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Int.J.Curr.Microbiol.App.Sci (2014) 3(9) 34-40

ISSN: 2319-7706 Volume 3 Number 9 (2014) pp. 34-40 http://www.ijcmas.com

Review Article Free radicals and in bacteria Z.N.Kashmiri* and S.A.Mankar Department of Zoology, Sindhu Mahavidyalaya, Nagpur 440 017, India *Corresponding author

A B S T R A C T

Aerobic bacteria use molecular (O2) for respiration or oxidation of nutrients K e y w o r d s to obtain energy. (ROS) are necessary for various Oxidative physiological functions but an imbalance in favor of reactive oxygen species results stress, in oxidative stress (OS). As in humans, the exposure of bacteria to ROS causes Reactive damage to a variety of macromolecules, resulting in and often in cell oxygen . However, ROS may also be considered to be beneficial compounds, as they species, function as signaling that to a coordinated response of bacteria under -stress conditions. This paper reviews about free radical, generation of reactive oxygen species in bacteria and defense mechanisms responses to oxidative stress.

Introduction 2. Types of free radicals 1. Free radicals Reactive oxygen species (ROS) represent Free radicals are a group of short-lived the collection of number of molecules and reactive chemical intermediates, which free radicals derived from molecular oxygen contain one or more electrons with unpaired in addition, there is another class of free and can oxidatively modify radicals that are derived called that they encounter. This reactive nitrogen species (RNS) (Sikka, causes them to react almost instantly with 2001). These reactive species are readily any substance in their vicinity (Warren et converted into reactive non-radical species al., 1987). Generally, free radicals attack the by enzymatic or non-enzymatic chemical nearest stable , "stealing" its reactions that in turn can give rise to new electron (Fig.1). When the "attacked" radicals (Table 1 and Fig. 2). molecule loses its electron, it becomes a free radical itself, beginning a chain reaction. 3. Cellular Source of ROS s Once the process is started, it can cascade and ultimately lead to the disrupting of In a living system there are various sources living cells. They are highly reactive and of ROS such as byproduct of cellular oxidize lipids, amino acids and respiration (presence of redox cycling carbohydrates as well as causing DNA compounds), synthesized by mutations. systems phagocytic cells, neutrophils and

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Int.J.Curr.Microbiol.App.Sci (2014) 3(9) 34-40 (NADPH oxidase, 5. Physiological role of ROS myeloperoxidases), exposure to ionizing , production: a) Chain reaction, a Aerobic bacteria use molecular oxygen (O2) free radical steals an electron from a nearby for respiration or oxidation of nutrients to compound forming a new free radical. Free obtain energy. Bacteria are in continuous radicals may steal electrons from cellular contact with ROS over the course of their structures or molecules, b) by normal life cycle generated both endogenously, as a cellular respiration electron transport product of aerobic metabolism, or system often oxygen is the terminal exogenously during ionizing ( ) and electron acceptor in the cell mitochondria to nonionizing (UV) irradiation leading to the ROS. Also smoking, herbicides, pesticides, production of a number of radical and fried foods as well as industrial peroxide species through the ionization of contaminants that are widespread in soils intracellular water (Cabiscol et al., 2000 and and on the surfaces of plants are sources of de Oru´e Lucana et al., 2012). Reactive by- ROS. Recent work has demonstrated that products of oxygen, such as ROS have a role in cell signaling, including; anion radical (O2 ), apoptosis; gene expression; and the (H2O2), and the highly reactive hydroxyl activation of cell signaling cascades. It radicals (·OH), are generated continuously should be noted that ROS can serve as both in cells grown aerobically (Cabiscol et al., intra- and intercellular messengers (Hancock 2000). These species cause damage to et al., 2001). proteins, lipids, and nucleotides, negatively impacting the organism (de Oru´e Lucana et 4. Oxidative stress al., 2012).

Oxidative stress (OS) is the term applied 6. Cellular defense or when oxidants outnumber . defense mechanisms against ROS Interference in the balance between the environmental production of reactive Living organisms have to build up oxygen species (ROS), including hydroxyl mechanisms to protect themselves against radicals ( OH) and hydrogen peroxide oxidative stress, with such as (H2O2), and the ability of biological systems and , small to readily detect and detoxify them, or repair proteins like thioredoxin and glutaredoxin, the resulting damage are oxidative stress (de and molecules such as . Bacterial Oru´e Lucana et al., 2012). genetic responses to oxidative stress are controlled by two major transcriptional It is a common condition caused by regulators (OxyR and SoxRS). ROS damage biological systems in aerobic conditions a variety of cellular macromolecules and such that antioxidants cannot scavenge the thus elicit adaptive oxidative stress free radicals. This causes an excessive responses in bacteria intended to permit generation of ROS, which damages cells, survival in the presence of this stressor tissues and organs. Highly reactive radicals (Storz and Imlay, 1999; James and Imlay, cause the oxidative damage of different 2008). Expression of a number of multidrug macromolecules proteins, DNA, and efflux systems is positively impacted by lipids leading to loss of function, an agents of oxidative stress, these efflux increased rate of mutagenesis, and systems possibly playing a role in ultimately cell death. ameliorating the effects of this stress.

