OXIDATIVE STRESS CONTROL

AGM-602 –Microbial Physiology and Regulation

BY
Namadara Sandhya
Course Teacher 1st year PhD
DR. K. VIJILA 202211004
PROFESSOR Department of Agriculture
Dept. of Agril. Microbiology, Microbiology
AGM 602
Oxidative stress
 Oxidative stress is a phenomenon caused by an imbalance
between production and accumulation of oxygen reactive
species (ROS) in cells and tissues and the ability of
biological systems to readily detect and detoxify them, or
repair the resulting damage are oxidative stress.
 Also it is the excess formation or insufficient removal of highly
reactive molecules such as reactive oxygen species (ROS)
 Highly reactive radicals cause the oxidative damage of
different macromolecules proteins, DNA, and lipids leading to
loss of function, an increased rate of mutagenesis, and
ultimately cell death.
ROS

Antioxidant
 PHYSIOLOGICAL FUNCTIONS OF ROS
• It is best not to think of oxygen radicals as "bad". They are generated in a
number of reactions essential to life and, as mentioned above, phagocytic cells
generate radicals to kill invading pathogens.
• Provide defense against infection in higher organisms
• Involved in the regulation and signal transduction of many antioxidant
enzymes. For example, addition of superoxide or hydrogen peroxide to a
variety of cultured cells leads to an increased rate of DNA replication and cell
proliferation - in other words, these radicals function as mitogens.
• Hydrogen peroxide activates the transcription factor which in turn initiates many
antioxidant genes transcription in E. coli and yeasts.
• 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)
OXIDANTS
Oxidants are normally formed as the products of aerobic metabolism
that can be produced at elevated rate under pathophysiological
conditions.
• Superoxide- this lacks to penetrate lipid membrane.
• Hydrogen peroxide- It has a signaling role.
• Hydroxyl radical - most reactive with biomolecules.

ANTIOXIDANTS
Antioxidant defense involves several strategies, both enzymatic and
non-enzymatic protecting cytosol nuclear and mitochondrial matrices
and extracellular fluid. They are:
• Catalase - reduces H2O2 to H2O and O2
• Superoxide Dismutase - DISMUTASE SUPEROXIDE TO H2O2
and molecular oxygen.
• Peroxiredoxin - reduces peroxides to corresponding alcohols
 PRODUCTION OF ROS
1. The reaction of oxidative enzymes with molecular oxygen can generate
superoxide (superoxide anion):
  O2 + e− + oxidative enzymes −−−→ O2
2. Superoxide is relatively unreactive with DNA and proteins. However, it may
interact in a number of enzymatic as well as spontaneous chemical reactions to
produce more highly reactive oxygen derivatives such as hydrogen peroxide and
hydroxyl radicals:
  O2−• + H2O2 −−−→ OH− + OH• + O2
3. Auto oxidation of reduced FAD or reduced flavoprotein gives rise to hydrogen
peroxide:
  FADH2 + O2 −−−→ FAD + H2O2
4. The enzyme NADPH oxidase can generate superoxide anion and hydrogen
peroxide:
  NADPH + H+ + 2O2 −−−→ NADP+ + O2−• + H2O2
5. Superoxide can release Fe2+ and Fe3+ from various cellular compounds and
ultimately give rise to hydroxyl radicals through the Fenton reaction:
  Fe2+ + H2O2 −−−→ Fe3+ + OH• + OH−
OXIDATIVE STRESS IN PROKARYOTES
• Oxidative stress can also induce adaptive
responses i.e. the ability of cells or
organisms to better resist the damaging
effects of a toxic agent when first pre-
exposed to a lower dose.

• For example, Nunoshiba et al. reported that

a sub-lethal concentration of H2O2 made E.
coli resistant
c
to lethal amounts
d
of several
different aldehydes .
 OXIDATIVE STRESS AT METABOLIC LEVEL
•Many effects of O2 are also observed at the metabolic level

•For e.g. In Lactococcus lactis, under anaerobic conditions, it has a fermentative

metabolism that enables the transformation of various types of carbohydrates into lactic acid.

•2NADH molecules, generated from the oxidation of glyceraldehydes-3-phosphate, are re-

oxidized to favour the reduction of pyruvate to lactic acid by the action of lactate
dehydrogenase (LDH).

