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  • 1. Response and tolerance/avoidance strategies of microorganisms to oxidative stress Karthikeyan Nanjappan Roll No: 10007 Division of Microbiology
  • 2. This seminar would answer the following questions....
    • What is oxidative stress?
    • Why should we study oxidative stress?
    • What causes oxidative stress?
    • What is the mechanism of oxidative stress?
    • Response strategies for oxidative stress in microbes?
    • Molecular biology and biochemistry of oxidative stress tolerance
    • Mechanisms present in different groups of microbes
    • Future thrust areas of research
  • 3. Introduction to Oxidative stress
    • Definition of oxidative stress
      • ‘ Interference in the balance between the production of Reactive Oxygen Species (ROS), including free radicals, oxides and peroxides and the ability of biological systems to readily detect their presence and detoxify ROS or repair the resulting damage’
      • (Groves and Lucana, 2010)
  • 4. Reactive Oxygen Species (ROS)
    • Highly reactive molecules derived from molecular oxygen through various reactions in the cell system
    • They have unpaired electrons which readily react with biomolecules
    • Some highly reactive and some are less reactive
    • Term used interchangeably to the intracellular free radicals
    • Balance is maintained in the cell system
    • Seven reactive oxygen species have been described elaborately
    (Groves and Lucana, 2010; Lushchak, 2011)
  • 5. ROS contd.,
    • Unavoidable by products of aerobic life style for e.g. H 2 O 2 , O 2 •−
    • During energy production, the consecutive addition of electrons to oxygen leads to ROS production uncoupled with ATP production
  • 6. Important ROS ROS Molecule Main sources Defense systems Superoxide (O 2 •− )
    • Leakage of electrons from ETC during autooxidation reactions, flavoenzymes
    Superoxide dismutases (SOD), Superoxide reductases (SOR) Hydrogen peroxide ( H 2 O 2 )
    • Product of superoxide dismutase,
    • Glucose oxidase,
    • Xanthine oxidase
    • During biodegradation of cellulose
    Glutathione peroxidase, Catalases, Peroxiredoxins (Prx) Hydroxyl radical (OH•)
    • Formed by Fentan reaction and decomposition of peroxynitrite
    • Transition metals involved
    Catalase-peroxidases Nitric Oxide (NO)
    • Endogenously from Arg and oxygen by nitric oxide synthases
    Glutathione /TrxR
  • 7. ROS Molecule Main sources Defense systems Hypochlorous acid (HOCl) By myeloperoxidase from H 2 O 2 Peroxynitrite anion (ONOO-) Formed during the reaction between O 2 •− and NO• Organic hydroperoxide (ROOH) Formed by radical reactions with cellular components such as lipids and nucleobases Alkylhydroperoxide Reductases (Ahp)
  • 8.
    • Super oxide
    • Produced by the addition of an electron with molecular oxygen
    • Not highly reactive
    • Cannot penetrate lipid membranes so confined to the site of production
    • Hydrogen peroxide
    • Not a free radical
    • But highly reactive due do its penetrability
    • Produces highly reactive HOCl by myeloperoxidases
    (Nordberg and Arner, 2001,Groves and Lucana, 2010)
  • 9.
    • Hydroxyl radical (•OH)
    • The most potent oxidant amongst ROS
    • Formed by Fenton reaction
    (Nordberg and Arner, 2001,Groves and Lucana, 2010)
    • Transition metals play a vital role in formation of hydroxyl radicals
    • These two reactions together called as Haber-Weiss reaction
  • 10. Physiological functions of ROS
    • Provide defense against infection in higher organisms
    • Involved in the regulation and signal transduction of many antioxidant enzymes
    • Hydrogen peroxide activates the transcription factor which in turn initiates many antioxidant genes transcription in E. coli and yeasts.
  • 11. Physiological functions of ROS contd.
