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Abstract Ribosomal RNA (rRNA) modifications are involved in multiple biological processes. KsgA is a 16S rRNA adenine dimethyltransferase that methylates at the adenines 1518 and 1519 (A1518/1519) positions, which are located near the ribosome decoding center. These methylations are conserved and important for ribosome biogenesis and protein translation. In this study, we demonstrated the absence of A1518/1519 methylation in the 16S rRNA of a Pseudomonas aeruginosa ksgA mutant. Biolog phenotypic microarrays were used to screen the phenotypes of the ksgA mutant against various antimicrobial agents. The loss of ksgA led to increased sensitivity to menadione, a superoxide generator, which was, at least in part, attributed to decreased in a superoxide dismutase (SOD) activity. Interestingly, the decrease in SOD activity in the ksgA mutant was linked to a decrease in the SodM protein levels, but not the sodM mRNA levels. Furthermore, the ksgA mutant strain exhibited sensitivity to hygromycin B and tylosin antibiotics. The tylosin-sensitive phenotype was correlated with decreased transcriptional levels of tufA, tufB, and tsf, which encode elongation factors. Additionally, the ksgA mutant showed resistance to kasugamycin. Collectively, these findings highlight the role of KsgA in oxidative stress responses and antibiotic sensitivity in P. aeruginosa.

Public toilets may increase the risk of COVID-19 infection via airborne transmission; however, related research is limited. We aimed to estimate SARS-CoV-2 infection risk through respiratory transmission using a quantitative microbial risk assessment framework by retrieving SARS-CoV-2 concentrations from the swab tests of 251 Thai patients. Three virus-generating scenarios were investigated: an infector breathing, breathing with a cough, and breathing with a sneeze. The infection risk (95th percentile) was as high as 10−1 with breathing and increased to 1 with a cough or a sneeze. No significant gender differences for toilet users (receptors) were noted. The highest risk scenario, namely breathing with a sneeze, was further evaluated for risk mitigation measures. Mitigation to a lower risk under 10−3 succeeded only when the infector and the receptor both wore N95 respirators or surgical masks. Ventilation of up to 20 air changes per hour (ACH) did not decrease the risk. However, an extended waiting time of 10 min between an infector and a receptor resulted in approximately 1.0-log10 further risk reduction when both wore masks with the WHO-recommended 12 ACH. The volume of expelled droplets, virus concentrations, and receptor dwell time were identified as the main contributors to transmission risk. HIGHLIGHTS The use of public toilets poses a risk of SARS-CoV-2 respiratory transmission.; No gender differences in risk by counteracting dwell times and inhalation rates.; Ventilation alone did not reduce risk at 20 ACH with immediate receptor entrance.; 10-min waiting time further mitigated risks beyond face masks at 12 ACH ventilation.; N95 and surgical masks offer the most effective risk mitigation to toilet users.;

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.

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.

Objectives: Antibacterial resistance (ABR) is a major global health security threat, with a disproportionate burden on lower-and middle-income countries (LMICs). It is not understood how ‘One Health’, where human health is co-dependent on animal health and the environment, might impact the burden of ABR in LMICs. Thailand's 2017 “National Strategic Plan on Antimicrobial Resistance” (NSP-AMR) aims to reduce AMR morbidity by 50% through 20% reductions in human and 30% in animal antibacterial use (ABU). There is a need to understand the implications of such a plan within a One Health perspective. Methods: A model of ABU, gut colonisation with extended-spectrum beta-lactamase (ESBL)-producing bacteria and transmission was calibrated using estimates of the prevalence of ESBL-producing bacteria in Thailand. This model was used to project the reduction in human ABR over 20 years (2020–2040) for each One Health driver, including individual transmission rates between humans, animals and the environment, and to estimate the long-term impact of the NSP-AMR intervention. Results: The model predicts that human ABU was the most important factor in reducing the colonisation of humans with resistant bacteria (maximum 65.7–99.7% reduction). The NSP-AMR is projected to reduce human colonisation by 6.0–18.8%, with more ambitious targets (30% reductions in human ABU) increasing this to 8.5–24.9%. Conclusions: Our model provides a simple framework to explain the mechanisms underpinning ABR, suggesting that future interventions targeting the simultaneous reduction of transmission and ABU would help to control ABR more effectively in Thailand.

