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1.
Microb Drug Resist ; 29(4): 115-126, 2023 Apr.
Article in English | MEDLINE | ID: mdl-36897754

ABSTRACT

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.


Subject(s)
Gram-Negative Bacterial Infections , Stenotrophomonas maltophilia , Humans , Anti-Bacterial Agents/pharmacology , Ceftazidime/pharmacology , Stenotrophomonas maltophilia/genetics , Drug Resistance, Bacterial/genetics , Gram-Negative Bacterial Infections/drug therapy , Microbial Sensitivity Tests , Ciprofloxacin/pharmacology
2.
PLoS One ; 17(8): e0272388, 2022.
Article in English | MEDLINE | ID: mdl-35913917

ABSTRACT

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.


Subject(s)
Stenotrophomonas maltophilia , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Cysteine/metabolism , Diamide/metabolism , Diamide/pharmacology , Gene Expression Regulation, Bacterial , Membrane Transport Proteins/genetics , Membrane Transport Proteins/metabolism , Promoter Regions, Genetic , Stenotrophomonas maltophilia/genetics , Stenotrophomonas maltophilia/metabolism
3.
FEMS Microbiol Lett ; 368(15)2021 08 10.
Article in English | MEDLINE | ID: mdl-34329426

ABSTRACT

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.


Subject(s)
Bacterial Proteins , Benzalkonium Compounds , Stenotrophomonas maltophilia , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Benzalkonium Compounds/pharmacology , Gene Expression Regulation, Bacterial , Membrane Transport Proteins/genetics , Operon , Phylogeny , Quaternary Ammonium Compounds/pharmacology , Stenotrophomonas maltophilia/classification , Stenotrophomonas maltophilia/drug effects , Stenotrophomonas maltophilia/genetics , Stenotrophomonas maltophilia/metabolism
4.
Can J Microbiol ; 67(6): 491-495, 2021 Jun.
Article in English | MEDLINE | ID: mdl-33151759

ABSTRACT

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.


Subject(s)
Benzalkonium Compounds/pharmacology , Disinfectants/pharmacology , Drug Resistance, Bacterial/genetics , Membrane Transport Proteins/genetics , Stenotrophomonas maltophilia/physiology , Genes, Bacterial , Humans , Membrane Transport Proteins/metabolism , Microbial Sensitivity Tests , Mutation , Quaternary Ammonium Compounds/pharmacology , Stenotrophomonas maltophilia/drug effects , Stenotrophomonas maltophilia/genetics
5.
Front Microbiol ; 11: 592153, 2020.
Article in English | MEDLINE | ID: mdl-33510718

ABSTRACT

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.

6.
Antonie Van Leeuwenhoek ; 112(5): 809-814, 2019 May.
Article in English | MEDLINE | ID: mdl-30467663

ABSTRACT

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.


Subject(s)
Bacterial Proteins/genetics , Disinfectants/pharmacology , Hydrogen Peroxide/pharmacology , Peroxiredoxins/genetics , Stenotrophomonas maltophilia/drug effects , Stenotrophomonas maltophilia/enzymology , Bacterial Proteins/metabolism , Drug Resistance, Bacterial , Gene Expression Regulation, Bacterial , Gene Silencing , Peroxiredoxins/metabolism , Stenotrophomonas maltophilia/genetics
7.
J Antimicrob Chemother ; 73(5): 1263-1266, 2018 05 01.
Article in English | MEDLINE | ID: mdl-29462315

ABSTRACT

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.


Subject(s)
Anti-Bacterial Agents/pharmacology , Bacterial Proteins/biosynthesis , Drug Resistance, Bacterial , Fluoroquinolones/pharmacology , Gene Expression , Membrane Transport Proteins/biosynthesis , Stenotrophomonas maltophilia/drug effects , Bacterial Proteins/genetics , Genetic Vectors , Membrane Transport Proteins/genetics , Microbial Sensitivity Tests , Plasmids , Stenotrophomonas maltophilia/metabolism , Transformation, Bacterial
8.
PLoS One ; 12(12): e0189066, 2017.
Article in English | MEDLINE | ID: mdl-29216242

ABSTRACT

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 <2 or 500 mM H2O2 in planktonic and biofilm bacteria, respectively. In this study, we first employed a bioinformatic approach to elucidate the potential regulatory breadth of OxyR by scanning the entire P. aeruginosa PAO1 genome for canonical OxyR promoter recognition sequences (ATAG-N7-CTAT-N7-ATAG-N7-CTAT). Of >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.


