Methylation at position 32 of tRNA catalyzed by TrmJ alters oxidative stress response in Pseudomonas aeruginosa

Bacteria respond to environmental stresses using a variety of signaling and gene expression pathways, with translational mechanisms being the least well understood. Here, we identified a tRNA methyltransferase in Pseudomonas aeruginosa PA14, trmJ, which confers resistance to oxidative stress. Analysis of tRNA from a trmJ mutant revealed that TrmJ catalyzes formation of Cm, Um, and, unexpectedly, Am. Defined in vitro analyses revealed that tRNA[superscript Met(CAU)] and tRNA[superscript Trp(CCA)] are substrates for Cm formation, tRNA[superscript Gln(UUG)], tRNA[superscript Pro(UGG)], tRNA[superscript Pro(CGG)] and tRNA[superscript His(GUG)] for Um, and tRNA[superscript Pro(GGG)] for Am. tRNA[superscript Ser(UGA)], previously observed as a TrmJ substrate in Escherichia coli, was not modified by PA14 TrmJ. Position 32 was confirmed as the TrmJ target for Am in tRNA[superscriptPro(GGG)] and Um in tRNA[superscript Gln(UUG)] by mass spectrometric analysis. Crystal structures of the free catalytic N-terminal domain of TrmJ show a 2-fold symmetrical dimer with an active site located at the interface between the monomers and a flexible basic loop positioned to bind tRNA, with conformational changes upon binding of the SAM-analog sinefungin. The loss of TrmJ rendered PA14 sensitive to H2O2 exposure, with reduced expression of oxyR-recG, katB-ankB, and katE. These results reveal that TrmJ is a tRNA:Cm32/Um32/Am32 methyltransferase involved in translational fidelity and the oxidative stress response.National Science Foundation (U.S.) (CHE-1308839)Agilent TechnologiesSingapore-MIT Alliance for Research and Technology (SMART

Pseudomonas aeruginosa GidA modulates the expression of catalases at the posttranscriptional level and plays a role in virulence

Pseudomonas aeruginosa gidA, which encodes a putative tRNA-modifying enzyme, is associated with a variety of virulence phenotypes. Here, we demonstrated that P. aeruginosa gidA is responsible for the modifications of uridine in tRNAs in vivo. Loss of gidA was found to have no impact on the mRNA levels of katA and katB, but it decreased KatA and KatB protein levels, resulting in decreased total catalase activity and a hydrogen peroxide-sensitive phenotype. Furthermore, gidA was found to affect flagella-mediated motility and biofilm formation; and it was required for the full virulence of P. aeruginosa in both Caenorhabditis elegans and macrophage models. Together, these observations reveal the posttranscriptional impact of gidA on the oxidative stress response, highlight the complexity of catalase gene expression regulation, and further support the involvement of gidA in the virulence of P. aeruginosa

Analyses of the Regulatory Mechanism and Physiological Roles of Pseudomonas aeruginosa OhrR, a Transcription Regulator and a Sensor of Organic Hydroperoxides▿

ohrR encodes an organic hydroperoxide sensor and a transcriptional repressor that regulates organic hydroperoxide-inducible expression of a thiol peroxidase gene, ohr, and itself. OhrR binds directly to the operators and represses transcription of these genes. Exposure to an organic hydroperoxide leads to oxidation of OhrR and to subsequent structural changes that result in the loss of the repressor's ability to bind to the operators that allow expression of the target genes. Differential induction of ohrR and ohr by tert-butyl hydroperoxide suggests that factors such as the repressor's dissociation constants for different operators and the chemical nature of the inducer contribute to OhrR-dependent organic hydroperoxide-inducible gene expression. ohrR and ohr mutants show increased and decreased resistance to organic hydroproxides, respectively, compared to a parental strain. Moreover, the ohrR mutant had a reduced-virulence phenotype in the Pseudomonas aeruginosa-Caenorhabditis elegans pathogenicity model

The FinR-regulated essential gene fprA, encoding ferredoxin NADP+ reductase: Roles in superoxide-mediated stress protection and virulence of Pseudomonas aeruginosa.

