Functional Assays of Oxidative Stress Using Genetically Engineered Escherichia coli Strains

Guadalupe Herrera1, Alicia Martínez1, José‐Enrique O'Cornor1, Manuel Blanco2

1 Universidad de Valencia, Valencia, Spain, 2 Instituto de Investigaciones Citológicas Fundación Valenciana de Investigaciones Biomédicas, Valencia, Spain
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 11.16
DOI:  10.1002/0471142956.cy1116s24
Online Posting Date:  May, 2003
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Abstract

Oxidative stress may be induced in bacteria by exogenous biocidal agents and is involved in endogenous metabolism. The oxyR operon is a main sensor of oxidative stress and oxyR‐deficient bacteria show enhanced sensitivity to oxidative stress and increased accumulation of intracellular reactive oxygen species (ROS). Flow cytometric functional assays in bacteria are limited by the impaired penetration of vital dyes trough the cell wall. Escherichia coli B WP2 strains possess an altered cell‐wall lipopolysaccharide that leads to increased membrane permeability. Flow cytometric analysis of WP2 strains is a convenient alternative for cytometric assays of bacterial function. This unit presents protocols for flow cytometric studies of intracellular oxidative stress in two E. coli B WP2 strains, wild‐type or deficient in the oxyR function, using ROS‐sensitive fluorogenic substrates. Support Protocols describe preparation of phage C21 stock for bacterial verification, verification of the WP2 phenotype, and verification of the deficiency in oxyR function.

     
 
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Table of Contents

  • Basic Protocol 1: Detection of Intracellular Superoxide Anion in E. Coli B WP2 Strains
  • Basic Protocol 2: Detection of Intracellular Peroxides In E. Coli B WP2 Strains
  • Reagents and Solutions
  • Commentary
  • Figures
     
 
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Materials

Basic Protocol 1: Detection of Intracellular Superoxide Anion in E. Coli B WP2 Strains

  Materials
  • Overnight bacterial culture (see recipe)
  • LB medium (see recipe)
  • Test substance(s)
  • 1 mg/ml plumbagin (see recipe)
  • PBS ( appendix 2A)
  • 1 mg/ml HE (dihydroethidium; see recipe)
  • 37°C incubator (Heraeus Sepatech)
  • Spectrophotometer
  • Flow cytometer with an air‐cooled argon laser (488 nm, 15 mW), equipped with light‐scatter detectors that measure forward (FS) and side scatter (SS) and with filter set to collect orange fluorescence (575 to 625 nm)

Basic Protocol 2: Detection of Intracellular Peroxides In E. Coli B WP2 Strains

  Materials
  • Overnight bacterial culture (see recipe)
  • LB medium (see recipe)
  • Test substance(s)
  • 1 M hydrogen peroxide (see recipe)
  • 100 mM t‐butyl hydroperoxide (see recipe)
  • PBS ( appendix 2A)
  • 1 mg/ml DCFH‐DA (2′,7′‐dichlorofluorescin diacetate; see recipe)
  • 37°C incubator (Heraeus Sepatech)
  • Spectrophotometer
  • Flow cytometer with an air‐cooled argon laser (488 nm, 15 mW), equipped with light‐scatter detectors that measure forward (FS) and side scatter (SS) and with filter set to collect green fluorescence (∼525 nm)

Support Protocol 1:

  Materials
  • Bacterial strains IC188 (WP2 uvrA/pKM101; ATCC), IC203 (WP2 uvrAΔoxyR30/pKM101; obtain from Manuel Blanco; ), and GY783 (C21‐sensitive; obtain from Manuel Blanco)
  • Phage C21 (obtain from Manuel Blanco; )
  • LB medium (see recipe)
  • Molten top agar (see recipe)
  • LA medium dishes (see recipe)
  • 1 M hydrogen peroxide (see recipe)
  • 37°C incubator (Heraeus Sepatech)
  • 6‐mm Whatman paper disks
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Figures