    • ROS cause oxidative damages in many important biomolecules
    • Creates mutation in genes as a result of damage in DNA molecule especially hydroxyl radical
    • Lipid peroxidation by the ROS creates many secondary molecules
    • Modify protein molecules by reacting with several amino acid residues rendering the protein functionally redundant
    (Nordberg and Arner, 2001)
  • 12. Mechanism of oxidative damage in cells: Endocellular (Storz and Imlay, 1999)
  • 13. Mechanism of Oxidative damage: Exocellular (Storz and Imlay, 1999)
  • 14. Response mechanisms in microorganisms
  • 15. Antioxidant enzymes
    • Superoxide dismutase (SOD)
      • First discovered ROS metabolizing enzyme
      • Several metal containing SODs characterised (Cu, Mn & Zn)
    • Superoxide reductase (SOR)
      • Discovered in sulfate reducing bacteria
      • Present in anaerobic archaea Pyrococcus furiosis and microaerophile Tryponema pallidum
      • Bacterium Tryponema pallidum lacking SOD utilizes SOR
      • Otherwise called as desulfoferrodoxin
  • 16. Antioxidant enzymes
    • Catalase - Peroxidase
      • Catalase Promote disportionation of H 2 O 2
      • Peroxidase use H 2 O 2 to oxidize number of compounds
    • Alkylhydroperoxide reductase (Ahp)
      • Possesses redox active cysteine (peroxide cysteine) that can be oxidized to a sulfenic acid by the peroxide substrate
      • This compensates catalase activity in katG mutants
    (Groves and Lucana, 2010)
  • 17. (Penninckx, 2000) Oxidative stress tolerance mechanism present in different groups of organisms Oxidative stress tolerance mechanism Organisms Glutathione (GSH) (L- γ -glutamyl-L- cysteinyl- glycine) Most microorganisms to humans More frequently in aerobic gram negative & less frequently in anaerobes and gram positive bacteria Mycothiol (an alternative thiol) Gram positive bacteria of the actinomycetes lineage L- γ -glutamyl-L- cysteine Halobacteria
  • 18. Antioxidant activities in E. coli Gene Activity Regulators sod A Manganese superoxide dismutase SoxRS, ArcAB, FNR, Fur, IHF fum C Fumarase C SoxRS, ArcAB, σ s acn A Aconitase A SoxRS, ArcAB, FNR, Fur, σ s zwf Glucose 6 phosphate dehydrogenase SoxRS fur Ferric uptake repressor SoxRS, OxyR mic F RNA regulator of omp F SoxRS, OmpR, LRP acr AB Multidrug efflux pump SoxRS tol C Outer membrane protein SoxRS fpr Ferridoxin reductase SoxRS fld A Flavodoxin SoxRS nfo Endonuclease IV SoxRS sod B Iron superoxide dismutase FNR, σ s sod C Cu-Zn superoxide dismutase kat G Hydroperoxidase I OxyR, σ s
  • 19. Antioxidant activities in E. coli (Storz and Imlay, 1999) Gene Activity Regulators ahp CF Alkyl hydroperoxide reductase OxyR gor A Glutathione reductase OxyR, σ s grx A Glutaredoxin 1 OxyR dps Non specific DNA binding protein OxyR, IHF, σ s oxy S Regulatory RNA OxyR kat E Hydroperoxidase II σ s xth A Exonuclease III Σ s pol A DNA polymerase I RecA, LexA rec A RecA msr A Methionine sulfoxide reductase hsl O Molecular chaperone
  • 20. Operation of SoxRS system in E. coli Luschak, 2011
  • 21. Operation of OxyR system in E. coli (Luschak, 2011)
  • 22. E. C oli contd.,
    • when E. coli grown on medium supplemented with 37 mM phosphate exhibited
      • higher viability
      • low NADH/NAD + ratio during stationary growth phase
    • Further,
    • Defense genes ( kat G and ahp C) and respiratory genes were activated during stationary phase
    • Critical phosphate concentration provided protection against endo and exogenous levels of oxidative stress
    • (Schwrig-Briccio et al., 2009)
  • 23. Moorella thermoacetica
    • Gram positive anaerobic acetogenic bacteria, it contains a membrane bound cytochrome bd oxidase that reduces low levels of oxygen (Das et al., 2005)
    • Bacillus subtilis
    • Showed nitric oxide (NO) induces the activation of cryoprotection system in B. subtilis
    • NO directly reactivates the catalase system using endogenous cysteine
    • (Gusarov and Nudler, 2005)
  • 24. Sulfate reducing bacteria
    • Desulfovibrio sp.