•Under aerobic conditions, increased expression & activities of NADH oxidase & NADH
peroxidase compete with LDH for NADH molecules which leads to mixed fermentation by
the action of pyruvate dehydrogenase (PDH).
•These changes also lead to the formation of H2O2, which causes a reduction of the growth
rate of lactococcus lactis, & eventually its death.

•Although, NADH peroxidase contributes to cellular H2O2 detoxification, it’s activities is

generally low (10-30 times lower than that of NADH oxidase) & endogenous & exogenous
H2O2 overcomes the cell’s capability to deal with this oxygen species.
Overview of damage caused by reactive oxygen species in Escherichia coli
REGULATION OF THE OXIDATIVE STRESS RESPONSE
• The gene regulatory response of Escherichia coli to superoxide and peroxide stress is
largely mediated through the induction of superoxide dismutases (SOD) and catalases,
respectively. Superoxide is dismutated to form hydrogen peroxide (H2O2) by superoxide
dismutase activity

• E. coli has three superoxide dismutases, namely MnSOD (sodA), FeSOD (sodB), and
CuZnSOD (sodC). Catalases, part of the peroxidase family of enzymes, subsequently
degrade H2O2 into H2O and O2 .E. coli has two catalases, hydroperoxidase I (HPI) and
hydroperoxidase II (HPII), encoded by katG and katE.

• E. coli superoxide dismutase and catalase genes are members of two major oxidative
stress regulons, the OxyR and SoxRS regulons, as well as the RpoS general stress regulon.
BACTERIA DEFENSE MECHANISM AGAINST OXIDATIVE STRESS

Peroxidases can also catalyse the

reduction of hydrogen peroxide by
organic reductants such as
glutathione or ascorbic acid:
 
H2O2 + 2RH −−−→ 2 H2O +2 Rox
BACTERIA DEFENSE MECHANISM AGAINST OXIDATIVE STRESS

Example:

E. coli produces a cytoplasmic Mn-SOD (SodA) and Fe-

SOD (SodB) that protect DNA and proteins from
oxidation.

Mutants deficient in these enzymes display enzyme

inactivation, growth deficiencies, and DNA damage.

A periplasmic Cu/Zn-SOD (SodC) protects the

periplasmic and membrane constituents from exogenous
superoxide.
BACTERIA DEFENSE MECHANISM AGAINST OXIDATIVE STRESS

Example:
Coxiella burnettii encodes for
two superoxide dismutase
enzymes (SodA, SodC) which
mediate the decomposition of
superoxide radicals (O2-) to
hydrogen peroxide (H2O2),

In the following step, H2O2 is

degraded to H2O and O2 by
catalase (KatE) or alkyl
hydroperoxide reductases (Ahp).
Presence of iron (Fe2+) can lead to
additional oxidative stress by
generation of hydroxyl radicals
(OH-) via the Fenton reaction.
•The presence of SOD and catalase in anaerobic organisms would
appear to be self-defeating since oxygen is a product of both
reactions.

•Anaerobes must rely on other mechanisms to eliminate oxygen

and prevent the formation of toxic oxygen species

•One method used by organisms that do not carry out oxygen-

dependent respiration involves a unique flavoprotein, NADH oxidase,
which catalyses the direct four-electron reduction of oxygen to water:

•NADH + H+ + 0.5 O2 −−−→ NAD+ + H2O

•This enzyme actually allows streptococci and other lactic acid

bacteria to use oxygen directly in the metabolism of carbohydrates
without the inherent problems of oxygen toxicity caused by the
formation of reactive oxygen derivatives.
 REGULATION OF THE OXIDATIVE STRESS
RESPONSE
The natural by-products of aerobic metabolism are the reactive
compounds superoxide (O2−) and hydrogen peroxide (H2O2). These
two species can lead to the generation of hydroxyl radicals (OH•),
which can damage biological macromolecules.
Research efforts have uncovered two regulons within this stress
response, although there are certainly more.
 
1. H2O2 response to oxyR Regulon

2. Superoxide response to soxRS Regulon.

 oxyR Regulon
• OxyR is a LysR-type transcriptional regulator, widespread in most Gram-
negative bacteria but it has also been reported in some Gram-positive
bacteria, such as S. aureus and S. Coelicolor.

• OxyR regulon spans more than 20 different genes, involved in several

molecular mechanisms of adaptive response to redox stress, such as H2O2
detoxification (katE and ahpCF), heme biosynthesis (hemH), reductant
supply (grxA, gorA, trxC), repression of iron import (fur, encoding Fur
regulator of ferric ion uptake) and others .

