Induction of Antimicrobial Resistance of Stenotrophomonas maltophilia by Exposure to Nonlethal Levels of Antibiotics.

The effects of the sequential subculture in the presence of a driving force on antimicrobial resistance of Stenotrophomonas maltophilia K279a were investigated. Stationary-phase cells were inoculated into the lysogeny broth medium, with and without antibiotic supplementation, and grown until the stationary phase before being subcultured into the same antibiotic-supplemented medium for six consecutive cycles. Thirty colonies from each cycle and treatment condition were selected and their antibiotic susceptibility profiles were determined. The sequential subculture of K279a for a number of cycles reduced susceptibility to diverse classes of antibiotics, including ciprofloxacin, amikacin, gentamicin, ceftazidime, co-trimoxazole, and chloramphenicol, regardless of the antibiotic used. Supplementation with antibiotics that is, ampicillin, kanamycin, ciprofloxacin, and ceftazidime, at sublethal concentrations significantly accelerated the development rate of strains that reduced susceptibility to other antibiotics. The patterns of reduced susceptibility were different depending on the antibiotic used for supplementation. Thus, without gene transfer, antibiotic-resistant strains of S. maltophilia can readily develop, especially after antibiotic treatments. Whole-genome sequence analysis of the selected antibiotic-resistant mutants identified gene mutations that might be responsible for antimicrobial resistance of S. maltophilia.

Analysis of the role of the QseBC two-component sensory system in epinephrine-induced motility and intracellular replication of Burkholderia pseudomallei.

Burkholderia pseudomallei is a facultative intracellular bacterial pathogen that causes melioidosis, a severe invasive disease of humans. We previously reported that the stress-related catecholamine hormone epinephrine enhances motility of B. pseudomallei, transcription of flagellar genes and the production of flagellin. It has been reported that the QseBC two-component sensory system regulates motility and virulence-associated genes in other Gram-negative bacteria in response to stress-related catecholamines, albeit disparities between studies exist. We constructed and whole-genome sequenced a mutant of B. pseudomallei with a deletion spanning the predicted qseBC homologues (bpsl0806 and bpsl0807). The ΔqseBC mutant exhibited significantly reduced swimming and swarming motility and reduced transcription of fliC. It also exhibited a defect in biofilm formation and net intracellular survival in J774A.1 murine macrophage-like cells. While epinephrine enhanced bacterial motility and fliC transcription, no further reduction in these phenotypes was observed with the ΔqseBC mutant in the presence of epinephrine. Plasmid-mediated expression of qseBC suppressed bacterial growth, complicating attempts to trans-complement mutant phenotypes. Our data support a role for QseBC in motility, biofilm formation and net intracellular survival of B. pseudomallei, but indicate that it is not essential for epinephrine-induced motility per se.

Monothiol Glutaredoxin Is Essential for Oxidative Stress Protection and Virulence in Pseudomonas aeruginosa.

Glutaredoxins (Grxs), ubiquitous redox enzymes belonging to the thioredoxin family, catalyze the reduction of thiol-disulfide exchange reactions in a glutathione-dependent manner. A Pseudomonas aeruginosa ΔgrxD mutant exhibited hypersensitivity to oxidative stress-generating agents, such as paraquat (PQ) and cumene hydroperoxide (CHP). In vitro studies showed that P. aeruginosa GrxD acts as an electron donor for organic hydroperoxide resistance enzyme (Ohr) during CHP degradation. The ectopic expression of iron-sulfur cluster ([Fe-S]) carrier proteins, including ErpA, IscA, and NfuA, complements the function of GrxD in the ΔgrxD mutant under PQ toxicity. Constitutively high expression of iscR, nfuA, tpx, and fprB was observed in the ΔgrxD mutant. These results suggest that GrxD functions as a [Fe-S] cluster carrier protein involved in [Fe-S] cluster maturation. Moreover, the ΔgrxD mutant demonstrates attenuated virulence in a Drosophila melanogaster host model. Altogether, the data shed light on the physiological role of GrxD in oxidative stress protection and virulence of the human pathogen, P. aeruginosa. IMPORTANCE Glutaredoxins (Grxs) are ubiquitous disulfide reductase enzymes. Monothiol Grxs, containing a CXXS motif, play an essential role in iron homeostasis and maturation of [Fe-S] cluster proteins in various organisms. We now establish that the human pathogen Pseudomonas aeruginosa GrxD is crucial for bacterial virulence, maturation of [Fe-S] clusters and facilitation of Ohr enzyme activity. GrxD contains a conserved signature monothiol motif (C29GFS), in which C29 is essential for its function in an oxidative stress protection. Our findings reveal the physiological roles of GrxD in oxidative stress protection and virulence of P. aeruginosa.