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Int.J.Curr.Microbiol.App.Sci (2014) 3(9) 34-40

Similarly, antioxidant mechanisms are complex chain reactions initiated. DNA is a recruited in response to antimicrobial main target; active species attack both the exposure, antimicrobials being known to base and the sugar moieties producing generate ROSs that are key to the often single- and double-strand breaks in the lethal effects of these agents (Dwyer et al., backbone, adducts of base and sugar groups, 2009; Kolodkin-Gal et al., 2008). As such and cross-links to other molecules, lesions oxidative stress responses have the potential that block replication (Sies and Menck, to contribute to antimicrobial resistance in a 1992, Sies, 1993). variety of ways. Presence of oxygen in the atmosphere led to Cells have a variety of defense mechanisms the development of defense mechanisms that to ameliorate the harmful effects of ROS. either kept the concentration of the O2- Superoxide dismutase catalyzes the derived radicals at acceptable levels or conversion of two superoxide anions into a repaired oxidative damages. plays a molecule of hydrogen peroxide (H2O2) and significant role in biology (transport, storage oxygen (O2) (McCord and Fridovich, 1968). and activation of molecular oxygen, As studies the biological targets for these reduction of ribonucleotides, activation and highly reactive oxygen species are DNA, decomposition of peroxides, and electron RNA, proteins and lipids. Much of the transport) and Fe2+ is required for the damage is caused by hydroxyl radicals growth of almost all living cells. Due to its generated from H2O2 via the Fenton potential damaging effects, in bacteria, iron reaction, which requires iron (or another solubilization and metabolism is strictly divalent metal , such as copper) and a regulated at two levels: source of reducing equivalents (possibly NADH) to regenerate the metal. Lipids are (i) the entrance to the cell by specific major targets during oxidative stress. Free membrane-bound receptors, and radicals can attack directly polyunsaturated fatty acids in membranes and initiate lipid (ii) inside the cell, by two proteins, peroxidation. bacterioferritin and , very similar to the eukaryotic ferritin, but presenting A primary effect of is a ferroxidase activity. Some molecules are decrease in membrane fluidity, which alters constitutively present and help to maintain membrane properties and can disrupt an intracellular reducing environment or to membrane-bound proteins significantly. scavenge chemically reactive oxygen. This effect acts as an amplifier, more Among these molecules are nonenzymatic radicals are formed, and polyunsaturated antioxidants such as NADPH and NADH fatty acids are degraded to a variety of pools, -carotene, ascorbic acid, - products. Some of them, such as , , and glutathione (GSH). GSH, are very reactive and can damage molecules present at high concentrations, maintain a such as proteins (Humpries and Sweda, strong reducing environment in the cell, and 1998). Unlike reactive free radicals, its reduced form is maintained by aldehydes are rather long lived and can using NADPH as a therefore diffuse from the site of their origin source of reducing power. In addition, and reach and attack targets which are specific enzymes decrease the steady-state distant from the initial free-radical event, levels of reactive oxygen (Cabiscol et al., acting as second toxic messengers of the 2000).

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Int.J.Curr.Microbiol.App.Sci (2014) 3(9) 34-40

Fig.1 Formation of a free radical (Agarwal et al., 2008)

Fig.2 Free radicals

Table.1 Examples of ROS and RNS

Reactive oxygen species Reactive nitrogen species

Superoxide anion (O2 - ) (NO )

Hydrogen peroxide (H2O2) Nitric dioxide (NO2 )

Hydroxyl radical (OH ) Peroxynitrite (ONOO-)

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Int.J.Curr.Microbiol.App.Sci (2014) 3(9) 34-40

