Table 1.
Efficiency of fprA deletion in P. aeruginosa strains carrying an extra copy of various genes.
Fig 1.
fprA is an essential gene in P. aeruginosa.
The viability of exponential-phase cultures of P. aeruginosa PAO1 and ΔfprA mutant strains harboring an extra copy of fprA or fdx1 was determined using viable cell count on LB agar plates incubated at either 30°C or 45°C. The viability is expressed as a percentage of the CFU of the tested strain over the CFU of the PAO1::Tn7T or PAO1/pTS control.
Fig 2.
Expression analysis finR and fprA in response to various stresses.
The expression levels of finR (A) and fprA (B) were determined using real-time RT-PCR. Exponential-phase cultures of P. aeruginosa 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 t-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.
Fig 3.
Expression analysis of fprA and finR in P. aeruginosa strains.
Expression levels of fprA (A) and finR (B) in PAO1 wild type (PAO1::Tn7T), ΔfinR mutant (ΔfinR::Tn7T) and the complemented mutant (ΔfinR::Tn7T-finR) 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.
Fig 4.
Characterization and binding of purified FinR to the finR-fprA promoter.
(A) Nucleotide sequence showing the finR-fprA promoter structure. +1 indicates the transcriptional start site, and the bold sequences are the putative -35 and -10 promoter motifs. CAT and ATG 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 finR-fprA 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.
Fig 5.
Determination of paraquat resistance levels in P. aeruginosa strains.
(A) Paraquat resistance levels in PAO1 containing the mnin-Tn7 vector control (PAO1::Tn7T, red) and ΔfinR mutants containing Tn7T (dotted green), Tn7T-finR (purple), Tn7T-fprA (dotted blue), Tn7T-fprB (yellow), or Tn7T-fdx1 (dotted black) were determined using plate sensitivity assays. (B) Paraquat (150 μM) resistance levels of P. aeruginosa 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.
Fig 6.
Virulence of P. aeruginosa strains.
The virulence of PAO1 containing the Tn7T vector control (PAO1::Tn7T) and ΔfinR mutants containing Tn7T, Tn7T-finR, Tn7T-fprA, Tn7T-fprB, or Tn7T-fdx1 were determined using the Drosophila melanogaster feeding method. The percent fly survival was scored after 18 hours of incubation. Data presented are means ± SD of three independent experiments. The asterisk indicates statistically significant difference (p < 0.01) compared with PAO1::Tn7T. LB represents feeding the flies with LB medium.
Table 2.
List of plasmids used in this study.
Table 3.
List of primers used in this study.