Contribution of Stenotrophomonas maltophilia MfsC transporter to protection against diamide and the regulation of its expression by the diamide responsive repressor DitR.

Stenotrophomonas maltophilia contains an operon comprising mfsB and mfsC, which encode membrane transporters in the major facilitator superfamily (MFS). The results of the topological analysis predicted that both MfsB and MfsC possess 12 transmembrane helices with the N- and C-termini located inside the cells. The deletion of mfsC increased the susceptibility to diamide, a chemical oxidizing agent, but not to antibiotics and oxidative stress-generating substances relative to wild-type K279a. Moreover, no altered phenotype was observed against all tested substances for the ΔmfsB mutant. The results of the expression analysis revealed that the mfsBC expression was significantly induced by exposure to diamide. The diamide-induced gene expression was mediated by DitR, a TetR-type transcriptional regulator encoded by smlt0547. A constitutively high expression of mfsC in the ditR mutant indicated that DitR acts as a transcriptional repressor of mfsBC under physiological conditions. Purified DitR was bound to three sites spanning from position + 21 to -57, corresponding to the putative mfsBC promoter sequence, thereby interfering with the binding of RNA polymerase. The results of electrophoretic mobility shift assays illustrated that the treatment of purified DitR with diamide caused the release of DitR from the mfsBC promoter region, and the diamide sensing mechanism of DitR required two conserved cysteine residues, Cys92 and Cys127. This suggests that exposure to diamide can oxidize DitR through the oxidation of cysteine residues, leading to its release from the promoter, thus allowing mfsBC transcription. Overall, MfsC and DitR play a role in adaptive resistance against the diamide of S. maltophilia.

The role of MfsR, a TetR-type transcriptional regulator, in adaptive protection of Stenotrophomonas maltophilia against benzalkonium chloride via the regulation of mfsQ.

A gene encoding the TetR-type transcriptional regulator mfsR is located immediately downstream of mfsQ and is transcribed in the same transcriptional unit. mfsQ encodes a major facilitator superfamily (MFS) efflux transporter contributing to the resistance of Stenotrophomonas maltophilia towards disinfectants belonging to quaternary ammonium compounds (QACs), which include benzalkonium chloride (BAC). Phylogenetic analysis revealed that MfsR is closely related to CgmR, a QAC-responsive transcriptional regulator belonging to the TetR family. MfsR regulated the expression of the mfsQR operon in a QAC-inducible manner. The constitutively high transcript level of mfsQ in an mfsR mutant indicated that MfsR functions as a transcriptional repressor of the mfsQR operon. Electrophoretic mobility shift assays showed that purified MfsR specifically bound to the putative promoter region of mfsQR, and in vitro treatments with QACs led to the release of MfsR from binding complexes. DNase I protection assays revealed that the MfsR binding box comprises inverted palindromic sequences located between motifs -35 and -10 of the putative mfsQR promoter. BAC-induced adaptive protection was abolished in the mfsR mutant and was restored in the complemented mutant. Overall, MfsR is a QACs-sensing regulator that controls the expression of mfsQ. In the absence of QACs, MfsR binds to the box located in the mfsQR promoter and represses its transcription. The presence of QACs derepresses MfsR activity, allowing RNA polymerase binding and transcription of mfsQR. This MfsR-MsfQ system enables S. maltophilia to withstand high levels of QACs.

mfsQ encoding an MFS efflux pump mediates adaptive protection of Stenotrophomonas maltophilia against benzalkonium chloride.