Capsid integrity quantitative PCR (qPCR), a molecular detection method for infectious viruses combining azo dye pretreatment with qPCR, has been widely used in recent years; however, variations in pretreatment conditions for various virus types can limit the efficacy of specific protocols. By identifying and critically synthesizing forty-one recent peer-reviewed studies employing capsid integrity qPCR for viruses in the last decade (2009–2019) in the fields of food safety and environmental virology, we aimed to establish recommendations for the detection of infectious viruses. Intercalating dyes are effective measures of viability in PCR assays provided the viral capsid is damaged; viruses that have been inactivated by other causes, such as loss of attachment or genomic damage, are less well detected using this approach. Although optimizing specific protocols for each virus is recommended, we identify a framework for general assay conditions. These include concentrations of ethidium monoazide, propidium monoazide or its derivates between 10 and 200 μM; incubation on ice or at room temperature (20 - 25 °C) for 5–120 min; and dye activation using LED or high light (500–800 Watts) exposure for periods ranging from 5 to 20 min. These simple steps can benefit the investigation of infectious virus transmission in routine (water) monitoring settings and during viral outbreaks such as the current COVID-19 pandemic or endemic diseases like dengue fever.

Talaromyces marneffei is a thermally dimorphic fungus that causes opportunistic systemic mycoses in patients with AIDS or other immunodeficiency syndromes. The purpose of this study was to develop an immunochromatographic strip test (ICT) based on a solid phase sandwich format immunoassay for the detection of T. marneffei antigens in clinical urine specimens. The T. marneffei yeast phase specific monoclonal antibody 4D1 (MAb4D1) conjugated with colloidal gold nanoparticle was used as a specific signal reporter. Galanthus nivalis Agglutinin (GNA) was adsorbed onto nitrocellulose membrane to serve as the test line. Similarly, a control line was created above the test line by immobilization of rabbit anti-mouse IgG. The immobilized GNA served as capturing molecule and as non-immune mediated anti-terminal mannose of T. marneffei antigenic mannoprotein. The MAb4D1-GNA based ICT showed specific binding activity with yeast phase antigen of T. marneffei, and it did not react with other common pathogenic fungal antigens. The limit of detection of this ICT for T. marneffei antigen spiked in normal urine was approximately 0.6 μg/ml. The diagnostic performance of the ICT was validated using 341 urine samples from patents with culture- confirmed T. marneffei infection and from a control group of healthy individuals and patients with other infections in an endemic area. The ICT exhibited 89.47% sensitivity, 100% specificity, and 97.65% accuracy. Our results demonstrate that the urine-based GNA-MAb4D1 based ICT produces a visual result within 30 minutes and that the test is highly specific for the diagnosis of T. marneffei infection. The findings validate the deployment of the ICT for clinical use.

Rapid economic development has caused industrial expansion into residential communities, leading to higher fecal pollution loads that could be discharged into aquatic environments. However, little is known regarding the potential microbial impact on human health. This study investigated microbial contamination from coastal industrial–residential community areas in nine sampling sites in waterways during three dry events. A general microbial source tracking (MST) marker, GenBac3, was detected in all samples from all three events, indicating continuing fecal pollution in the area, mostly from human sewage contamination. This was shown by the human-specific genetic marker crAssphage (88.9%) and human polyomavirus (HPyVs; 92.6%) detection. Enteric human adenovirus (HAdV40/41) showed three positive results only from residential sites in the first event. No spatial difference was observed for MST markers and traditional fecal indicators (total coliforms and Escherichia coli) in each event. Still, a significantly lower abundance of GenBac3, HPyVs, and total coliforms in the first sampling event was detected. Spearman’s rho analysis indicated a strong correlation among certain pairs of microbial parameters. Multivariate analysis revealed two clusters of samples separated by land use type (industrial vs. residential). According to factor analysis of mixed data, the land use parameter was more associated with physicochemical parameters (i.e., salinity, conductivity, water temperature, and dissolved oxygen). A Quantitative Microbial Risk Assessment (QMRA) was then conducted to estimate the annual infection risks of HAdV40/41 for non-potable water reuse purposes using predicted concentrations from crAssphage and HPyVs. The highest risks (95th percentiles) were ranked by food crop irrigation, aquaculture, and toilet flushing, at 10–1, 10–2, and 10–3 per person per year (pppy). Required treatment levels to achieve a 10–4 pppy annual infection risk were estimated. QMRA-based water treatment scenarios were suggested, including chlorination for toilet flushing reuse and depth filtration prior to chlorination for aquaculture and food crop irrigation. Microbial monitoring combined with a QMRA could provide better insights into fecal pollution patterns and the associated risks, facilitating effective water quality management and appropriate prior treatments for water reuse.

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.