• Additionally, OxyR upregulates the expression of OxyS, a small regulatory

RNA that directs peroxide stress response .Although OxyS does not directly
affect the combat against excessive H2O2, this sRNA seems to play a role in
protection from H2O2-induced mutagenesis .

• Additionally, depletion of OxyS has shown to result in considerably higher

levels of H2O2 in E. coli
 H2O2 response to oxyR Regulon
• In E. coli, in which the normal intracellular H 2O2 level is ∼20 nM, OxyR is present in its reduced
form . Rapid oxidation of OxyRred occurs when intracellular H2O2 levels reach ∼100 nM, which is well
below the level at which growth is inhibited (∼2 μM) 

• Regulation by OxyR in E. coli begins by sensing the levels of H2O2 at a specific cysteine residue in the
protein (C199). Under regular conditions, OxyR is present in its reduced form .

• increased levels of H2O2 result in the rapid oxidation of OxyR: the peroxide molecule reacts with the
thiol group of C199, leading to interaction with the C208-SH to form an intramolecular disulphide
bond, inducing conformational changes which alter the DNA binding properties of OxyR, allowing
effective interaction with RNA polymerase .

• This molecular mechanism is then reversed through feedback regulation, since oxidized OxyR
induces the expression of the grxA and gor genes, encoding glutaredoxin 1 and glutathione
reductase, which act to reduce OxyR .

• Although the OxyR system has been mostly studied in E. coli, different bacteria have evolved in the
direction to adapt their particular OxyR regulon to better suit their environmental niches: they may
display differences in the molecular mechanism of H2O2 regulation, the number of OxyR homologs
or the type of genes present in their regulon.
• it affect DNA binding affinity and promoter conformation as well as render OxyR capable of
interacting with RNA polymerase. Transcription activation involves the direct interaction of OxyR
with the alpha subunit C-terminal domain (α-CTD) of RNA polymerase.
•Oxidized OxyR is reduced, using reduced glutathione (GSH) as the electron donor, via the
glutaredoxin (grxA)/glutathione reductase (gor) system, with reducing equivalents ultimately
supplied by NAD(P)H.
•Red and green boxes indicate RNA polymerase σ 70 −35 and −10 promoter elements,
respectively.
• Blue boxes indicate OxyR DNA binding contacts. Activation can also occur via oxidative
modification of the sensing cysteine alone.
 H2O2 response to oxyR Regulon

•By contrast to the E. coli model, OxyR has a dual function, acting as
not only an activator of peroxidescavenging enzymes under oxidative
stress conditions, but also a repressor of the same target genes under
nonstress conditions in some bacterial species such as Xanthomonas
campestris , Corynebacterium glutamicum ect ,.Although both the
reduced and oxidized forms of OxyR bind to the target promoter
regions .

•In vitro DNA-binding analyses reveal nonspecific DNA-binding

activity of the oxidized form of the OxyR protein in contrast to the
specific binding to the four target promoter regions under reducing
conditions.
 PerR System
• the most prevalent system for preventing peroxide oxidative stress in Gram-
positive bacteria is the PerR system. PerR belongs to the ferric uptake
regulator (Fur) superfamily of metalloregulatory transcription factors, firstly
identified in Bacillus subtilis where it is found to be the key regulator of the
H2O2 response .
• PerR has also been identified in some Gram-negative bacteria, response to
metal ions inside the cell through metal-catalyzed oxidation.
• Each subunit contains a structural site that binds irreversibly to zinc (Zn2+)
and a regulatory metal binding site . In B.subtilis, PerR binds either to
manganese (Mn2+) or to ferrous iron (Fe2+), which is preferred in most
conditions . Regulation by PerR is based on the oxidation of ferrous iron
(Fe2+) into ferric iron (Fe3+)
• The Fe(II)-bound form of PerR is responsive to H2O2, but the Mn(II)- bound
form is sensitive to peroxide and exerts repression in the presence of
inducing levels of peroxide. Thus, PerR activity is connected to metal ion
homeostasis in B. subtilis
Transcriptional regulator PerR regulates the inducible peroxide resistance response.
(A) Under normal conditions, PerR has a bound zinc ion (Zn 2+) and generally represses
transcription of PerR-regulated genes by directly binding conserved sequences,
known as Per boxes, in the promoter of each target gene.
(B) Under conditions of peroxide stress (H2O2), a ferrous ion (Fe2+) is bound, resulting
in the oxidation of histidine residues and conformational changes within PerR.
(C) Conformational changes reduce the DNA-binding affinity of PerR, resulting in the
dissociation of PerR from the PerR boxes, and transcription of the target gene.
Among these is the mrgA gene, a homolog of
the gene encoding Dps, a nonspecific DNA
binding protein that also sequesters Fe,
thereby protecting DNA from damaging
Fenton chemistry.
mrgA, katA (catalase), ahpCF
(alkylhydroperoxide reductase), and heme
biosynthesis genes are induced in the
presence of peroxide, but are repressed by
PerR when Mn(II) is added
 Superoxide response to soxRS Regulon
• The SoxRS regulon is a response mechanism to O2− and to redox-cycling
compounds, but not to H2O2 .

