Escherichia coli YqhD Exhibits Aldehyde Reductase Activity and Protects from the Harmful Effect of Lipid Peroxidation-derived Aldehydes
2008; Elsevier BV; Volume: 283; Issue: 12 Linguagem: Inglês
10.1074/jbc.m708846200
ISSN1083-351X
AutoresJosé M. Pérez, Felipe Arenas, Gonzalo A. Pradenas, Juan M. Sandoval, Claudio C. Vásquez,
Tópico(s)Microbial Metabolic Engineering and Bioproduction
ResumoEvidence that Escherichia coli YqhD is involved in bacterial response to compounds that generate membrane lipid peroxidation is presented. Overexpression of yqhD results in increased resistance to the reactive oxygen species-generating compounds hydrogen peroxide, paraquat, chromate, and potassium tellurite. Increased tolerance was also observed for the lipid peroxidation-derived aldehydes butanaldehyde, propanaldehyde, acrolein, and malondialdehyde and the membrane-peroxidizing compound tert-butylhydroperoxide. Expression of yqhD was also associated with changes in the concentration of intracellular peroxides and cytoplasmic protein carbonyl content and with a reduction in intracellular acrolein levels. When compared with the wild type strain, an yqhD mutant exhibited a sensitive phenotype to all these compounds and also augmented levels of thiobarbituric acid-reactive substances, which may indicate an increased level of lipid peroxidation. Purified YqhD catalyzes the in vitro reduction of acetaldehyde, malondialdehyde, propanaldehyde, butanaldehyde, and acrolein in a NADPH-dependent reaction. Finally, yqhD transcription was induced in cells that had been exposed to conditions favoring lipid peroxidation. Taken together these results indicate that this enzyme may have a physiological function by protecting the cell against the toxic effect of aldehydes derived from lipid oxidation. We speculate that in Escherichia coli YqhD is part of a glutathione-independent, NADPH-dependent response mechanism to lipid peroxidation. Evidence that Escherichia coli YqhD is involved in bacterial response to compounds that generate membrane lipid peroxidation is presented. Overexpression of yqhD results in increased resistance to the reactive oxygen species-generating compounds hydrogen peroxide, paraquat, chromate, and potassium tellurite. Increased tolerance was also observed for the lipid peroxidation-derived aldehydes butanaldehyde, propanaldehyde, acrolein, and malondialdehyde and the membrane-peroxidizing compound tert-butylhydroperoxide. Expression of yqhD was also associated with changes in the concentration of intracellular peroxides and cytoplasmic protein carbonyl content and with a reduction in intracellular acrolein levels. When compared with the wild type strain, an yqhD mutant exhibited a sensitive phenotype to all these compounds and also augmented levels of thiobarbituric acid-reactive substances, which may indicate an increased level of lipid peroxidation. Purified YqhD catalyzes the in vitro reduction of acetaldehyde, malondialdehyde, propanaldehyde, butanaldehyde, and acrolein in a NADPH-dependent reaction. Finally, yqhD transcription was induced in cells that had been exposed to conditions favoring lipid peroxidation. Taken together these results indicate that this enzyme may have a physiological function by protecting the cell against the toxic effect of aldehydes derived from lipid oxidation. We speculate that in Escherichia coli YqhD is part of a glutathione-independent, NADPH-dependent response mechanism to lipid peroxidation. Oxidative stress is detrimental to a number of cellular macromolecules including proteins, nucleic acids, and membrane lipids. In eukaryotic organisms oxidative modification of cellular constituents has been implicated in the etiology of different pathological conditions such as diabetes, cataracts, pulmonary emphysema, arthritis, cancer, and aging (1Mates J.M. Toxicology. 