Subject(s)
Bacterial Proteins/genetics , Genes, Bacterial , Pseudomonas aeruginosa/drug effects , Pseudomonas aeruginosa/genetics , tert-Butylhydroperoxide/pharmacology , DNA Footprinting , Electrophoretic Mobility Shift Assay , Reverse Transcriptase Polymerase Chain Reaction
9.
PLoS One ; 11(12): e0168791, 2016.
Article in English | MEDLINE | ID: mdl-28036400

ABSTRACT

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.


Subject(s)
Agrobacterium tumefaciens/genetics , Bacterial Proteins/genetics , Esterases/genetics , Agrobacterium tumefaciens/metabolism , Amino Acid Sequence , Gene Expression Regulation, Bacterial/genetics , Hydrogen Peroxide/metabolism , Oxidation-Reduction , Promoter Regions, Genetic , Repressor Proteins/genetics , Sequence Alignment , Transcription Factors/genetics
11.
PLoS One ; 10(4): e0123699, 2015.
Article in English | MEDLINE | ID: mdl-25915643

ABSTRACT

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.


Subject(s)
Anti-Bacterial Agents/pharmacology , Bacterial Proteins/genetics , Drug Resistance, Microbial , Gene Expression Regulation, Bacterial , Membrane Transport Proteins/genetics , Paraquat/pharmacology , Stenotrophomonas maltophilia/genetics , Transcription Factors/genetics , Amino Acid Sequence , Bacterial Proteins/metabolism , Base Sequence , Membrane Transport Proteins/metabolism , Molecular Sequence Data , Promoter Regions, Genetic , Protein Binding , Stenotrophomonas maltophilia/drug effects , Stenotrophomonas maltophilia/metabolism , Transcription Factors/metabolism , Transcriptional Activation
12.
Can J Microbiol ; 61(5): 343-50, 2015 May.
Article in English | MEDLINE | ID: mdl-25825971

ABSTRACT

The exposure of Xanthomonas campestris pv. campestris to sublethal concentrations of a sodium hypochlorite (NaOCl) solution induced the expression of genes that encode peroxide scavenging enzymes within the OxyR and OhrR regulons. Sensitivity testing in various X. campestris mutants indicated that oxyR, katA, katG, ahpC, and ohr contributed to protection against NaOCl killing. The pretreatment of X. campestris cultures with oxidants, such as hydrogen peroxide (H2O2), t-butyl hydroperoxide, and the superoxide generator menadione, protected the bacteria from lethal concentrations of NaOCl in an OxyR-dependent manner. Treating the bacteria with a low concentration of NaOCl resulted in the adaptive protection from NaOCl killing and also provided cross-protection from H2O2 killing. Taken together, the results suggest that the toxicity of NaOCl is partially mediated by the generation of peroxides and other reactive oxygen species that are removed by primary peroxide scavenging enzymes, such as catalases and AhpC, as a part of an overall strategy that protects the bacteria from the lethal effects of NaOCl.


Subject(s)
Peroxides/metabolism , Regulon , Sodium Hypochlorite/pharmacology , Xanthomonas campestris/drug effects , Catalase/metabolism , Gene Expression Regulation, Bacterial , Hydrogen Peroxide/pharmacology , Xanthomonas campestris/genetics , Xanthomonas campestris/metabolism
13.
Microbiology (Reading) ; 160(Pt 2): 458-466, 2014 Feb.
Article in English | MEDLINE | ID: mdl-24385479

