1 mM 2,2’-dipyridyl (DIPY), high salts (0.5 M NaCl and 0.5 M KCl), or 0.04% NaOCl for 15 minutes prior to RNA preparation for real-time RT-PCR analysis. Relative expression was analyzed using the 16S rRNA gene as the normalizing gene and was expressed as the fold expression relative to the level of uninduced (UN) PAO1. Data shown are means ± SD of three independent experiments.</p

List of plasmids used in this study.

<p>List of plasmids used in this study.</p

Expression analysis of <i>fprA</i> and <i>finR</i> in <i>P</i>. <i>aeruginosa</i> strains.

<p>Expression levels of <i>fprA</i> (A) and <i>finR</i> (B) in PAO1 wild type (PAO1::Tn7T), Δ<i>finR</i> mutant (Δ<i>finR</i>::Tn7T) and the complemented mutant (Δ<i>finR</i>::Tn7T-<i>finR</i>) grown under uninduced, 0.5 mM paraquat (PQ), or 0.04% NaOCl (NaOCl) induced conditions. Relative expression was analyzed using the 16S rRNA gene as the normalizing gene and is expressed as fold expression relative to the level of uninduced PAO1. Data shown are means ± SD of three independent experiments. The asterisks indicate statistically significant differences (p < 0.01) compared with the uninduced condition.</p

List of primers used in this study.

<p>List of primers used in this study.</p