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Reactive chlorine species (RCS), particularly hypochlorous acid (HOCl), are powerful antimicrobial oxidants generated by biological pathways and chemical syntheses. Pseudomonas aeruginosa is an important opportunistic pathogen that has adapted mechanisms for protection and survival in harsh environments, including RCS exposure. Based on previous transcriptomic studies of HOCl exposure in P. aeruginosa, we found that the expression of PA0565, or rcsA, which encodes an alkyl hydroperoxidase D-like protein, exhibited the highest induction among the RCS-induced genes. In this study, the rcsA expression was dominant under HOCl stress and highly increased under HOCl31 related stress conditions. Functional analysis of RcsA showed that the distinguishing core amino acid resides Cys60, Cys63, and His67 were required for the degradation of sodium hypochlorite (NaOCl), suggesting an extended motif in the AhpD family. After producing allelic exchange mutagenesis in the P. aeruginosa rcsA, the P. aeruginosa rcsA-deletion mutant showed a significantly decreased HOCl resistance. Ectopic expression of P. aeruginosa rcsA led to significantly increased NaOCl resistance in Escherichia coli. Moreover, the pathogenicity of the rcsA mutant decreased dramatically in both Caenorhabditis elegans and Drosophila melanogaster host-model systems compared to the wild type. Finally, the Cys60, Cys63, and His67 variants of RcsA were unsuccessful to complement to phenotypes of the rcsA mutant. Overall, our data indicate the importance of P. aeruginosa RcsA in defense against HOCl stress under disinfections and during infections of hosts, which involve the catalytic Cys60, Cys63, and His67 residues. [ABSTRACT FROM AUTHOR]

Pseudomonas aeruginosa has two genes encoding ferredoxin NADP(+) reductases, denoted fprA and fprB. We show here that P. aeruginosa fprA is an essential gene. However, the ΔfprA mutant could only be successfully constructed in PAO1 strains containing an extra copy of fprA on a mini-Tn7 vector integrated into the chromosome or carrying it on a temperature-sensitive plasmid. The strain containing an extra copy of the ferredoxin gene (fdx1) could suppress the essentiality of FprA. Other ferredoxin genes could not suppress the requirement for FprA, suggesting that Fdx1 mediates the essentiality of FprA. The expression of fprA was highly induced in response to treatments with a superoxide generator, paraquat, or sodium hypochlorite (NaOCl). The induction of fprA by these treatments depended on FinR, a LysR-family transcription regulator. In vivo and in vitro analysis suggested that oxidized FinR acted as a transcriptional activator of fprA expression by binding to its regulatory box, located 20 bases upstream of the fprA -35 promoter motif. This location of the FinR box also placed it between the -35 and -10 motifs of the finR promoter, where the reduced regulator functions as a repressor. Under uninduced conditions, binding of FinR repressed its own transcription but had no effect on fprA expression. Exposure to paraquat or NaOCl converted FinR to a transcriptional activator, leading to the expression of both fprA and finR. The ΔfinR mutant showed an increased paraquat sensitivity phenotype and attenuated virulence in the Drosophila melanogaster host model. These phenotypes could be complemented by high expression of fprA, indicating that the observed phenotypes of the ΔfinR mutant arose from the inability to up-regulate fprA expression. In addition, increased expression of fprB was unable to rescue essentiality of fprA or the superoxide-sensitive phenotype of the ΔfinR mutant, suggesting distinct mechanisms of the FprA and FprB enzymes. [ABSTRACT FROM AUTHOR]

IscR is a global transcription regulator responsible for governing various physiological processes during growth and stress responses. The IscR-mediated regulation of the Pseudomonas aeruginosa isc operon, which is involved in iron-sulphur cluster ([Fe-S]) biogenesis, was analysed. The expression of iscR was highly induced through the exposure of the bacteria to various oxidants, such as peroxides, redox-cycling drugs, intracellular iron-chelating agents, and high salts. Two putative type 1 IscR-binding sites were found around RNA polymerase recognition sites, in which IscR-promoter binding could preclude RNA polymerase from binding to the promoter and resulting in repression of the isc operon expression. An analysis of the phenotypes of mutants and cells with altered gene expression revealed the diverse physiological roles of this regulator. High-level IscR strongly inhibited anaerobic, but not aerobic, growth. iscR contributes significantly to the bacteria overall resistance to oxidative stress, as demonstrated through mutants with increased sensitivity to oxidants, such as peroxides and redox-cycling drugs. Moreover, the regulator also plays important roles in modulating intracellular iron homeostasis, potentially through sensing the levels of [Fe-S]. The increased expression of the isc operon in the mutant not only diverts iron away from the available pool but also reduces the total intracellular iron content, affecting many iron metabolism pathways leading to alterations in siderophores and haem levels. The diverse expression patterns and phenotypic changes of the mutant support the role of P. aeruginosa IscR as a global transcriptional regulator that senses [Fe-S] and directly represses or activates the transcription of genes affecting many physiological pathways. [ABSTRACT FROM AUTHOR]

IscR is a global transcription regulator responsible for governing various physiological processes during growth and stress responses. The IscR-mediated regulation of the Pseudomonas aeruginosa isc operon, which is involved in iron-sulphur cluster ([Fe-S]) biogenesis, was analysed. The expression of iscR was highly induced through the exposure of the bacteria to various oxidants, such as peroxides, redox-cycling drugs, intracellular iron-chelating agents, and high salts. Two putative type 1 IscR-binding sites were found around RNA polymerase recognition sites, in which IscR-promoter binding could preclude RNA polymerase from binding to the promoter and resulting in repression of the isc operon expression. An analysis of the phenotypes of mutants and cells with altered gene expression revealed the diverse physiological roles of this regulator. High-level IscR strongly inhibited anaerobic, but not aerobic, growth. iscR contributes significantly to the bacteria overall resistance to oxidative stress, as demonstrated through mutants with increased sensitivity to oxidants, such as peroxides and redox-cycling drugs. Moreover, the regulator also plays important roles in modulating intracellular iron homeostasis, potentially through sensing the levels of [Fe-S]. The increased expression of the isc operon in the mutant not only diverts iron away from the available pool but also reduces the total intracellular iron content, affecting many iron metabolism pathways leading to alterations in siderophores and haem levels. The diverse expression patterns and phenotypic changes of the mutant support the role of P. aeruginosa IscR as a global transcriptional regulator that senses [Fe-S] and directly represses or activates the transcription of genes affecting many physiological pathways. [ABSTRACT FROM AUTHOR]