The persistence of Stenotrophomonas maltophilia, especially in hospital environments where disinfectants are used intensively, is one of the important factors that allow this opportunistic pathogen to establish nosocomial infections. In the present study, we illustrated that S. maltophilia possesses adaptive resistance to the disinfectant benzalkonium chloride (BAC). This BAC adaptation was abolished in the ΔmfsQ mutant, in which a gene encoding an efflux transporter belonging to the major facilitator superfamily (MFS) was deleted. The ΔmfsQ mutant also showed increased susceptibility to BAC and chlorhexidine gluconate compared with a parental wild type. The expression of mfsQ increased upon exposure to quaternary ammonium compounds, including BAC. Thus, the results of this study suggest that mfsQ plays a role in both adaptive and nonadaptive protection of S. maltophilia from the toxicity of the disinfectant BAC.

Identification of Burkholderia pseudomallei Genes Induced During Infection of Macrophages by Differential Fluorescence Induction.

Burkholderia pseudomallei, the causative agent of melioidosis, can survive and replicate in macrophages. Little is known about B. pseudomallei genes that are induced during macrophage infection. We constructed a B. pseudomallei K96243 promoter trap library with genomic DNA fragments fused to the 5' end of a plasmid-borne gene encoding enhanced green fluorescent protein (eGFP). Microarray analysis showed that the library spanned 88% of the B. pseudomallei genome. The recombinant plasmids were introduced into Burkholderia thailandensis E264, and promoter fusions active during in vitro culture were removed. J774A.1 murine macrophages were infected with the promoter trap library, and J774A.1 cells containing fluorescent bacteria carrying plasmids with active promoters were isolated using flow cytometric-based cell sorting. Candidate macrophage-induced B. pseudomallei genes were identified from the location of the insertions containing an active promoter activity. A proportion of the 138 genes identified in this way have been previously reported to be involved in metabolism and transport, virulence, or adaptation. Novel macrophage-induced B. pseudomallei genes were also identified. Quantitative reverse-transcription PCR analysis of 13 selected genes confirmed gene induction during macrophage infection. Deletion mutants of two macrophage-induced genes from this study were attenuated in Galleria mellonella larvae, suggesting roles in virulence. B. pseudomallei genes activated during macrophage infection may contribute to intracellular life and pathogenesis and merit further investigation toward control strategies for melioidosis.

Over-Expression of Hypochlorite Inducible Major Facilitator Superfamily (MFS) Pumps Reduces Antimicrobial Drug Susceptibility by Increasing the Production of MexXY Mediated by ArmZ in Pseudomonas aeruginosa.

Pseudomonas aeruginosa, a well-known cause of nosocomial infection, is frequently antibiotic resistant and this complicates treatment. Links between oxidative stress responses inducing antibiotic resistance through over-production of RND-type efflux pumps have been reported in P. aeruginosa, but this has not previously been associated with MFS-type efflux pumps. Two MFS efflux pumps encoded by mfs1 and mfs2 were selected for study because they were found to be sodium hypochlorite (NaOCl) inducible. Antibiotic susceptibility testing was used to define the importance of these MFS pumps in antibiotic resistance and proteomics was used to characterize the resistance mechanisms involved. The results revealed that mfs1 is NaOCl inducible whereas mfs2 is NaOCl, N-Ethylmaleimide and t-butyl hydroperoxide inducible. Deletion of mfs1 or mfs2 did not affect antibiotic or paraquat susceptibility. However, over-production of Mfs1 and Mfs2 reduced susceptibility to aminoglycosides, quinolones, and paraquat. Proteomics, gene expression analysis and targeted mutagenesis showed that over-production of the MexXY RND-type efflux pump in a manner dependent upon armZ, but not amgRS, is the cause of reduced antibiotic susceptibility upon over-production of Mfs1 and Mfs2. mexXY operon expression analysis in strains carrying various lengths of mfs1 and mfs2 revealed that at least three transmembrane domains are necessary for mexXY over-expression and decreased antibiotic susceptibility. Over-expression of the MFS-type efflux pump gene tetA(C) did not give the same effect. Changes in paraquat susceptibility were independent of mexXY and armZ suggesting that it is a substrate of Mfs1 and Mfs2. Altogether, this is the first evidence of cascade effects where the over-production of an MFS pump causes over-production of an RND pump, in this case MexXY via increased armZ expression.

