Azoreductase in Staphylococcus aureus

Wen Zou1, Carl E. Cerniglia1, Huizhong Chen1

1 National Center for Toxicological Research/FDA, Jefferson, Arkansas
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 4.28
DOI:  10.1002/0471140856.tx0428s41
Online Posting Date:  August, 2009
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Abstract

Azoreductase(s) catalyze a NAD(P)H‐dependent reaction in bacteria to metabolize azo dyes to colorless aromatic amines. Azoreductases from bacteria represent a novel family of enzymes with little similarity to other reductases. This unit will describe the current methods for measuring azoreductase from Staphylococcus aureus, which has been suggested to serve as a model strain to study the azo dye degradation by human skin microflora. Curr. Protoc. Toxicol. 41:4.28.1‐4.28.9. © 2009 by John Wiley & Sons, Inc.

Keywords: azoreductase; azo dye; Staphylococcus aureus; enzyme activity; metabolites

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Measuring the Enzymatic Activity of Azoreductase
  • Basic Protocol 2: Identification of Azoreductase Expression in S. aureus
  • Basic Protocol 3: HPLC Analysis of Azo Dye and Metabolites Excreted in the Culture
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Measuring the Enzymatic Activity of Azoreductase

  Materials
  • S. aureus (ATCC no. 25923) growing as colonies on TSA plate
  • Brain heart infusion (BHI) broth (Remel)
  • 25 mM (1000×) methyl red stock solution (see recipe)
  • 25 mM potassium phosphate buffer, pH 7.1 ( appendix 2A)
  • Glass beads (212 to 300 µm; Sigma)
  • Recombinant E. coli expressing an S. aureus azoreductase gene (azo1): e.g., BL21‐Gold(DE3)pLysS harboring pAZO1 (Chen et al., ) grown on LB‐ampicillin‐chloramphenicol plate
  • LB medium: dissolve 25.0 g powdered LB medium (Sigma) in ∼800 ml H 2O and make up the final volume to 1000 ml; autoclave and store up to 2 months at 4°C
  • 50 mg/ml ampicillin stock solution (Sigma)
  • 50 mg/ml chloramphenicol stock solution (Sigma, in 100% ethanol)
  • 0.5 M isopropyl‐1‐thio‐β‐galactopyranoside (IPTG, Roche)
  • 10 mM (100×) NADPH stock solution (see recipe)
  • Bicinchoninic acid protein assay kit (Thermo Scientific)
  • 5‐ml and 15‐ml polypropylene round‐bottle tubes (Becton Dickinson)
  • 50‐ml disposable centrifuge tubes (Fisher Scientific)
  • Innova 4330 incubator/shaker (New Brunswick Scientific)
  • Centrifuges (Centrifuge 5810R and 5415R, Eppendorf)
  • 500‐ml Erlenmeyer flask
  • Vibracell VCX 400 model sonifier (Sonics & Materials)
  • DU 800 Spectrophotometer (Beckman Coulter) and 1‐cm path length glass spectrophotometer cuvettes

Basic Protocol 2: Identification of Azoreductase Expression in S. aureus

  Materials
  • Female rabbits (New Zealand white, 2.5 kg body weight)
  • Recombinant S. aureus Azo1, purified in the laboratory (Chen et al. )
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • Complete Freund's adjuvant (CFA; e.g., Sigma; also see Cooper and Patterson, )
  • Bicinchoninic acid protein assay kit (Thermo Scientific)
  • 12.5% (w/v) sodium dodecyl sulfate‐polyacrylamide electrophoresis (SDS‐PAGE) gel (BioRad)
  • Coomassie brilliant blue R‐250 (Thermo Scientific; also see unit 2.3)
  • Superblock (Thermo Scientific)
  • Anti–recombinant S. aureus Azo1antiserum (Liu et al., )
  • PBS‐T buffer (Fisher)
  • ImmunoPure goat anti–rabbit IgG, peroxidase conjugated (Thermo Scientific)
  • SuperSignal West Pico Chemiliminescent Substrate (Thermo Scientific)
  • Nitrocellulose membrane (Thermo Scientific)
  • Additional reagents and equipment for producing polyclonal antibodies (Cooper and Patterson, ), obtaining blood via the ear vein in rabbits (Donovan and Brown, ), testing for Azo1 activity (Liu et al., ), preparing crude protein extract of S. aureus ( protocol 1), SDS‐PAGE ( appendix 3F), transfer of proteins to nitrocellulose membrane (unit 2.3), and Coomassie blue staining of gels (unit 2.3)

Basic Protocol 3: HPLC Analysis of Azo Dye and Metabolites Excreted in the Culture

  Materials
  • S. aureus (ATCC no. 25923) growing as colonies on TSA plate
  • Brain heart infusion (BHI) broth (Remel)
  • 25 mM (1000×) methyl red stock solution (see recipe)
  • 1 M HCl
  • Ethyl acetate (Fisher)
  • Acetonitrile (Fisher)
  • 25 mM sodium phosphate/citrate buffer, pH 3.0 (prepare 25 mM sodium phosphate and titrate to pH 3 with citric acid)
  • Authentic standards for metabolites:
    • N,N′‐Dimethyl‐p‐phenylenediamine (Sigma)
    • 2‐Aminobenzoic acid (Sigma)
  • 5‐ and 10‐ml culture tubes
  • 50‐ml conical centrifuge tubes
  • Rotary evaporator R‐200 (Buchi)
  • 0.2 µm pore‐sized syringe filters (Millipore)
  • Hewlett‐Packard HPLC 1050
  • Reversed‐phase Inertsil 5 µm ODS‐2 column (4.6 × 250 mm, Varian)
  • Micromass Quattro Ultima mass spectrometer (Waters) equipped with electron spray ionization source operated in positive mode (Bafana et al., )
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Figures