2000; 153: 83-104Crossref PubMed Scopus (1157) Google Scholar, 2Halliwell B. Gutteridge J. 3rd Ed. Free Radicals in Biology and Medicine. Oxford University Press, NY1999Google Scholar). The electron-transport chain provides a constant supply of reactive oxygen species (ROS). 4The abbreviations used are: ROSreactive oxygen speciesMDAmalondialdehydet-BOOHtert-butylhydroperoxideTBARthiobarbituric acid-reactive substanceqRTquantitative real timeCpcrossing pointwtwild type. Hydrogen peroxide (H2O2), superoxide (O2·¯), and hydroxyl radical (OH·) are typical side products of the aerobic metabolism. ROS compounds are also formed during cell exposure to redox-cycling chemicals like paraquat or metals and metalloids like chromate and potassium tellurite (K2TeO3) (3Imlay J. Annu. Rev. Microbiol. 2003; 57: 395-418Crossref PubMed Scopus (1646) Google Scholar, 4Pérez J.M. Calderón I.L. Arenas F.A. Fuentes D.E. Pradenas G.A. Fuentes E.L. Sandoval J.M. Castro M.E. Elías A.O. Vásquez C.C. Plos One. 2007; 2: e211Crossref PubMed Scopus (167) Google Scholar, 5Itoh M. Nakamura M. Suzuki T. Hawai K. Horitsu H. Takamizawa K. J. Biochem. 1995; 117: 780-786Crossref PubMed Scopus (39) Google Scholar). To alleviate ROS-generated oxidative damage, Escherichia coli cells induce the synthesis of a variety of antioxidant enzymes including catalases and superoxide dismutases (3Imlay J. Annu. Rev. Microbiol. 2003; 57: 395-418Crossref PubMed Scopus (1646) Google Scholar, 6Fridovich I. Annu. Rev. Biochem. 1995; 64: 97-112Crossref PubMed Scopus (2733) Google Scholar). reactive oxygen species malondialdehyde tert-butylhydroperoxide thiobarbituric acid-reactive substance quantitative real time crossing point wild type. Except for mechanisms involved in membrane peroxidation, prokaryotic and eukaryotic cells use similar mechanisms to respond to oxidative stress. Lipid oxidation is a common consequence of the activity of free radicals on cell membrane components. Oxidation of polyunsaturated fatty acids by HO2, the protonated form of superoxide anion, or OH· leads to the formation of lipid peroxides (7Esterbauer H. Zollner H. Schaur R. Vigo-Pelfrey C. Membrane Lipid Oxidation. 1. CRC Press, Inc., Boca Raton, FL1990: 239-268Google Scholar). Enzymatic or chemical degradation of lipid peroxides results in the generation of toxic breakdown products like short-chain (C3-C9) aldehydes such as 2-alkenals (α,β-unsaturated aldehydes), 2-propenal (acrolein), 4-hydroxynonenal, and malondialdehyde (MDA) (7Esterbauer H. Zollner H. Schaur R. Vigo-Pelfrey C. Membrane Lipid Oxidation. 1. CRC Press, Inc., Boca Raton, FL1990: 239-268Google Scholar, 8Esterbauer H. McBrien D.C.H. Slater T.F. Free Radicals, Lipid Peroxidation, and Cancer. Academia Press Ltd, London1982: 101Google Scholar). The toxicity of these "reactive aldehydes" lies in their ability to form Michael adducts with thiol and amino groups of proteins, thus affecting several cellular processes (9Esterbauer H. Zollner H. Scholz N. Z. Naturforsch. 1975; 30: 466-473Crossref PubMed Scopus (247) Google Scholar). For example, inactivation of glucose-6-phosphate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, and cytochrome c oxidase by reactive aldehydes that modify lysine and cysteine residues located in enzyme active sites has been reported (10Szweda L. Uchida K. Stadtman R. J. Biol. Chem. 1993; 268: 3342-3347Abstract Full Text PDF PubMed Google Scholar, 11Uchida K. Stadtman E. J. Biol. Chem. 1993; 268: 6288-6393Abstract Full Text PDF PubMed Google Scholar, 12Chen J. Schenker S. Frosto T. Henderson G. Biochim. Biophys. Acta. 1998; 1380: 336-344Crossref PubMed Scopus (129) Google Scholar). Reactive aldehydes can