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

Mapping of the OspR binding sites on the <i>Pseudomonas aeruginosa gpx</i> promoter region.

<p>Gel shift assay of purified OspR protein with various <i>gpx</i> promoter fragments. The concentrations of OspR protein added in the binding reactions are indicated above each lane. The unbound promoter fragment was designated p_gpx, and the protein-DNA complex was designated p_gpx-OspR.</p

Expression analysis of <i>gpx</i> and <i>ospR</i> in the presence of oxidants.

<p>Quantitative real-time PCR was performed to measure relative expression levels (2^-ΔΔCt) of <i>gpx</i> (A) and <i>ospR</i> (B) in wild-type <i>Pseudomonas aeruginosa</i> with various oxidant treatments for 15 min. The uninduced sample was set to 1. UN, uninduced; CHP500, 500 μM CHP; tBOOH500, 500 μM tBOOH; LOOH100, 100 μM linoleic hydroperoxide; H₂O₂500, 500 μM H₂O₂; H₂O₂1000, 1000 μM H₂O₂; H₂O₂4000, 4000 μM H₂O₂; MD50, 50 μM menadione; MD500, 500 μM menadione; PQ50, 50 μM paraquat; PQ100, 100 μM paraquat; PQ500, 500 μM paraquat. Significant differences (<i>P</i> < 0.05) between the treated samples and uninduced sample are denoted with asterisks.</p

Regulation of <i>ohr</i> by OspR, the alteration of <i>ohr</i> expression in the <i>ospR</i> mutant and the alteration of <i>ospR</i> expression in the <i>ohrR</i> mutant.

<p>Quantitative real-time PCR was performed to measure relative expression levels (2^-ΔΔCt) of <i>gpx</i> (A) and <i>ohr</i> (B) in various strains. (C) Expression of <i>gpx</i> in wild-type and <i>ohrR</i> mutant cultures treated with various concentrations of CHP (mM) for 15 min. (D) Expression of <i>ohr</i> in wild-type and <i>ospR</i> mutant cultures treated with various concentrations of CHP (mM) for 15 min. The expression in the wild-type strain was set to 1. Significant differences (<i>P</i> < 0.05) between samples are denoted with asterisks. For (C) and (D), means were compared between wild-type strain and mutant strain at the same condition.</p

Regulation of Organic Hydroperoxide Stress Response by Two OhrR Homologs in <i>Pseudomonas aeruginosa</i>

<div><p><i>Pseudomonas aeruginosa ohrR</i> and <i>ospR</i> are gene homologs encoding oxidant sensing transcription regulators. OspR is known to regulate <i>gpx</i>, encoding a glutathione peroxidase, while OhrR regulates the expression of <i>ohr</i> that encodes an organic peroxide specific peroxiredoxin. Here, we show that <i>ospR</i> mediated <i>gpx</i> expression, like <i>ohrR</i> and <i>ohr</i>, specifically responds to organic hydroperoxides as compared to hydrogen peroxide and superoxide anion. Furthermore, the regulation of these two systems is interconnected. OspR is able to functionally complement an <i>ohrR</i> mutant, i.e. it regulates <i>ohr</i> in an oxidant dependent manner. In an <i>ohrR</i> mutant, in which <i>ohr</i> is derepressed, the induction of <i>gpx</i> expression by organic hydroperoxide is reduced. Likewise, in an <i>ospR</i> mutant, where <i>gpx</i> expression is constitutively high, oxidant dependent induction of <i>ohr</i> expression is reduced. Moreover, <i>in vitro</i> binding assays show that OspR binds the <i>ohr</i> promoter, while OhrR binds the <i>gpx</i> promoter, albeit with lower affinity. The binding of OhrR to the <i>gpx</i> promoter may not be physiologically relevant; however, OspR is shown to mediate oxidant-inducible expression at both promoters. Interestingly, the mechanism of OspR-mediated, oxidant-dependent induction at the two promoters appears to be distinct. OspR required two conserved cysteines (C24 and C134) for oxidant-dependent induction of the <i>gpx</i> promoter, while only C24 is essential at the <i>ohr</i> promoter. Overall, this study illustrates possible connection between two regulatory switches in response to oxidative stress.</p></div