• This system is controlled by two proteins, which activate consecutively two

stages of transcription .

• SoxR is a homodimer, each with an iron-sulfur cluster [2Fe-2S] center and

whose activity depends on its oxidation state.
• Only the oxidized form ([2Fe-2S]2+) is active and able to induce transcription
of soxS.

• SoxS is a second transcriptional activator that induces the transcription of more

than 100 genes with functions in antioxidant defense, damage repair and
maintenance of central metabolisms that allow the cell to survive under
oxidative stress.
• Some of the genes regulated by SoxS are sodA, nfo, zwf, fumC, acnA, fur and
ntrA .
•After oxidative stress, SoxR is again reduced and inactivated through
reducing systems encoded by rseC and rsxABCDGE operon.

•Therefore, these systems maintain SoxR in its reduced state, preventing

activation of the SoxRS regulon .

•Also, when oxidative stress decreases, SoxS is rapidly degraded by

proteolysis.

• The transcription factor SoxR is widely distributed in Actinobacteria and

Proteobacteria.

• However, while in enteric bacteria SoxR activates the expression of SoxS

which in turn controls the expression of a diverse class of genes involved in the
stress response, in nonenteric bacteria (like Pseudomonas aeruginosa or
Streptomyces coelicolor) SoxS is absent, and SoxR directly activates a small
sets of genes in response to redox-cycling agents
The soxRS regulon. The soxRS locus is composed of the divergently transcribed soxR and soxS
genes. The SoxR protein is produced constitutively and is activated upon exposure to
superoxide-generating agents or nitric oxide (NO).
The oxidized form of SoxR enhances the transcription of the soxS gene, the product of which is
also a transcriptional activator.
The SoxS protein activates transcription of genes that increase the resistance to oxidants.
Additionally, activation of the SoxS-regulated genes increases the resistance to antibiotics and
macrophage-generated NO. Abbreviation: G6PD, glucose-6-P- dehydrogenase.  
 RpoS, general stress response regulator

RpoS respond to oxidative stress.

a toxin-antitoxin system, MqsR-MqsA, was determined to regulate rpoS levels

during oxidative stress .
 It was determined that, by mutating a palindromic sequence in the rpoS
promoter, MqsA no longer repressed expression from the promoter.

Therefore, under normal growth conditions, MqsA binds to the palindromic

sequence, 5’-ACCTTGCAGGT-3’, preventing rpo transcription .
Under oxidative stress however, Lon protease expression increases 8-fold ,
degrading MqsA and allowing for transcription .

RpoS is also essential for maintaining a lower frequency of DNA mutations in

longterm starved populations.
Transcriptomic investigation reveals that RpoS regulates oxidative stress
response genes, including those that encode for ferrodoxin, rubredoxin,
desulfoferredoxin ferrous iron-binding protein, and superoxide dismutase
References
 Seixas AF, Quendera AP, Sousa JP, Silva AFQ, Arraiano CM, Andrade JM.
Bacterial Response to Oxidative Stress and RNA Oxidation. Front
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35082839; PMCID: PMC8784731.

 Sarah M. Chiang, Herb E. Schellhorn(2012). Regulators of oxidative stress

response genes in Escherichia coli and their functional conservation in
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 Z.N.Kashmiri* and S.A.Mankar(2014). Free radicals and oxidative stress in

bacteria ISSN: 2319-7706 Volume 3 Number 9 (2014) pp. 34-40

 SPENCER B. FARR'* AND TOKIO KOGOMA2 (1991). Oxidative Stress

Responses in Escherichia coli and Salmonella typhimurium. American
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