also generate DNA adducts causing an important increase of the rate of mutagenesis in E. coli and Salmonella (13Yang I. Hossain M. Miller H. Khullar S. Johnson F. Grollam A. Moriya M. J. Biol. Chem. 2001; 276: 9071-9076Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 14Neudecker T. Eder E. Deininger C. Henschler C. Mutat. Res. 1991; 264: 193-196Crossref PubMed Scopus (9) Google Scholar). Mammalian cells use three distinct systems to cope with the harmful effect of reactive aldehydes. The glutathione S-transferase pathway fueled by the GSH or thioredoxin redox cycle conjugates aldehydes with glutathione (GSH), the aldo-keto reductase system reduces aldehydes to alcohols, and alcohol and aldehyde dehydrogenases oxidize aldehydes to carboxyl groups. In animals, the main detoxification pathway is GSH-dependent, and it has been shown that the alcohol/aldehyde dehydrogenase and glutathione S-transferase pathways account for 10 and 55% of aldehyde elimination by hepatocytes, respectively (15Hartley D. Ruth J. Petersen D. Arch. Biochem. Biophys. 1995; 316: 197-205Crossref PubMed Scopus (223) Google Scholar). Because the major peroxidation substrate, polyunsaturated fatty acid, is found at very low levels in bacterial membranes, peroxidation of prokaryotic membranes has been a matter of controversy. Increasing experimental evidence suggesting that lipid peroxidation is physiologically important in microorganisms has been reported during the last few years. Bacterial accumulation of reactive aldehydes has been demonstrated in cells exposed to tert-butylhydroperoxide (t-BOOH), hydrogen peroxide (H2O2), potassium tellurite (K2TeO3), and titanium oxide (TiO2) (4Pérez J.M. Calderón I.L. Arenas F.A. Fuentes D.E. Pradenas G.A. Fuentes E.L. Sandoval J.M. Castro M.E. Elías A.O. Vásquez C.C. Plos One. 2007; 2: e211Crossref PubMed Scopus (167) Google Scholar, 16Yoon S. Park J. Yang J. Park J. J. Biochem. Mol. Biol. 2002; 35: 297-301PubMed Google Scholar, 17Maness P. Smolinski S.Z. Blake D. Huang Z. Wolfrum E. Jacoby W. Appl. Environ. Microbiol. 1999; 65: 4094-4098Crossref PubMed Google Scholar, 18Semchyshyn H. Bagnyukoba T. Storey K. Lushchak V. Cell Biol. Int. 2005; 29: 898-902Crossref PubMed Scopus (76) Google Scholar). It has been proposed that members of the ubiquitous thiol peroxidase thiol-dependent antioxidant/AhpC family participate in protecting E. coli against alkyl and lipid hydroperoxides (19Cha M.K. Kim H.K. Kim I.H. J. Bacteriol. 2004; 178: 5610-5614Crossref Google Scholar) such as alkyl hydroperoxidase reductase, bacterioferrin comigratory protein, and periplasmic thiol peroxidase. However, direct experimental evidence for specific defense systems against lipid peroxidation-derived aldehydes in bacteria have not been reported to date. Sulzenbacher et al. (20Sulzenbacher G. Alvarez K. van den Heuvel R. Versluis C. Spinelli S. Campanacci V. Valencia C. Cambillau C. Eklund H. Tegoni M. J. Mol. Biol. 2004; 342: 489-502Crossref PubMed Scopus (85) Google Scholar) determined the crystal structure of a new E. coli dehydrogenase, YqhD, whose physiological substrates could not be determined. Based mainly on the bound coenzyme, NADP(OH)2, they proposed a putative relationship between YqhD and oxidative stress. Genomic and proteomic studies have shown that YqhD is expressed in E. coli after exposure to stress conditions such as cold shock (21Phadtare S. Inouye M. J. Bacteriol. 2004; 186: 7007-7014Crossref PubMed Scopus (151) Google Scholar), nutrient starving (22Hua Q. Yang C. Oshima T. Mori H. Shimizu K. Appl. Environ. Microbiol. 2004; 70: 2354-2366Crossref PubMed Scopus (142) Google Scholar), and oxidative stress generated by the Curvularia peroxidase system (23Hansen E. Schembri M. Klemm P. Schäfer T. Molin S. Gram L. Appl. Environ. Microbiol. 