ABSTRACT

Copper (Cu)-based biocides are currently used as control measures for both fungal and bacterial diseases in agricultural fields. In this communication, we show that exposure of the bacterial plant pathogen Xanthomonas campestris to nonlethal concentrations of Cu(2+) ions (75 µM) enhanced expression of genes in OxyR, OhrR and IscR regulons. High levels of catalase, Ohr peroxidase and superoxide dismutase diminished Cu(2+)-induced gene expression, suggesting that the production of hydrogen peroxide (H2O2) and organic hydroperoxides is responsible for Cu(2+)-induced gene expression. Despite high expression of antioxidant genes, the CuCl2-treated cells were more susceptible to H2O2 killing treatment than the uninduced cells. This phenotype arose from lowered catalase activity in the CuCl2-pretreated cells. Thus, exposure to a nonlethal dose of Cu(2+) renders X. campestris vulnerable to H2O2, even when various genes for peroxide-metabolizing enzymes are highly expressed. Moreover, CuCl2-pretreated cells are sensitive to treatment with the redox cycling drug, menadione. No physiological cross-protection response was observed in CuCl2-treated cells in a subsequent challenge with killing concentrations of an organic hydroperoxide. As H2O2 production is an important initial plant immune response, defects in H2O2 protection are likely to reduce bacterial survival in plant hosts and enhance the usefulness of copper biocides in controlling bacterial pathogens.


Subject(s)
Anti-Bacterial Agents/toxicity , Antioxidants/metabolism , Copper/toxicity , Gene Expression Regulation, Bacterial/drug effects , Hydrogen Peroxide/toxicity , Oxidative Stress , Xanthomonas campestris/drug effects , Microbial Sensitivity Tests , Xanthomonas campestris/genetics
14.
Arch Microbiol ; 195(9): 671-4, 2013 Sep.
Article in English | MEDLINE | ID: mdl-23907582

ABSTRACT

The presence of the widely used selectable antibiotic marker, tetA(C), unexpectedly increased the sensitivity of Pseudomonas aeruginosa PAO1 to the superoxide-generating herbicide, paraquat. A DNA fragment spanning the first 99 amino acids of TetA(C) was sufficient to confer paraquat sensitivity. The TetA(C)-induced paraquat sensitive phenotype was observed in other Gram-negative bacteria such as Agrobacterium tumefaciens, Salmonella enterica ser. Typhimurium and Xanthomonas campestris suggesting that this is a general property of tetA(C). This finding serves as a cautionary note for those using tetA(C) as a selectable marker for genetic manipulations in studies using paraquat either as a superoxide stress generator or a redox cycling drug.


Subject(s)
Antiporters/metabolism , Bacterial Proteins/metabolism , Herbicides/pharmacology , Paraquat/metabolism , Pseudomonas aeruginosa/drug effects , Pseudomonas aeruginosa/metabolism , Bacterial Proteins/genetics , Oxidation-Reduction , Paraquat/pharmacology , Superoxides/metabolism
15.
J Bacteriol ; 194(2): 209-17, 2012 Jan.
Article in English | MEDLINE | ID: mdl-22056938

ABSTRACT

In Xanthomonas campestris pv. campestris, SoxR likely functions as a sensor of redox-cycling drugs and as a transcriptional regulator. Oxidized SoxR binds directly to its target site and activates the expression of xcc0300, a gene that has protective roles against the toxicity of redox-cycling compounds. In addition, SoxR acts as a noninducible repressor of its own expression. X. campestris pv. campestris requires SoxR both for protection against redox-cycling drugs and for full virulence on a host plant. The X. campestris model of the gene regulation and physiological roles of SoxR represents a novel variant of existing bacterial SoxR models.


Subject(s)
Bacterial Proteins/metabolism , Transcription Factors/metabolism , Xanthomonas campestris/metabolism , Bacterial Proteins/genetics , Base Sequence , Host-Pathogen Interactions , Molecular Sequence Data , Oxidation-Reduction , Plant Diseases/microbiology , Promoter Regions, Genetic , Raphanus/microbiology , Stress, Physiological , Transcription Factors/genetics , Virulence , Xanthomonas campestris/genetics , Xanthomonas campestris/pathogenicity
16.
Infect Immun ; 80(3): 921-8, 2012 Mar.
Article in English | MEDLINE | ID: mdl-22184416

