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Response and tolerance/avoidance strategies of microorganisms to
oxidative stress
Karthikeyan Nanjappan
Roll No: 10007
Division of Microbiology
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
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)
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)
ROS contd.,
• Unavoidable by products of aerobic life style for e.g. H2O2 , O2
•−
• During energy production, the consecutive addition of electrons to oxygen leads to ROS production uncoupled with ATP production
ROS Molecule Main sources Defense systems
Superoxide (O2
•−)Leakage of electrons from ETC during autooxidation reactions, flavoenzymes
Superoxide dismutases (SOD), Superoxide reductases (SOR)
Hydrogen peroxide (H2O2)
Product of superoxide dismutase,Glucose oxidase,Xanthine oxidaseDuring biodegradation of cellulose
Glutathione peroxidase,Catalases,Peroxiredoxins(Prx)
Hydroxyl radical (OH•)
Formed by Fentan reaction and decomposition of peroxynitriteTransition metals involved
Catalase-peroxidases
Nitric Oxide(NO)
Endogenously from Arg and oxygen by nitric oxide synthases
Glutathione /TrxR
Important ROS
ROS Molecule Main sources Defense systems
Hypochlorous acid (HOCl)
By myeloperoxidase from H2O2
Peroxynitrite anion (ONOO-)
Formed during the reaction between O2
•− and NO•
Organic hydroperoxide (ROOH)
Formed by radical reactions with cellular components such as lipids and nucleobases
AlkylhydroperoxideReductases (Ahp)
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)
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
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.
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)
Mechanism of oxidative damage in cells: Endocellular
(Storz and Imlay, 1999)
Mechanism of Oxidative damage: Exocellular
(Storz and Imlay, 1999)
Response mechanisms in microorganisms
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
Antioxidant enzymes
• Catalase - Peroxidase– Catalase Promote disportionation of H2O2
– Peroxidase use H2O2 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)
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 (Penninckx, 2000)
Oxidative stress tolerance mechanism present in different groups of organisms
Antioxidant activities in E. coliGene Activity Regulators
sodA Manganese superoxide dismutase SoxRS, ArcAB, FNR, Fur, IHF
fumC Fumarase C SoxRS, ArcAB, σs
acnA Aconitase A SoxRS, ArcAB, FNR, Fur, σs
zwf Glucose 6 phosphate dehydrogenase SoxRS
fur Ferric uptake repressor SoxRS, OxyR
micF RNA regulator of ompF SoxRS, OmpR, LRP
acrAB Multidrug efflux pump SoxRS
tolC Outer membrane protein SoxRS
fpr Ferridoxin reductase SoxRS
fldA Flavodoxin SoxRS
nfo Endonuclease IV SoxRS
sodB Iron superoxide dismutase FNR, σs
sodC Cu-Zn superoxide dismutase
katG Hydroperoxidase I OxyR, σs
Antioxidant activities in E. coliGene Activity Regulators
ahpCF Alkyl hydroperoxide reductase OxyR
gorA Glutathione reductase OxyR, σs
grxA Glutaredoxin 1 OxyR
dps Non specific DNA binding protein OxyR, IHF, σs
oxyS Regulatory RNA OxyR
katE Hydroperoxidase II σs
xthA Exonuclease III Σs
polA DNA polymerase I RecA, LexA
recA RecA
msrA Methionine sulfoxide reductase
hslO Molecular chaperone
(Storz and Imlay, 1999)
Operation of SoxRS system in E. coli
Luschak, 2011
Operation of OxyR system in E. coli
(Luschak, 2011)
E.Coli 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)
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)
Sulfate reducing bacteriaDesulfovibrio 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
Proteome analysis of Desulfovibrio vulgaris
36 protein spots found less abundant19 protein spots found more intense Under oxidative conditions
(Fournier et al., 2006)
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)
nox1 H2O2 forming NADH oxidase
nox2 H2O forming NADH oxidase
Higuchi et al., 2000
YeastsSaccharomyces 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 GRX5
Thioredoxin Glutathione/ glutaredoxins
Glutathione is a fundamental molecule
for dehydration tolerance in microbes
Reacts with ROS and protein groups
provides membrane protection
Yeasts contd.,The indicators of oxidative stress in S. cerevisiae
Elevated glutathione content
Increased lipid peroxidation damage (Garre et al., 2011)
Cystofilobasidium infirmominiatumAn 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)
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)
CyanobacteriaCyanobacterium 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)
ConclusionsReactive 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
Future Thrust areas of researchUnderstanding 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
Thanks for the
Attention!!!!!