Videos

Literature Cited

Literature Cited
   Alvarez‐Barrientos, A.M., Arroyo, J., Cantón, R., Nombela, C., and Sánchez‐Pérez, M. 2000. Applications of flow cytometry to clinical microbiology. Clin. Microbiol. Rev. 13:167‐195.
   Blanco, M., Urios, A., and Martínez, A. 1998a. New Escherichia coli WP2 tester strains highly sensitive to reversion by oxidative mutagens. Mutat. Res. 413:95‐101.
   Blanco, M., Martínez, A., Urios, A., Herrera, G., and O'Connor, J.E. 1998b. Detection of oxidative mutagenesis by isoniazid and other hydrazine derivatives in Escherichia coli WP2 tester strain IC203, deficient in OxyR: Strong protective effects of rat liver S9. Mutat. Res. 417:39‐46.
   Carter, W.O., Narayanan, P.K., and Robinson, J.P. 1994. Intracellular hydrogen peroxide and superoxide anion detection in endothelial cells. J. Leukoc. Biol. 55:253–258.
   Davey, H.M. and Kell, D.B. 1996. Flow cytometry and cell sorting of heterogeneous microbial populations: The importance of single‐cell analysis. Microbiol. Rev. 60:641‐696.
   Droge, W. 2002. Free radicals in the physiological control of cell function. Physiol. Rev. 82:47‐95.
   Gorman, A.M., Samali, A., McGowan, A.J., and Cotter, T.G. 1997. Use of flow cytometry techniques in studying mechanisms of apoptosis in leukemic cells. Cytometry 29:97‐105.
   Harvey, E.M., Merchant, K., and Stamler, J.S. 2000. Nitrosation and oxidation in the regulation of gene expression. FASEB J. 14:1889‐1900.
   Herrera, G., Urios, A., Aleixandre, V., and Blanco, M. 1993. Mutability by polyciclic hydrocarbons is improved in derivatives of Escherichia coli WP2 uvrA with increased permeability. Mutat. Res. 301:1‐5.
   Herrera, G., Martínez, A., Blanco, M., and O'Connor, J.E. 2002. Assessment of Escherichia coli B with enhanced permeability to fluorochromes for flow cytometric assays of bacterial cell function. Cytometry 49:62‐69.
   Janson, P.E., Lindberg, B., and Wollin, R. 1981. Structural studies on the hexose region of the core in lipopolysaccharides from Enterobacteriaceae. Eur. J. Biochem. 115:571‐577.
   Jernaes, M.W. and Steen, H.B. 1994. Staining of Escherichia coli for flow cytometry: Influx and efflux of ethidium bromide. Cytometry 17:302‐309.
   Martínez, A., Urios, A., and Blanco, M. 2000. Mutagenicity of 80 chemicals in Escherichia coli tester strains IC203, deficient in OxyR, and its oxyR+ parent WP2 uvrA/pKM101: Detection of 31 oxidative mutagens. Mutat. Res. 467:41‐53.
   Nikaido, H. and Vaara, M. 1987. Outer membrane. In Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology. (F.C. Neidhardt, J.L. Ingraham, K.B. Low, B. Magasanik, M. Schaecchter, and H.E. Umbarger, eds.) pp.7‐22. American Society for Microbiology, Washington, D.C.
   O'Connor, J.E., Callaghan, R.C., Escudero, M., Herrera, G., Martínez, A., Monteiro, M.C., and Montolíu, H. 2001. The relevance of flow cytometry for biochemical analysis. IUBMB Life 51:231‐239.
   Organization for Economic Cooperation and Development. 1998. OECD Guideline for Testing of Chemicals: Bacterial Reverse Mutation Test. In Ninth Addendum to the OECD Guidelines for the Testing of Chemicals. pp. 1‐11. OECD, Paris.
   Pomposiello, P.J. and Demple, B. 2002. Global adjustment of microbial physiology during free radical stress. Adv. Microb.Physiol. 46:319‐341.
   Rothe, G. and Valet, G. 1990. Flow cytometric analysis of respiratory burst activity in phagocytes with hydroethidine and 2′,7′‐dichlorofluorescin. J. Leukoc. Biol. 47:440‐448.
   Storz, G., Tartaglia, L.A., and Ames, B.N. 1990. Transcriptional regulator of oxidative stress–inducible genes: Direct activation by oxidation. Science 248:189‐194.
   Tarpey, M.M. and Fridovich, I. 2001. Methods of detection of vascular reactive species nitric oxide, superoxide, hydrogen peroxide, and peroxynitrite. Circ. Res. 89:224‐236.
   Zheng, M. and Storz, G. 2000. Redox sensing by prokaryotic transcription factors. Biochem. Pharmacol. 59:1‐6.
   Zheng, M., Wang, X., Templeton, L.J., Smulski, D.R., LaRosa, R.A., and Storz, G. 2001. DNA microarray‐mediated transcriptional profiling of the Escherichia coli response to hidrogen peroxide. J. Bacteriol. 183:4562‐4570.
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