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.

The Pseudomonas aeruginosa PAO1 thiol peroxidase homolog (Tpx) belongs to a family of enzymes implicated in the removal of toxic peroxides. We have shown the expression of tpx to be highly inducible with redox cycling/superoxide generators and diamide and weakly inducible with organic hydroperoxides and hydrogen peroxide (H 2 O 2 ). The PAO1 tpx pattern is unlike the patterns for other peroxide-scavenging genes in P. aeruginosa. Analysis of the tpx promoter reveals the presence of a putative IscR binding site located near the promoter. The tpx expression profiles in PAO1 and the iscR mutant, together with results from gel mobility shift assays showing that purified IscR specifically binds the tpx promoter, support the role of IscR as a transcriptional repressor of tpx that also regulates the oxidant-inducible expression of the gene. Recombinant Tpx has been purified and biochemically characterized. The enzyme catalyzes thioredoxin-dependent peroxidation and can utilize organic hydroperoxides and H 2 O 2 as substrates. The ⌬tpx mutant demonstrates differential sensitivity to H 2 O 2 only at moderate concentrations (0.5 mM) and not at high (20 mM) concentrations, suggesting a novel protective role of tpx against H 2 O 2 in P. aeruginosa. Altogether, P. aeruginosa tpx is a novel member of the IscR regulon and plays a primary role in protecting the bacteria from submillimolar concentrations of H 2 O 2 .

Pseudomonas aeruginosa PAO1 contains gshA and gshB genes, which encode enzymes involved in glutathione (GSH) biosynthesis. Challenging P. aeruginosa with hydrogen peroxide, cumene hydroperoxide, and t-butyl hydroperoxide increased the expression of gshA and gshB. The physiological roles of these genes in P. aeruginosa oxidative stress, bacterial virulence, and biofilm formation were examined using P. aeruginosa ΔgshA, ΔgshB, and double ΔgshAΔgshB mutant strains. These mutants exhibited significantly increased susceptibility to methyl viologen, thiol-depleting agent, and methylglyoxal compared to PAO1. Expression of functional gshA, gshB or exogenous supplementation with GSH complemented these phenotypes, which indicates that the observed mutant phenotypes arose from their inability to produce GSH. Virulence assays using a Drosophila melanogaster model revealed that the ΔgshA, ΔgshB and double ΔgshAΔgshB mutants exhibited attenuated virulence phenotypes. An analysis of virulence factors, including pyocyanin, pyoverdine, and cell motility (swimming and twitching), showed that these levels were reduced in these gsh mutants compared to PAO1. In contrast, biofilm formation increased in mutants. These data indicate that the GSH product and the genes responsible for GSH synthesis play multiple crucial roles in oxidative stress protection, bacterial virulence and biofilm formation in P. aeruginosa.

P. aeruginosa (PAO1) has two putative genes encoding ferredoxin NADP(+) reductases, denoted fprA and fprB. Here, the regulation of fprB expression and the protein’s physiological roles in [4Fe-4S] cluster biogenesis and stress protection are characterized. The fprB mutant has defects in [4Fe-4S] cluster biogenesis, as shown by reduced activities of [4Fe-4S] cluster-containing enzymes. Inactivation of the gene resulted in increased sensitivity to oxidative, thiol, osmotic and metal stresses compared with the PAO1 wild type. The increased sensitivity could be partially or completely suppressed by high expression of genes from the isc operon, which are involved in [Fe-S] cluster biogenesis, indicating that stress sensitivity in the fprB mutant is partially caused by a reduction in levels of [4Fe-4S] clusters. The pattern and regulation of fprB expression are in agreement with the gene physiological roles; fprB expression was highly induced by redox cycling drugs and diamide and was moderately induced by peroxides, an iron chelator and salt stress. The stress-induced expression of fprB was abolished by a deletion of the iscR gene. An IscR DNA-binding site close to fprB promoter elements was identified and confirmed by specific binding of purified IscR. Analysis of the regulation of fprB expression supports the role of IscR in directly regulating fprB transcription as a transcription activator. The combination of IscR-regulated expression of fprB and the fprB roles in response to multiple stressors emphasizes the importance of [Fe-S] cluster homeostasis in both gene regulation and stress protection.

During the translation process, transfer RNA (tRNA) carries amino acids to ribosomes for protein synthesis. Each codon of mRNA is recognized by a specific tRNA, and enzyme-catalysed modifications to tRNA regulate translation. TtcA is a unique tRNA-thiolating enzyme that requires an iron-sulfur ([Fe-S]) cluster to catalyse thiolation of tRNA. In this study, the physiological functions of a putative ttcA in Pseudomonas aeruginosa, an opportunistic human pathogen that causes serious problems in hospitals, were characterized. A P. aeruginosa ttcA-deleted mutant was constructed, and mutant cells were rendered hypersensitive to oxidative stress, such as hydrogen peroxide (H2O2) treatment. Catalase activity was lower in the ttcA mutant, suggesting that this gene plays a role in protecting against oxidative stress. Moreover, the ttcA mutant demonstrated attenuated virulence in a Drosophila melanogaster host model. Site-directed mutagenesis analysis revealed that the conserved cysteine motifs involved in [Fe-S] cluster ligation were required for TtcA function. Furthermore, ttcA expression increased upon H2O2 exposure, implying that enzyme levels are induced under stress conditions. Overall, the data suggest that P. aeruginosa ttcA plays a critical role in protecting against oxidative stress via catalase activity and is required for successful bacterial infection of the host.