Fig.3 Overview of damage caused by reactive oxygen species in Escherichia coli

enzymes. DNA repair enzymes (Demple Two superoxide dismutases (SOD), which and Harrison, 1994) include endonuclease convert O2 to H2O2 and O2, have been IV, which is induced by oxidative stress, described in Escherichia coli (fig.3) an and exonuclease III, which is induced in iron-containing enzyme, whose expression the stationary phase and in starving cells. is modulated by intracellular iron levels Both enzymes act on duplex DNA (Niederhoffer et al., 1990), and a cleaning up DNA 3' termini. Prokaryotic containing SOD, the cells contain catalysts able to repair predominant enzyme during aerobic directly some covalent modifications to growth, whose expression is the primary structure of proteins. One of transcriptionally regulated by at least six the most frequent modifications is the control systems (Compan and Touati, reduction of oxidized disulfide bonds: (i) 1993). A third SOD activity with thioredoxin reductase transfers electrons properties like eukaryotic CuZn-SOD has from NADPH to thioredoxin via a flavin been found in the E. coli periplasmic space carrier, (ii) glutaredoxin is also able to (Benov and Fridovich, 1994). In E. coli, reduce disulfide bonds, but using GSH as H2O2 is removed by two an electron donor and, (iii) protein (yielding H2O and O2): hydroperoxidase I disulfide isomerase facilitates disulfide (HPI), which is present during aerobic exchange reactions with large inactive growth and transcriptionally controlled at protein substrates, besides having different levels (Finn and Condon, 1975), chaperone activity. Oxidation of and hydroperoxidase II (HPII), which is methionine to can be induced during stationary phase (von repaired by methionine sulfoxide Ossowski et al., 1991). Glutathione reductase. Recent experimental data peroxidase and DT-diaphorase are also described that surface exposed methionine scavenging enzymes. residues surrounding the entrance to the are preferentially oxidized Secondary defenses include DNA-repair without loss of catalytic activity, and systems and proteolytic and lipolytic 38 Int.J.Curr.Microbiol.App.Sci (2014) 3(9) 34-40

suggested that methionine residues could Oxidative stress in bacteria and function as a last-chance antioxidant protein damage by reactive oxygen defense system for proteins (Levine et al., species. Int. Microbiol., 3: 3 8. 1996). Carlioz, A., Touati, D. (1986). Isolation of superoxide dismutase mutants in Reactive oxygen species (ROS) damage Escherichia coli: is superoxide DNA, RNA, proteins and lipids, resulting dismutase necessary for aerobic life? in cell death when the level of ROS EMBO J., 5: 623 630. exceeds an organism s detoxification and Compan, I., Touati, D. (1993). Interaction repair capabilities. Despite this danger, of six global transcription regulators bacteria growing aerobically generate in expression of manganese ROS as a metabolic by-product, a risk superoxide dismutase in Escherichia balanced by an increased efficiency and coli K-12. J. Bacteriol., 175:1687 yield of energy from growth substrates. 1696. Two possible mechanisms can be used to de Oru´e Lucana, D.O., Wedderhoff, I., manipulate bacterial ROS metabolism and Groves, M.R. (2012). ROS-mediated increase sensitivity of bacteria to oxidative signalling in bacteria: zinc- attack i) amplification of endogenous ROS containing cys-x-x-cys redox centres production and ii) impairment of and iron-based oxidative stress. J. detoxification and repair systems. Whereas Sig. Trans., p. 9. removal of their detoxification and repair doi:10.1155/2012/605905. system has been shown to make bacteria Demple, B., Harrison, L. (1994). Repair of more susceptible to oxidants (Carlioz and oxidative damage to DNA: Touati, 1986; Loewen, 1984), antibiotics enzymology and biology. Annu. Rev. (Dwyer et al., 2007) and immune attack Biochem., 63: 915 948. (Hebrard et al., 2009 and Liu, 2008), Dwyer, D.J., Kohanski, M.A., Hayete, B., manipulation of endogenous bacterial Collins, J.J. (2007). Gyrase inhibitors ROS production remains largely induce an oxidative damage cellular unexplored. death pathway in Escherichia coli. Mol. Syst. Biol., 3: 91. References Dwyer, D.J., Kolanski, M.A., Collins, J.J. (2009). Role of reactive oxygen Agarwal, A., Cocuzza, M., Abdelrazik, H., species in antibiotic-mediated cell Sharma, R.K. (2008). Oxidative death. Cell, 135: m679 690. stress measurement in patients with Finn, G.J., Condon, S. (1975). Regulation male or female factor infertility. In: of catalase synthesis in Handbook of Chemiluminescent typhimurium. J. Bacteriol., 123: Methods in Oxidative Stress 570 579. Assessment, Kerala Transworld Hancock, J.T., Desikan, R., Neill, S.J. Research Network, pp. 195 218. (2001). Role of reactive oxygen Benov, L.T., Fridovich, I. (1994). species in cell signaling pathways. Escherichia coli expresses a copper- Biochemical and Biomedical Aspects and zinc-containing superoxide of Oxidative Modification, 29(2): dismutase. J. Biol. Chem., 269: 345 350. 25310 25314. Hebrard, M., Viala, J.P., Meresse, S., Cabiscol, E., Tamarit, J., Ros, J. (2000) Barras, F., Aussel, L. (2009).

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