2004; 70: 1749-1757Crossref PubMed Scopus (12) Google Scholar). YqhD has several features similar to AdhE, an alcohol/aldehyde dehydrogenase involved in fermentative metabolism in E. coli for which an antioxidant function under aerobic conditions has been proposed (24Echave P. Tamarit J. Cabiscol E. Ros J. J. Biol. Chem. 2003; 32: 30193-30198Abstract Full Text Full Text PDF Scopus (91) Google Scholar). We have found that YqhD is induced under tellurite exposure, suggesting that it is involved in bacterial response to oxidative stress. In this work we show that E. coli YqhD is an aldehyde reductase involved in resistance to ROS-generating compounds and reactive aldehydes. Evidence to confirm that YqhD catalyzes the NADPH-dependent reduction of various membrane peroxidation-derived aldehydes, including acrolein and MDA, is provided. Increased levels of thiobarbituric acid-reactive substances (TBARs) and acrolein are found in ΔyqhD cells as compared with the parental, isogenic, wild type strain cells. Expression of YqhD resulted in diminished concentrations of cytoplasmic peroxides and oxidized proteins. This antioxidant-like effect was only observed in the presence of oxygen, a condition required for lipid peroxidation. Finally, we determined that yqhD transcription was induced upon exposure to H2O2, K2TeO3, paraquat, t-BOOH, and low temperatures, conditions known to promote membrane lipid peroxidation. Results suggest that YqhD is part of a specific defense mechanism against reactive aldehydes generated by membrane peroxidation in E. coli. To our knowledge, this is the first communication describing that such mechanism might be used by bacteria to cope with the toxicity of membrane lipid peroxidation-derived aldehydes. Strains and Plasmids—E. coli strains and plasmids used in this study are listed in Table 1. E. coli BW25113 ΔyqhD, harboring a KanR insertion in the chromosomal yqhD gene, was kindly provided by the NARA Institute, Japan.TABLE 1Bacterial strains, plasmids, and primers used in this studyStrainRelevant genotypeSource or referenceE. coli TOP10F- mcrA Δ(mrr-hsdRMS-mcrBC) ϕ80lacZΔM15 ΔlacX74 deoR nupG recA1 araD139 Δ(ara-leu)7697 galU galK rpsL(StrR) endA1 λ-Invitrogen®E. coli BW25113laclq rmBT14 ΔlacZWJ16 hsdRS14 ΔaraBADAH33 ΔrhaBADLD78Datsenko and Wanner (46Datsenko K.A. Wanner B.L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6640-6645Crossref PubMed Scopus (11205) Google Scholar)E. coli pBADE. coli BW25113 harboring pBADThis workE. coli pBAD yqhDE. coli BW25113 harboring pBAD yqhDThis workE. coli ΔyqhDE. coli BW25113 ΔyqhD (yqhD::KanR)NARA Institute, JapanE. coli ΔyqhD pBADE. coli BW25113 ΔyqhD harboring pBADThis workE. coli ΔyqhD pBAD yqhDE. coli BW25113 ΔyqhD harboring pBAD yqhDThis workE. coli pDEST17 yqhDE. coli BL21 (DE3) harboring pDest17 yqhD plasmidSulzenbacher et al. (20Sulzenbacher G. Alvarez K. van den Heuvel R. Versluis C. Spinelli S. Campanacci V. Valencia C. Cambillau C. Eklund H. Tegoni M. J. Mol. Biol. 2004; 342: 489-502Crossref PubMed Scopus (85) Google Scholar)PlasmidsPlasmid featurespBAD TOPOExpression vector, ApRInvitrogen®pBAD yqhDpBAD harboring E. coli yqhD geneThis workpGEMTCloning vector, ApRPROMEGA®pGEMT yqhDppGEMT harboring E. coli yqhD gene plus 300-bp upstream of yqhD translation start codonThis workpDEST17 yqhDpDest17 harboring E. coli yqhD gene. Adds 6X His to YqhD and allows iso propyl 1-thio-β-d-galactopyranoside-induced over expression of YqhDSulzenbacher et al. (20Sulzenbacher G. Alvarez K. van den Heuvel R. Versluis C. Spinelli S. Campanacci V. Valencia C. Cambillau C. Eklund H. Tegoni M. J. Mol. Biol. 2004; 342: 489-502Crossref PubMed Scopus (85) Google Scholar)PrimersForward (F) or Reverse (R), to amplify5′-3′ SequenceyqhD