TrmB, a tRNA m7G46 methyltransferase, plays a role in hydrogen peroxide resistance and positively modulates the translation of katA and katB mRNAs in Pseudomonas aeruginosa.

Cellular response to oxidative stress is a crucial mechanism that promotes the survival of Pseudomonas aeruginosa during infection. However, the translational regulation of oxidative stress response remains largely unknown. Here, we reveal a tRNA modification-mediated translational response to H2O2 in P. aeruginosa. We demonstrated that the P. aeruginosa trmB gene encodes a tRNA guanine (46)-N7-methyltransferase that catalyzes the formation of m7G46 in the tRNA variable loop. Twenty-three tRNA substrates of TrmB with a guanosine residue at position 46 were identified, including 11 novel tRNA substrates. We showed that loss of trmB had a strong negative effect on the translation of Phe- and Asp-enriched mRNAs. The trmB-mediated m7G modification modulated the expression of the catalase genes katA and katB, which are enriched with Phe/Asp codons at the translational level. In response to H2O2 exposure, the level of m7G modification increased, consistent with the increased translation efficiency of Phe- and Asp-enriched mRNAs. Inactivation of trmB led to decreased KatA and KatB protein abundance and decreased catalase activity, resulting in H2O2-sensitive phenotype. Taken together, our observations reveal a novel role of m7G46 tRNA modification in oxidative stress response through translational regulation of Phe- and Asp-enriched genes, such as katA and katB.

Transcriptional regulation of the Pseudomonas aeruginosa iron-sulfur cluster assembly pathway by binding of IscR to multiple sites.

Iron-sulfur ([Fe-S]) cluster proteins have essential functions in many biological processes. [Fe-S] homeostasis is crucial for bacterial survival under a wide range of environmental conditions. IscR is a global transcriptional regulator in Pseudomonas aeruginosa; it has been shown to regulate genes involved in [Fe-S] cluster biosynthesis, iron homeostasis, resistance to oxidants, and pathogenicity. Many aspects of the IscR transcriptional regulatory mechanism differ from those of other well-studied systems. This study demonstrates the mechanisms of IscR Type-1 binding to its target sites that mediate the repression of gene expression at the isc operon, nfuA, and tpx. The analysis of IscR binding to multiple binding sites in the promoter region of the isc operon reveals that IscR first binds to the high-affinity site B followed by binding to the low-affinity site A. The results of in vitro IscR binding assays and in vivo analysis of IscR-mediated repression of gene expression support the role of site B as the primary site, while site A has only a minor role in the efficiency of IscR repression of gene expression. Ligation of an [Fe-S] cluster to IscR is required for the binding of IscR to target sites and in vivo repression and stress-induced gene expression. Analysis of Type-1 sites in many bacteria, including P. aeruginosa, indicates that the first and the last three AT-rich bases were among the most highly conserved bases within all analyzed Type-1 sites. Herein, we first propose the putative sequence of P. aeruginosa IscR Type-1 binding motif as 5'AWWSSYRMNNWWWTNNNWSGGNYWW3'. This can benefit further studies in the identification of novel genes under the IscR regulon and the regulatory mechanism model of P. aeruginosa IscR as it contributes to the roles of an [Fe-S] cluster in several biologically important cellular activities.

Inactivation of ahpC renders Stenotrophomonas maltophilia resistant to the disinfectant hydrogen peroxide.

Inactivation of ahpC, encoding alkyl hydroperoxide reductase, rendered Stenotrophomonas maltophilia more resistant to H2O2; the phenotype was directly correlated with enhanced total catalase activity, resulting from an increased level of KatA catalase. Plasmid-borne expression of ahpC from pAhpCsm could complement all of the mutant phenotypes. Mutagenesis of the proposed AhpC peroxidactic and resolving cysteine residues to alanine (C47A and C166A) on the pAhpCsm plasmid diminished its ability to complement the ahpC mutant phenotypes, suggesting that the mutagenized ahpC was non-functional. As mutations commonly occur in bacteria living in hostile environment, our data suggest that point mutations in ahpC at codons required for the enzyme function (such as C47 and C166), the AhpC will be non-functional, leading to high resistance to the disinfectant H2O2.