ABSTRACT

Helicobacter cinaedi, a common human intestinal bacterium, has been implicated in various enteric and systemic diseases in normal and immunocompromised patients. Protection against oxidative stress is a crucial component of bacterium-host interactions. Alkyl hydroperoxide reductase C (AhpC) is an enzyme responsible for detoxification of peroxides and is important in protection from peroxide-induced stress. H. cinaedi possesses a single ahpC, which was investigated with respect to its role in bacterial survival during oxidative stress. The H. cinaedi ahpC mutant had diminished resistance to organic hydroperoxide toxicity but increased hydrogen peroxide resistance compared with the wild-type (WT) strain. The mutant also exhibited an oxygen-sensitive phenotype and was more susceptible to killing by macrophages than the WT strain. In vivo experiments in BALB/c and BALB/c interleukin-10 (IL-10)(-/-) mice revealed that the cecal colonizing ability of the ahpC mutant was significantly reduced. The mutant also had diminished ability to induce bacterium-specific immune responses in vivo, as shown by immunoglobulin (IgG2a and IgG1) serum levels. Collectively, these data suggest that H. cinaedi ahpC not only contributes to protecting the organism against oxidative stress but also alters its pathogenic properties in vivo.


Subject(s)
Bacterial Proteins/metabolism , Helicobacter Infections/microbiology , Helicobacter/pathogenicity , Interleukin-10/immunology , Microbial Viability , Oxidative Stress , Peroxidases/metabolism , Stress, Physiological , Animals , Bacterial Load , Bacterial Proteins/genetics , Cecum/microbiology , Female , Gene Deletion , Helicobacter/drug effects , Helicobacter/enzymology , Host-Pathogen Interactions , Hydrogen Peroxide/toxicity , Interleukin-10/deficiency , Macrophages/immunology , Macrophages/microbiology , Male , Mice , Mice, Inbred BALB C , Mice, Knockout , Oxygen/toxicity , Peroxidases/genetics , Peroxiredoxins/genetics , Peroxiredoxins/metabolism , Virulence Factors/genetics , Virulence Factors/metabolism
17.
Curr Microbiol ; 63(2): 232-7, 2011 Aug.
Article in English | MEDLINE | ID: mdl-21710133

ABSTRACT

Xanthomonas campestris pv. campestris causes black rot in cruciferous crops. Hydrogen peroxide (H(2)O(2)) production and accumulation is an important initial response in plant defense against invading microbes. The role of genes involved in the bacterial H(2)O(2) protection system in pathogenicity was evaluated. Mutants of katA (encoding a monofunctional catalase) and, to a lesser extent, katG (encoding a catalase-peroxidase) and oxyR (encoding a H(2)O(2) sensor and a transcription regulator), are hypersensitive to H(2)O(2) treatments that mimic the plant H(2)O(2) burst. These data correlate with the results of pathogenicity testing that show katA, katG, and oxyR mutants are avirulent on a compatible plant. Moreover, exposure to H(2)O(2) (1, 2, and 4 mM) highly induces the expression of genes in the OxyR regulon, including katA, katG, and ahpC. The avirulent phenotype of the oxyR mutant is partly because of its inability to mount an adaptive response upon exposure to an H(2)O(2) burst. Our data provide insights into important roles of a transcription regulator and other genes involved in peroxide stress protection in the virulence of X. campestris pv. campestris.


Subject(s)
Hydrogen Peroxide/toxicity , Oxidative Stress , Plant Diseases/microbiology , Regulon , Xanthomonas campestris/pathogenicity , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Catalase/metabolism , Mutant Proteins/genetics , Mutant Proteins/metabolism , Peroxidase/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism , Virulence , Xanthomonas campestris/drug effects , Xanthomonas campestris/genetics , Xanthomonas campestris/metabolism
18.
FEMS Microbiol Lett ; 317(1): 60-6, 2011 Apr.
Article in English | MEDLINE | ID: mdl-21219417

ABSTRACT

Xanthomonas campestris pv. campestris, a soil-borne plant-pathogenic bacterium, is exposed to multiple stresses in the environment and during interaction with a host plant. The roles of hydrogen peroxide (H(2) O(2) )-protective genes (katA, katG, and ahpC) and a peroxide sensor/transcription regulator (oxyR) in the viability of X. campestris pv. campestris at an elevated temperature were evaluated. The single katA and katG mutants showed moderate decreased survival after the heat treatment, while the double katA-katG and oxyR mutants were the most vulnerable to the heat treatment compared with a wild-type strain. However, ahpC provided no protective function against the heat treatment. Flow cytometric analysis revealed an increased accumulation of peroxide in cells treated with heat. Altogether, the data revealed a crucial role of genes in the H(2) O(2) detoxification system for protection against lethal heat shock in X. campestris pv. campestris.