FF, yqhDCCGGAAAAGAGAATTATGATGCCAGGCTCyqhD RR, yqhDAAGCTTTTAGCGGGCGGCTTCGTATATACyqhD RaPrimers used in qRT-PCR experiments. Primers were designed to generate a 300-bp product.R, yqhDACGCTGCCGCCGCCGTAGGTAATsodAFaPrimers used in qRT-PCR experiments. Primers were designed to generate a 300-bp product.F, sodACCTGCCATCCCTGCCGTATGCTTAsodARaPrimers used in qRT-PCR experiments. Primers were designed to generate a 300-bp product.R, sodATCGATAGCCGCTTTCAGGTCACCCsoxSFaPrimers used in qRT-PCR experiments. Primers were designed to generate a 300-bp product.F, soxSTTACAGGCGGTGGCGATAATCGCTsoxSRaPrimers used in qRT-PCR experiments. Primers were designed to generate a 300-bp product.R, soxSCGAGCATATTGACCAGCCGCTTAA16sFaPrimers used in qRT-PCR experiments. Primers were designed to generate a 300-bp product.F, 16 S RNAATGACCAGCCACACTGGAAC16sRaPrimers used in qRT-PCR experiments. Primers were designed to generate a 300-bp product.R, 16 S RNATGACTTAACAAACCGCCTGCgapAFaPrimers used in qRT-PCR experiments. Primers were designed to generate a 300-bp product.F, gapAGTAAAGTTGGTATTAACGGTTTTGGgapARaPrimers used in qRT-PCR experiments. Primers were designed to generate a 300-bp product.R, gapAGGTGATGTGTTTACGAGCAGa Primers used in qRT-PCR experiments. Primers were designed to generate a 300-bp product. Open table in a new tab Cloning and Expression of yqhD and Protein Purification—Plasmid pBAD yqhD was constructed by cloning the PCR-amplified E. coli yqhD gene fragment into the expression vector pBAD TOPO (Invitrogen). E. coli BW25113 chromosomal DNA and primers described in Table 1 was used to amplify the yqhD gene. The PCR product was ligated to the cloning vector, and the resulting plasmid pBAD yqhD was introduced into E. coli Top10 by electroporation. Insert orientation was confirmed by NcoI digestion, and gene integrity was determined by nucleotide sequencing. Plasmid pBAD yqhD was then mobilized into E. coli BW25113 to carry out the resistance studies reported in this work. E. coli BL21(DE3) cells harboring plasmid pDEST17 yqhD (a generous gift of Dr. C. Cambillau, CNRS, Marseille, France) were used to purify YqhD. After 5 h of induction with isopropyl 1-thio-β-d-galactopyranoside (1 mm), cells were harvested, resuspended in buffer 50 mm Tris-HCl, pH 7.0, and subjected to sonic disruption. Nucleic acids were precipitated with streptomycin sulfate (2%, w/v) on ice. After centrifuging at 13,000 × g for 5 min, the supernatant was loaded onto a Ni2+ column (nickel-nitrilotriacetic acid). Proteins were eluted with 0.5 m imidazole and fractionated by SDS-PAGE to estimate protein purity. Assay for YqhD Dehydrogenase—The reaction mixture contained 50 mm potassium phosphate buffer, pH 7.0, 2 mm NADPH, a defined aldehyde (acetaldehyde, propanal, or butanal), or alcohol (butanol, propanol or isopropanol) as substrate and purified YqhD (10 μg/ml). The mixture was incubated for 2 min at 37 °C, and NADPH oxidation was determined at 340 nm using a UV-Visible Agilent 8453 spectrophotometer. One unit of enzyme activity was defined as the amount of protein that oxidizes 1 pmol of NADPH/min at 37 °C. Minimal Inhibitory Concentrations—Cells from overnight cultures were diluted 100-fold with fresh LB medium. Ten μl of this dilution were used to inoculate 1 ml of LB medium containing the appropriate antibiotics (Kan and/or Amp) and the toxics to be tested. Cells were grown for 48 h at 37 °C with shaking. Peroxide Intracellular Levels—The oxidant-sensitive probe 2′,7′-dichlorodihydrofluorescein diacetate was used to measure peroxide levels in cells exposed to potassium tellurite (0.5 μg/ml), hydrogen peroxide (1 mm), paraquat (50 μg/ml), or chromate (1 mm) (24Echave P. Tamarit J. Cabiscol E. Ros J. J. Biol. Chem. 2003; 32: 30193-30198Abstract Full