Videos

Literature Cited

   Bafana, A., Chakrabarti, T., and Devi, S.S. 2008. Azoreductase and dye detoxification activities of Bacillus velezensis strain AB. Appl. Microbiol. Biotechnol. 77:1139‐1144.
   Bin, Y., Jiti, Z., Jing, W., Cuihong, D., Hongman, H., Zhiyong, S., and Yongming, B. 2004. Expression and characteristics of the gene encoding azoreductase from Rhodobacter sphaeroides AS1.1737. FEMS Microbiol. Lett. 236:129‐136.
   Blumel, S., Knackmuss, H. J., and Stolz, A. 2002. Molecular cloning and characterization of the gene coding for the aerobic azoreductase from Xenophilus azovorans KF46F. Appl. Environ. Microbiol. 68:3948‐3955.
   Chen, H. 2006. Recent advances in azo dye degrading enzyme research. Curr. Prot. Pept. Sci. 7:101‐111.
   Chen, H., Wang, R.F., and Cerniglia, C.E. 2004. Molecular cloning, overexpression, purification, and characterization of an aerobic FMN‐dependent azoreductase from Enterococcus faecalis. Prot. Expr. Purif. 34:302‐310.
   Chen, H., Hopper, S.L., and Cerniglia, C.E. 2005. Biochemical and molecular characterization of an azoreductase from Staphylococcus aureus, a tetrameric NADPH‐dependent flavoprotein. Microbiology 151:1433‐1441.
   Chung, K.T., Stevens, S.E. Jr., and Cerniglia, C.E. 1992. The reduction of azo dyes by the intestinal microflora. Crit. Rev. Microbiol. 18:175‐190.
   Cooper, H.M. and Patterson, Y. 2008. Production of polyclonal antisera. Curr. Protoc. Immunol. 82:2.4.1‐2.4.10.
   Donovan, J. and Brown, P. 2006. Blood collection. Curr. Protoc. Immunol. 73:1.7.1‐1.7.9.
   Isik, M. and Sponza, D.T. 2004. Monitoring of toxicity and intermediates of C.I. Direct Black 38 azo dye through decolorization in an anaerobic/aerobic sequential reactor system. J. Hazard Mater. 114:29‐39.
   Liu, Z.J., Chen, H., Shaw, N., Hopper, S.L., Chen, L., Chen, S., Cerniglia, C.E., and Wang, B.C. 2007. Crystal structure of an aerobic FMN‐dependent azoreductase (AzoA) from Enterococcus faecalis. Arch. Biochem. Biophys. 463:68‐77.
   Moutaouakkil, A., Zeroual, Y., Zohra Dzayri, F., Talbi, M., Lee, K., and Blaghen, M. 2003. Purification and partial characterization of azoreductase from Enterobacter agglomerans. Arch. Biochem. Biophys. 413:139‐146.
   Nagase, N., Sasaki, A., Yamashita, K., Shimizu, A., Wakita, Y., Kitai, S., and Kawano, J. 2002. Isolation and species distribution of staphylococci from animal and human skin. J. Vet. Med. Sci. 64:245‐250.
   Nakanishi, M., Yatome, C., Ishida, N., and Kitade, Y. 2001. Putative ACP phosphodiesterase gene (acpD) encodes an azoreductase. J. Biol. Chem. 276:46394‐46399.
   Nakayama, T., Kimura, T., Kodama, M., and Nagata, C. 1983. Generation of hydrogen peroxide and superoxide anion from active metabolites of naphthylamines and aminoazo dyes: Its possible role in carcinogenesis. Carcinogenesis 4:765‐769.
   Platzek, T., Lang, C., Grohmann, G., Gi, U.S., and Baltes, W. 1999. Formation of a carcinogenic aromatic amine from an azo dye by human skin bacteria in vitro. Hum. Exp. Toxicol. 18:552‐559.
   Wong, P.K. and Yuen, P.Y. 1998. Decolourization and biodegradation of N,N′‐dimethyl‐p‐phenylenediamine by Klebsiella pneumoniae RS‐13 and Acetobacter liquefaciens S‐1. J. Appl. Microbiol. 85:79‐87.
   Xu, H., Heinze, T.M., Chen, S., Cerniglia, C.E., and Chen, H. 2007. Anaerobic metabolism of 1‐amino‐2‐naphthol‐based azo dyes (Sudan dyes) by human intestinal microflora. Appl. Environ. Microbiol. 73:7759‐7762.
   Zille, A., Gornacka, B., Rehorek, A., and Cavaco‐Paulo, A. 2005. Degradation of azo dyes by Trametes villosa laccase over long periods of oxidative conditions. Appl. Environ. Microbiol. 71:6711‐6718.
   Zimmermann, T., Kulla, H.G., and Leisinger, T. 1982. Properties of purified Orange II azoreductase, the enzyme initiating azo dye degradation by Pseudomonas KF46. Eur. J. Biochem. 129:197‐203.
Key Reference
   Chen et al., 2005. See above
  Describes biochemical and molecular characterization of Azo1 from S. aureus.
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