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!!!!!