Pseudomonas aeruginosa glutathione biosynthesis genes play multiple roles in stress protection, bacterial virulence and biofilm formation.

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.

Pseudomonas aeruginosa ttcA encoding tRNA-thiolating protein requires an iron-sulfur cluster to participate in hydrogen peroxide-mediated stress protection and pathogenicity.

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.

Pseudomonas aeruginosa nfuA: Gene regulation and its physiological roles in sustaining growth under stress and anaerobic conditions and maintaining bacterial virulence.

The role of the nfuA gene encoding an iron-sulfur ([Fe-S]) cluster-delivery protein in the pathogenic bacterium Pseudomonas aeruginosa was investigated. The analysis of nfuA expression under various stress conditions showed that superoxide generators, a thiol-depleting agent and CuCl2 highly induced nfuA expression. The expression of nfuA was regulated by a global [2Fe-2S] cluster containing the transcription regulator IscR. Increased expression of nfuA in the ΔiscR mutant under uninduced conditions suggests that IscR acts as a transcriptional repressor. In vitro experiments revealed that IscR directly bound to a sequence homologous to the Escherichia coli Type-I IscR-binding motifs on a putative nfuA promoter that overlapped the -35 element. Binding of IscR prevented RNA polymerase from binding to the nfuA promoter, leading to repression of the nfuA transcription. Physiologically, deletion of nfuA reduced the bacterial ability to cope with oxidative stress, iron deprivation conditions and attenuated virulence in the Caenorhabditis elegans infection model. Site-directed mutagenesis analysis revealed that the conserved CXXC motif of the Nfu-type scaffold protein domain at the N-terminus was required for the NfuA functions in conferring the stress resistance phenotype. Furthermore, anaerobic growth of the ΔnfuA mutant in the presence of nitrate was drastically retarded. This phenotype was associated with a reduction in the [Fe-S] cluster containing nitrate reductase enzyme activity. However, NfuA was not required for the maturation of [Fe-S]-containing proteins such as aconitase, succinate dehydrogenase, SoxR and IscR. Taken together, our results indicate that NfuA functions in [Fe-S] cluster delivery to selected target proteins that link to many physiological processes such as anaerobic growth, bacterial virulence and stress responses in P. aeruginosa.

Inactivation of bpsl1039-1040 ATP-binding cassette transporter reduces intracellular survival in macrophages, biofilm formation and virulence in the murine model of Burkholderia pseudomallei infection.

Burkholderia pseudomallei, a gram-negative intracellular bacillus, is the causative agent of a tropical infectious disease called melioidosis. Bacterial ATP-binding cassette (ABC) transporters import and export a variety of molecules across bacterial cell membranes. At present, their significance in B. pseudomallei pathogenesis is poorly understood. We report here characterization of the BPSL1039-1040 ABC transporter. B. pseudomallei cultured in M9 medium supplemented with nitrate, demonstrated that BPSL1039-1040 is involved in nitrate transport for B. pseudomallei growth under anaerobic, but not aerobic conditions, suggesting that BPSL1039-1040 is functional under reduced oxygen tension. In addition, a nitrate reduction assay supported the function of BPSL1039-1040 as nitrate importer. A bpsl1039-1040 deficient mutant showed reduced biofilm formation as compared with the wild-type strain (P = 0.027) when cultured in LB medium supplemented with nitrate under anaerobic growth conditions. This reduction was not noticeable under aerobic conditions. This suggests that a gradient in oxygen levels could regulate the function of BPSL1039-1040 in B. pseudomallei nitrate metabolism. Furthermore, the B. pseudomallei bpsl1039-1040 mutant had a pronounced effect on plaque formation (P < 0.001), and was defective in intracellular survival in both non-phagocytic (HeLa) and phagocytic (J774A.1 macrophage) cells, suggesting reduced virulence in the mutant strain. The bpsl1039-1040 mutant was found to be attenuated in a BALB/c mouse intranasal infection model. Complementation of the bpsl1039-1040 deficient mutant with the plasmid-borne bpsl1039 gene could restore the phenotypes observed. We propose that the ability to acquire nitrate for survival under anaerobic conditions may, at least in part, be important for intracellular survival and has a contributory role in the pathogenesis of B. pseudomallei.