Typically, biological systems are protected from the toxic effect of free radicals by antioxidant defense. Extracts from orchids have been reported to show high levels of exogenous antioxidant activity including Bulbophyllum orchids but so far, there have been no reports on antioxidant enzymes. Therefore, differences in protein expression from leaves and pseudobulbs of Bulbophyllum morphologlorum Kraenzl and Dendrobium Sonia Earsakul were studied using two-dimensional gel electrophoresis and mass spectrometry (LC/MS/MS). Interestingly, the largest group of these stress response proteins were associated with antioxidant defense and temperature stress, including superoxide dismutase (Cu–Zn) and heat shock protein 70. The high expression of this antioxidant enzyme from Bulbophyllum morphologlorum Kraenzl was confirmed by activity staining on native-PAGE, and the two Cu/Zn-SODs isoenzymes were identified as Cu/Zn-SOD 1 and Cu/Zn-SOD 2 by LC/MS/MS. The results suggested that Bulbophyllum orchid can be a potential plant source for medicines and natural antioxidant supplements.

The occurrence of antibiotic resistance bacteria has become a major threat to public health. We have recently discovered a transcriptional activator that belongs to MarR family, EstR, and an esterase B (EstB) with a newly proposed de-arenethiolase activity from Sphingobium sp. SM42. De-arenethiolase activity involves the removal of the small aromatic side chain of cephalosporin antibiotics as an excellent leaving group by the enzymatic CS bond cleavage. Here, we report the regulation of estB through EstR as an activator in response to a third generation cephalosporin, cefoperazone, antibiotic. Cefoperazone induced the expression of estB in wild type Sphingobium sp., but not in the estR knockout strain, and the induction was restored in the complemented strain. Moreover, we revealed the importance of EstB localization in periplasm. Since EsB has the ability to inactivate selected β-lactam antibiotics in vitro, it is possible that the enzyme works at the periplasmic space of Gram negative bacteria similar to β-lactamases. EstB was genetically engineered by incorporating NlpA binding motif, or OmpA signal sequence, or SpyTag-SpyCatcher to the estB gene to mobilize it to different compartments of periplasm; inner membrane, outer membrane, and periplasmic space, respectively. Surprisingly, we found that Sphingobium sp. SM42 and E. coli expressing EstB at the periplasm were more sensitive to cefoperazone. The possible drug enhancement mechanism by enzyme was proposed. This work might lead to a novel strategy to tackle antibiotic resistance problem.

We reported modified endpoint PCR results analyzed by universal and human-, swine-, and cattle-specific Bacteroidales gene markers with human sewage and animal fecal samples (i.e., swine, cattle, chicken, goat, sheep, buffalo, and duck) from Tha Chin and Chao Phraya watersheds. Annealing locations of PCR primers were illustrated by maps of 16s rRNA Bacteroidales genes. We also summarized previously published work on the performance of the PCR assays. For further discussion of the data presented here, please refer to Somnark et al., Performance evaluation of Bacteroidales genetic markers for human and animal microbial source tracking in tropical agricultural watersheds, Environ. Pollut. 236 (2018) 100–110. Keywords: Endpoint PCR, Fecal pollution, Microbial source tracking, Bacteroidales, Sensitivity, Specificity, Water quality

Bacteriophages of the Bacteroides fragilis strains HSP40 and RYC2056 are used as indicators of human-specific and general (non-host specific) fecal pollution in water bodies. However, conventional anaerobic cultivation methods require 1-2 days of incubation. To overcome this limitation, in this study, we developed a DNA-based method to simultaneously detect representative bacteriophages (B40-8 and B56-3) that infect B. fragilis strains HSP40 and RYC2056, respectively. Both phages yielded a 224-bp amplicon with the primer pair BT5414/BT5415, and an additional 152-bp PCR product was observed for B40-8 with the primer pair BT5579/BT5580. The detection limits for B40-8 and B56-3 were 10-5 and 10-4 ng of pure DNA, and 1 and 50 ng of DNA template when 5 and 5,000 PFU/mL were spiked into distilled water, respectively. The assay exhibited a higher sensitivity for sewage samples, with < 0.1 and 15 PFU/mL of phages infecting HSP40 and RYC2056, respectively. The assay did not produce false positive results for the Bacteroides phages PG76, HB13, and GA17 or for the enterococcal phages AIM06 and SR14. The assay also detected RYC2056 phages that were isolated from sewage samples and the phage B40-8 when it was spiked into raw sewage. Thus, the newly developed PCR assay demonstrated potential for the environmental monitoring of Bacteroides bacteriophages, decreasing the analysis time to a few hours.