Subject(s)
Hot Temperature , Microbial Viability/drug effects , Microbial Viability/radiation effects , Oxidative Stress , Stress, Physiological , Xanthomonas campestris/enzymology , Xanthomonas campestris/physiology , Biotransformation , Hydrogen Peroxide/metabolism , Hydrogen Peroxide/toxicity , Oxidants/metabolism , Oxidants/toxicity , Xanthomonas campestris/drug effects , Xanthomonas campestris/radiation effects
19.
FEMS Microbiol Lett ; 313(1): 75-80, 2010 Dec.
Article in English | MEDLINE | ID: mdl-21029152

ABSTRACT

Copper (Cu)-based biocides are important chemical controls for both fungal and bacterial diseases in crop fields. Here, we showed that Cu ions at a concentration of 100 µM enhanced t-butyl hydroperoxide (tBOOH) and hydrogen peroxide (H(2) O(2) ) killing of Xanthomonas campestris pv. campestris through different mechanisms. The addition of an antilipid peroxidation agent (α-tocopherol) and hydroxyl radical scavengers (glycerol and dimethyl sulphoxide) partially protected the bacteria from the Cu-enhanced tBOOH and H(2) O(2) killing, respectively. Inactivation of the alkyl hydroperoxide reductase gene rendered the mutant vulnerable to lethal doses of copper sulphate, which could be alleviated by the addition of an H(2) O(2) scavenger (pyruvate) and α-tocopherol. Taken together, the data suggest that Cu ions influence the killing effect of tBOOH through the stimulation of lipid peroxidation, while hydroxyl radical production is the underlying mechanism responsible for the Cu-ion-enhanced H(2) O(2) killing effects.


Subject(s)
Copper/pharmacology , Hydrogen Peroxide/toxicity , Ions/pharmacology , Xanthomonas campestris/drug effects , Xanthomonas campestris/metabolism , Bacterial Proteins/genetics , Drug Resistance, Bacterial/genetics , Xanthomonas campestris/genetics
20.
J Bacteriol ; 191(16): 5159-68, 2009 Aug.
Article in English | MEDLINE | ID: mdl-19502402

ABSTRACT

The copper resistance determinant copARZ, which encodes a CPx-type copper ATPase efflux protein, a transcriptional regulator, and a putative intracellular copper chaperone, was functionally characterized for the phytopathogenic bacterium Agrobacterium tumefaciens. These genes are transcribed as an operon, and their expression is induced in response to increasing copper and silver ion concentrations in a copR-dependent fashion. Analysis of the copARZ promoter revealed a putative CopR binding box located within the spacer of the -35 and -10 promoter motifs. In vitro, purified CopR could specifically bind to the box. The inactivation of the copARZ operon or copZ reduces the level of resistance to copper but not to other metal ions. Also, the copARZ operon mutant shows increased sensitivity to the superoxide generators menadione and plumbagin. In addition, the loss of functional copZ does not affect the ability of copper ions to induce the copARZ promoter, indicating that CopZ is not involved in the copper-sensing mechanism of CopR. Altogether, the results demonstrate a crucial role for the copARZ operon as a component of the copper resistance machinery in A. tumefaciens.


Subject(s)
Agrobacterium tumefaciens/drug effects , Agrobacterium tumefaciens/genetics , Bacterial Proteins/physiology , Copper/pharmacology , Operon/physiology , Trace Elements/pharmacology , Bacterial Proteins/genetics , Binding Sites/genetics , DNA Footprinting , DNA Transposable Elements/genetics , Gene Expression Regulation, Bacterial/drug effects , Gene Expression Regulation, Bacterial/genetics , Genome, Bacterial/genetics , Genome, Bacterial/physiology , Mutation , Naphthoquinones/pharmacology , Operon/genetics , Promoter Regions, Genetic/genetics , Reverse Transcriptase Polymerase Chain Reaction , Vitamin K 3/pharmacology
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