Text Full Text PDF Scopus (91) Google Scholar). Cells were grown under aerobic conditions in LB medium amended with the compounds to be tested to an A600 ∼ 0.3. After washing with 10 mm potassium phosphate buffer, pH 7.0, cells were incubated in the same buffer containing 10 μm 2′,7′-dichlorodihydrofluorescein diacetate (dissolved in dimethyl sulfoxide) for 30 min. Cells were washed, suspended in the same buffer, and disrupted by sonication. Cell extracts (100 μl) were mixed with 1 ml of phosphate buffer, and fluorescence intensity was measured using an Applied Biosystems Cytofluor 4000 multi-well plate reader (excitation, 490 nm; emission, 519 nm). Fluorescence emission values were normalized to protein concentration. Protein Carbonylation—Protein carbonyl content was determined according to Semchyshyn et al. (18Semchyshyn H. Bagnyukoba T. Storey K. Lushchak V. Cell Biol. Int. 2005; 29: 898-902Crossref PubMed Scopus (76) Google Scholar). Nucleic acid-free extracts (100 μl) prepared from E. coli BW25113 cells treated and untreated for 30 min with K2TeO3 (0.5 μg/ml) or H2O2 (100 μm) were mixed with four volumes of 10 mm dinitrophenylhydrazine (dissolved in 2 m HCl) and incubated for 1 h at room temperature with vortexing every 10–15 min. Proteins were precipitated by adding 500 μl of 20% trichloroacetic acid and sedimented at 14,000 × g for 5 min. The pellet was washed at least 3 times with 1:1 ethanol:ethyl acetate to remove any unreacted dinitrophenylhydrazine. Finally, the pellet was dissolved at 37 °C in 450 μl of 50 mm dithiothreitol in 6 m guanidine HCl. Carbonyl content was determined spectrophotometrically at 370 nm using a molar absorption coefficient of 22,000 m–1 cm–1 (18Semchyshyn H. Bagnyukoba T. Storey K. Lushchak V. Cell Biol. Int. 2005; 29: 898-902Crossref PubMed Scopus (76) Google Scholar). TBARs—TBARs were determined in cell extracts as described by Semchyshyn et al. (18Semchyshyn H. Bagnyukoba T. Storey K. Lushchak V. Cell Biol. Int. 2005; 29: 898-902Crossref PubMed Scopus (76) Google Scholar). Cells were grown at 37 °C in LB media to an A600 ∼ 0.6 and exposed for 30 min to a particular toxic compound. Cell suspensions (1.0 ml) were then precipitated by the addition of 1.0 ml of 20% (w/v) trichloroacetic acid and centrifuged, and supernatants were mixed with 2.0 ml of a saturated solution of thiobarbituric acid dissolved in 0.1 m HCl and 10 mm butylated hydroxytoluene. Samples were heated at 100 °C for 60 min, and 1.5-ml aliquots were removed, chilled on ice, and mixed with 1.5 ml of n-butanol. After centrifugation at 4,000 × g for 10 min, the organic fraction was removed, and the A535 was measured. TBARs content was determined using a molar absorption coefficient of 156 mm–1 cm–1 (18Semchyshyn H. Bagnyukoba T. Storey K. Lushchak V. Cell Biol. Int. 2005; 29: 898-902Crossref PubMed Scopus (76) Google Scholar, 25Rice-Evans C. Diplock A. Symins M. Buerdon R. van Knippenberg P.H. Laboratory techniques in Biochemistry and Molecular Biology. 22. Elsevier Science Publishers B. V., Amsterdam1991: 147-149Google Scholar). RNA Purification and Quantitative Real Time (qRT)-PCR—RNA purification was carried out using the Qiagen RNeasy kit (Qiagen). E. coli BW25113 cultures were diluted 1:100, inoculated in 200 ml of LB medium, and incubated at 37 °C with shaking to an A600 ∼ 0.6. Cultures were then amended with 0.5 μg/ml K2TeO3, 0.1 mm H2O2, 50 μg/ml paraquat, or 0.05% t-BOOH and incubated for 30 min. Cells were sedimented at 13,000 × g for 3 min and used for RNA purification. Two μl of total RNA (∼1 μg/μl) from control and treated cells were used in qRT-PCR experiments. Reactions (20 μl) used the LightCycler® RNA Amplification kit SYBR Green I (Roche Applied Science) and contained Lightcycler® 0.4 