BackgroundAggregatibacter actinomycetemcomitans, a periodontal pathogen, secretes a cytolethal distending toxin (AaCDT) that causes host cell cycle arrest and cell death. Although CDT could be an important virulence factor, it is unclear how it enters the nucleus to exert its cytotoxicity.ObjectiveTo investigate the mechanisms of AaCDT by genome-wide screening for host mutations that confer resistance to the catalytic subunit, AaCdtB, in a Saccharomyces cerevisiae model.MethodsWe transformed the yeast haploid deletion library, a collection of yeast mutants with single gene deletions of virtually all non-essential ORFs in the genome, with plasmids carrying galactose-inducible AaCdtB. Yeast mutants that showed resistance to AaCdtB were selected and rescreened by a spotting assay. AaCdtB expression was confirmed by western blot analysis; any mutants that showed no or weak expression of AaCdtB were omitted from the analysis. The lists of genes whose mutations confer resistance to AaCdtB were analyzed for Gene Ontology (GO) term enrichments. Localization of AaCdtB-EGFP was examined using fluorescent microscopy. Nuclear localization relative to EGFP control was calculated and compared to wild-type.ResultsOut of approximately 5,000 deletion mutants, we isolated 243 mutants that are resistant to AaCdtB. GO analyses indicated that genes associated with organic anion transport are significantly enriched (16 genes). Furthermore, several genes associated with the nucleus and endoplasmic reticulum (ER) were identified. Localization studies of AaCdtB, in mutants with the deletion of genes associated with the GO term organic anion transport, showed lower nuclear localization than wild-type. The results suggest that these genes may be required for AaCdtB translocation into the nucleus and its cytotoxicity.ConclusionThe genome-wide screen in the yeast deletion library allowed us to identify a large number of host genes required for AaCdtB cytotoxicity. Further investigation could lead to more insights into the mechanisms of CdtB intoxication.

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.

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.

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.

The LysR member of bacterial transactivators, OxyR, governs transcription of genes involved in the response to H2O2 and organic (alkyl) hydroperoxides (AHP) in the Gram-negative pathogen, Pseudomonas aeruginosa. We have previously shown that organisms lacking OxyR are rapidly killed by 100 potential OxyR-controlled genes, 40 were strategically selected that were not predicted to be involved in the direct response to oxidative stress (e.g., catalase, peroxidase, etc.) and screened such genes by RT-PCR analysis for potentially positive or negative control by OxyR. Differences were found in 7 of 40 genes when comparing an oxyR mutant vs. PAO1 expression that was confirmed by ß-galactosidase reporter assays. Among these, phnW, encoding 2-aminoethylphosphonate:pyruvate aminotransferase, exhibited reduced expression in the oxyR mutant compared to wild-type bacteria. Electrophoretic mobility shift assays indicated binding of OxyR to the phnW promoter and DNase I footprinting analysis also revealed the sequences to which OxyR bound. Interestingly, a phnW mutant was more susceptible to t-butyl-hydroperoxide (t-BOOH) treatment than wild-type bacteria. Although we were unable to define the direct mechanism underlying this phenomenon, we believe that this may be due to a reduced efficiency for this strain to degrade t-BOOH relative to wild-type organisms because of modulation of AHP gene transcription in the phnW mutant.

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.

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.

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.

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.

Stenotrophomonas maltophilia MfsA (Smlt1083) is an efflux pump in the major facilitator superfamily (MFS). Deletion of mfsA renders the strain more susceptible to paraquat, but no alteration in the susceptibility levels of other oxidants is observed. The expression of mfsA is inducible upon challenge with redox cycling/superoxide-generating drug (paraquat, menadione and plumbagin) treatments and is directly regulated by SoxR, which is a transcription regulator and sensor of superoxide-generating agents. Analysis of mfsA expression patterns in wild-type and a soxR mutant suggests that oxidized SoxR functions as a transcription activator of the gene. soxR (smlt1084) is located in a head-to-head fashion with mfsA, and these genes share the -10 motif of their promoter sequences. Purified SoxR specifically binds to the putative mfsA promoter motifs that contain a region that is highly homologous to the consensus SoxR binding site, and mutation of the SoxR binding site abolishes binding of purified SoxR protein. The SoxR box is located between the putative -35 and -10 promoter motifs of mfsA; and this position is typical for a promoter in which SoxR acts as a transcriptional activator. At the soxR promoter, the SoxR binding site covers the transcription start site of the soxR transcript; thus, binding of SoxR auto-represses its own transcription. Taken together, our results reveal for the first time that mfsA is a novel member of the SoxR regulon and that SoxR binds and directly regulates its expression.

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.