    • Dissimilatory sulfate reducing bacterium
    • Strict anaerobes living in the marine sediments and microbial mats
    • Also found in oxic photosynthetic zones of microbial mats
    • Key enzymes are sensitive
    • Cells become elongate under oxic environments
    • Avoidance / tolerance mechanism
    • Forms aggregates resulting higher tolerance
    • Migrates into deeper layers
    • Many species reduces oxygen
    • Membrane bound cytochrome bd oxidase found
    • Utilizes superoxide reductase (SOR) enzyme
  • 25. Proteome analysis of Desulfovibrio vulgaris
    • 36 protein spots found less abundant
    • 19 protein spots found more intense
    Under oxidative conditions (Fournier et al., 2006)
  • 26. Lactic Acid Bacteria
    • LAB are aerotolerant anaerobe, grow in the presence of air, despite
      • Lack cytochromes and other heme containing proteins
      • Lack catalase
    • The protection mechanism involves two kinds of NADH oxygenase genes ( nox )
    • nox 1 H 2 O 2 forming NADH oxidase
    • nox 2 H 2 O forming NADH oxidase
    Higuchi et al., 2000
  • 27. Yeasts
    • Saccharomyces cerevisiae :
    • Important industrial organism in many commercial fermentations
    • Active dry yeasts are used commercially
    • Encounters oxidative stress during fermentation and ADY production
    • How S. cerevisiae adapts to oxidative stress?
    • Contains two genes TRR -1 and GRX 5
    Thioredoxin Glutathione/ glutaredoxins
    • Glutathione is a fundamental molecule for dehydration tolerance in microbes
    • Reacts with ROS and protein groups provides membrane protection
  • 28. Yeasts contd.,
    • The indicators of oxidative stress in S. cerevisiae
        • Elevated glutathione content
        • Increased lipid peroxidation damage (Garre et al. , 2011)
    • Cystofilobasidium infirmominiatum
    • An antagonist yeast used as bio-control against P. expansum
    • Post harvest bio-control agent in many fruits against fungi
    • Addition of glycine betaine in the medium at 1 mM conc. In the medium resulted in
        • Increased viability of yeast cells in the cut wounds of apple
        • Reduced accumulation of ROS in yeast cells
        • Reduced protein oxidation
        • Increased bio-control against Penicillium expansum
        • Increased Catalase, SOD, Glutathione peroxidase (GPx)
    (Jia Liu et al., 2011)
  • 29. Regulatory mechanism in S. cerevisiae to oxidative stress Gpx3- Glutathione peroxidase NES-Nuclear export sequence Yap 1- yeast activator protein Crm- cysteine rich motif (Lushchak, 2011)
  • 30. Cyanobacteria Cyanobacterium Synechocystis sp PCC 6803 has the similar sequences of gene coding for Glutaredoxin (Grx). The gene expression study conducted on E.coli confirmed that the amino acid sequence homology with glutaredoxin of other organisms. (Li et al., 2005)
  • 31. Conclusions
    • Reactive oxygen species are inevitable consequences of cellular oxidative metabolism leading to oxidative stress on microbes and other organisms endogenously and exogenously
    • Organisms have developed mechanisms counteract the oxidative stress in their environment
    • Even anaerobic organisms too have well organised tolerance mechanisms
    • Some of the components of ROS are involved in regulatory activities of antioxidant genes
    • Addition of some osmoregulants such as glycine betaine confers the microbe tolerance to oxidative stress
  • 32. Future Thrust areas of research
    • Understanding of the basic mechanisms of oxidative stress in microbes of our interest
    • Plant antioxidants which could confer tolerance/resistance to oxidative stress in microbes should be identified and studied
    • Techniques which exert less oxidative stress on commercial microbes should be identified and evaluated
    • Developing oxidation stress tolerant microbes would enhance the performance of microbes in agriculture and industry
  • 33. Thanks for the Attention!!!!!