μl of RT-PCR Enzyme Mix, 4 μl of SYBR Green I Reaction Mix, 3.2 μl of 25 mm MgCl2, 5.4 μl of H2O PCR grade, 4 μl of resolution solution, and 0.5 pmol of each specific primer. Amplification products were detected by increase of fluorescence. Crossing points (Cp) are inversely proportional to the RNA content and were determined using the software included in LightCycler® 2.0. Primers used to amplify yqhD are shown in Table 1. Induction of gene expression was expressed as the difference between the crossing points of each RT-PCR determination (Cp–Cptoxic). Positive numbers reflect higher amounts of the particular, specific mRNA in cells exposed to the toxic condition. Acrolein Concentration—Acrolein concentration was determined as described by Slininger and Bothast (26Slininger P.J. Bothast R.J. Appl. Environ. Microbiol. 1985; 50: 1444-1450Crossref PubMed Google Scholar). Acrolein reacts with tryptophan yielding a purple complex absorbing at 560 nm. Commercially available acrolein (Sigma) was used to standardize the assay and to make a calibration curve. "Tryptophan solution" consisted of tryptophan (2.05 g), 4.17 ml of 8.9 m HCl, and 2.5 ml of toluene in 1 liter of bi-distilled water. Approximately 300 μl of crude extracts (or acrolein standard) were mixed with 600 μl of 8.9 m HCl and 1.5 ml of tryptophan solution. Duplicated samples were incubated for 20 min at 40 °C until a purple color developed and became stable. A560 was determined spectrophotometrically. YqhD Mediates Resistance to ROS Elicitors in E. coli—To determine whether YqhD plays a role in resistance to oxidative damage, minimal inhibitory concentrations for different ROS-generating compounds for E. coli strains lacking (ΔyqhD) and overexpressing the yqhD gene (pBAD yqhD) were determined (Table 2). Although cells overexpressing yqhD showed increased tolerance to compounds whose toxicity involves superoxide generation like paraquat and potassium tellurite (4Pérez J.M. Calderón I.L. Arenas F.A. Fuentes D.E. Pradenas G.A. Fuentes E.L. Sandoval J.M. Castro M.E. Elías A.O. Vásquez C.C. Plos One. 2007; 2: e211Crossref PubMed Scopus (167) Google Scholar, 27Calderón I.L. Arenas F.A. Pérez J.M. Fuentes D.E. Araya M.A. Saavedra C.P. Tantaleán J.C. Pichuantes S.E. Youderian P.A. Vásquez C.C. Plos ONE. 2006; 1: e70Crossref PubMed Scopus (73) Google Scholar), the hydroxyl radical-generating chromate anion (5Itoh M. Nakamura M. Suzuki T. Hawai K. Horitsu H. Takamizawa K. J. Biochem. 1995; 117: 780-786Crossref PubMed Scopus (39) Google Scholar), and hydrogen peroxide, E. coli ΔyqhD showed increased sensitivity to all these compounds (Table 2). To assess whether YqhD-mediated resistance phenotype was also related to cytoplasm thiol depletion (especially GSH), the effect of YqhD on E. coli resistance to oxidant compounds that involve thiol oxidation like diamide and cadmium chloride (28Wang A. Crowley D. J. Bacteriol. 2005; 187: 3259-3266Crossref PubMed Scopus (102) Google Scholar) was determined. All E. coli strains tested exhibited similar resistance levels to these compounds, confirming the idea that YqhD protects E. coli from ROS-generated damage.TABLE 2YqhD mediates resistance to potassium tellurite and other ROS elicitors in E. coliStrainK2TeO3H2O2ParaquatK2CrO7CdCl2DiamideE. coli pBAD1.2553001.25250350E. coli pBAD yqhD241010005250350E. coli ΔyqhD0.31.21000.15250350 Open table in a new tab YqhD Is a NADPH-dependent Aldehyde Reductase—YqhD exhibits 40–50% sequence similarity with a number of bacterial alcohol dehydrogenases. However, it did not show any detectable enzymatic activity when tested with short-chain alcohols as substrates (20Sulzenbacher G. Alvarez K. van den Heuvel R. Versluis C. Spinelli S. Campanacci V. Valencia C. Cambillau C. Eklund