Overexpression of Stenotrophomonas maltophilia major facilitator superfamily protein MfsA increases resistance to fluoroquinolone antibiotics.

Background: Stenotrophomonas maltophilia is an opportunistic human pathogen causing nosocomial infections worldwide. S. maltophilia infection is of particular concern due to its inherent resistance to currently used antibiotics. Proton motive force-driven transporters of the major facilitator superfamily frequently contribute to the efflux of substances, including antibiotics, across cell membranes. Methods: An mfsA expression plasmid (pMfsA) was constructed and transferred into bacterial strains by electroporation. The antibiotic susceptibility levels of S. maltophilia strains were determined using standard methods. Results and conclusions: S. maltophilia MfsA is an efflux pump associated with paraquat resistance. We show here that plasmid-mediated overexpression of mfsA in WT S. maltophilia K279a increased resistance not only to paraquat but also to second-generation fluoroquinolone antibiotics, i.e. ciprofloxacin, norfloxacin, levofloxacin and ofloxacin. Ciprofloxacin was used as a representative drug. Addition of the proton motive force inhibitor carbonyl cyanide-m-chlorophenylhydrazone increases susceptibility to ciprofloxacin. Taken together these results suggest that MsfA is a novel fluoroquinolone efflux pump of S. maltophilia. Moreover, heterologous expression of mfsA in other Gram-negative pathogenic bacteria conferred resistance to paraquat as well as to fluoroquinolones. Thus, if this determinant was horizontally transferred, it could cause the spread of fluoroquinolone resistance among bacterial species.

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.

Agrobacterium tumefaciens estC, Encoding an Enzyme Containing Esterase Activity, Is Regulated by EstR, a Regulator in the MarR Family.

Analysis of the A. tumefaciens genome revealed estC, which encodes an esterase located next to its transcriptional regulator estR, a regulator of esterase in the MarR family. Inactivation of estC results in a small increase in the resistance to organic hydroperoxides, whereas a high level of expression of estC from an expression vector leads to a reduction in the resistance to organic hydroperoxides and menadione. The estC gene is transcribed divergently from its regulator, estR. Expression analysis showed that only high concentrations of cumene hydroperoxide (CHP, 1 mM) induced expression of both genes in an EstR-dependent manner. The EstR protein acts as a CHP sensor and a transcriptional repressor of both genes. EstR specifically binds to the operator sites OI and OII overlapping the promoter elements of estC and estR. This binding is responsible for transcription repression of both genes. Exposure to organic hydroperoxide results in oxidation of the sensing cysteine (Cys16) residue of EstR, leading to a release of the oxidized repressor from the operator sites, thereby allowing transcription and high levels of expression of both genes. The estC is the first organic hydroperoxide-inducible esterase-encoding gene in alphaproteobacteria.

Regulation of Organic Hydroperoxide Stress Response by Two OhrR Homologs in Pseudomonas aeruginosa.

Pseudomonas aeruginosa ohrR and ospR are gene homologs encoding oxidant sensing transcription regulators. OspR is known to regulate gpx, encoding a glutathione peroxidase, while OhrR regulates the expression of ohr that encodes an organic peroxide specific peroxiredoxin. Here, we show that ospR mediated gpx expression, like ohrR and ohr, 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 ohrR mutant, i.e. it regulates ohr in an oxidant dependent manner. In an ohrR mutant, in which ohr is derepressed, the induction of gpx expression by organic hydroperoxide is reduced. Likewise, in an ospR mutant, where gpx expression is constitutively high, oxidant dependent induction of ohr expression is reduced. Moreover, in vitro binding assays show that OspR binds the ohr promoter, while OhrR binds the gpx promoter, albeit with lower affinity. The binding of OhrR to the gpx 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 gpx promoter, while only C24 is essential at the ohr promoter. Overall, this study illustrates possible connection between two regulatory switches in response to oxidative stress.