H. Tegoni M. J. Mol. Biol. 2004; 342: 489-502Crossref PubMed Scopus (85) Google Scholar). In our hands, purified YqhD did not show NADP+-dependent dehydrogenase activity on methanol, ethanol, propanol, butanol, or isopropanol (not shown). YqhD showed enzymatic activity with short-chain aldehydes including a few with unsaturations or hydroxylations. Km values ranged from 0.67 mm for butanaldehyde to 28.5 mm for acetaldehyde. The kcat value was similar for all substrates (Table 3).TABLE 3YqhD is a NADPH-dependent aldehyde reductaseSubstrateKmkcatkcat/Kmmms-1s-1 m-1Acetaldehyde28.4753.51880Propanaldehyde3.3145.0613613Butanaldehyde0.6759.588018Acrolein4.8162.512994Malondialdehyde1.7860.133764 Open table in a new tab yqhD Elimination Increases TBARs Levels in E. coli—TBARs are routinely used to estimate malondialdehyde content. They can also indicate the presence of a series of reactive aldehydes that are mainly generated during lipid peroxidation (17Maness P. Smolinski S.Z. Blake D. Huang Z. Wolfrum E. Jacoby W. Appl. Environ. Microbiol. 1999; 65: 4094-4098Crossref PubMed Google Scholar, 18Semchyshyn H. Bagnyukoba T. Storey K. Lushchak V. Cell Biol. Int. 2005; 29: 898-902Crossref PubMed Scopus (76) Google Scholar, 25Rice-Evans C. Diplock A. Symins M. Buerdon R. van Knippenberg P.H. Laboratory techniques in Biochemistry and Molecular Biology. 22. Elsevier Science Publishers B. V., Amsterdam1991: 147-149Google Scholar). We have previously shown that E. coli cells exposed to H2O2 or K2TeO3 have increased levels of TBARs (4Pérez J.M. Calderón I.L. Arenas F.A. Fuentes D.E. Pradenas G.A. Fuentes E.L. Sandoval J.M. Castro M.E. Elías A.O. Vásquez C.C. Plos One. 2007; 2: e211Crossref PubMed Scopus (167) Google Scholar). E. coli ΔyqhD exhibited increased content of TBARs even when H2O2 or K2TeO3 were not added to the culture, suggesting that YqhD may function in controlling the amount of membrane peroxidation products that are generated during the normal basal metabolism. E. coli ΔyqhD showed 2-(H2O2) and 4-fold (K2TeO3) TBARs increases when compared with the wild type strain, indicating that YqhD affects the content of reactive aldehydes within the cell (Fig. 1). Elimination of yqhD Alters the Intracellular Level of Peroxides—The oxidation-sensitive probe 2′,7′-dichlorodihydrofluorescein diacetate was used to estimate the level of cytoplasmic peroxides in E. coli pBAD yqhD and E. coli ΔyqhD. Probe activation was monitored in the presence or absence of the ROS-generating compounds K2TeO3, H2O2, paraquat, and chromate. E. coli cells lacking the yqhD gene showed an important increase in probe activation as compared with the wild type counterpart even in the absence of any compound. Overexpression of yqhD resulted in decreased peroxide levels in untreated cells. Peroxide levels were similar in wild type and pBAD yqhD cells (Fig. 2). YqhD Protects E. coli from Protein Oxidation—Formation of carbonyl groups in proteins, a consequence of ROS and reactive aldehydes, is a commonly used marker to estimate oxidative stress. Anti-dinitrophenylhydrazine Western blotting was used to estimate the extent of protein oxidation. Analysis of cytoplasmic protein extracts revealed that protein carbonylation in E. coli ΔyqhD was slightly higher than that of wild type and E. coli pBAD yqhD even in unstressed cells (Fig. 3A). These results agree with those obtained spectrophotometrically with cells grown aerobically in LB medium and exposed to K2TeO3 or H2O2 (Fig. 3B). E. coli ΔyqhD showed increased protein oxidation even when grown in the absence of these compounds (Fig. 3B). Genetic complementation of the deletion mutant restored protein carbonyl content to wild type levels, whereas overexpression of yqhD resulted in decreased pro
Referência(s)