3 Introduction Aerobic organisms use molecular oxygen (O2) for respiration or oxidation of nutrients to obtain energy. Reactive by-products of oxygen, such as superoxide anion radical (O2���), hydrogen peroxide (H2O2), and the highly reactive hydroxyl radicals (��OH), are generated continuously in cells grown aerobically. Most of such products derive from sequential univalent reductions of molecular oxygen catalyzed by several membrane���associated respiratory chain enzymes. Experimental data indicate that, in Escherichia coli, the respiratory chain can account for as much as 87% of the total H2O2 production [23]. The leakage of single electrons from the bacterial respiratory chain was observed at the NADH dehydrogenase and ubiquinone sites, and was similar to that observed in eukaryotic mitochondria. Environmental agents such as ionizing, near- UV radiation, or numerous compounds that generate intra- cellular O2��� (redox-cycling agents such as menadione and paraquat) can cause oxidative stress, which arises when the concentration of active oxygen increases to a level that exceeds the cell���s defense capacity. Some immune cells which use the NADPH oxidase enzyme, upon invasion by pathogenic bacteria, also exploit oxidative stress as a weapon during phagocytosis. The biological targets for these highly reactive oxygen species are DNA, RNA, proteins and lipids. Much of the damage is caused by hydroxyl radicals generated from H2O2 via the Fenton reaction, which requires iron (or another divalent metal ion, such as copper) and a source of reducing equivalents (possibly NADH) to regenerate the metal. Lipids are major targets during oxidative stress. Free radicals can attack directly polyunsaturated fatty acids in membranes and initiate lipid peroxidation. A primary effect of lipid peroxidation is a decrease in membrane fluidity, which alters membrane properties and can disrupt membrane-bound proteins significantly. This effect acts as an amplifier, more radicals are formed, and polyunsaturated fatty acids are degraded to a variety of products. Some of them, such as aldehydes, are very reactive and can damage molecules such as proteins [28]. Unlike reactive free radicals, aldehydes are rather long lived and can therefore diffuse from the site of their origin and reach and attack targets which are distant from the initial free-radical event, acting as ���second toxic messengers��� of the complex chain reactions initiated. Among the many different aldehydes which can form during lipid peroxidation, the most intensively studied are malonaldehyde (MDA) and 4-hydroxyalkenals, in particular 4-hydroxynonenal (HNE) [17]. DNA is also a main target active species attack both the base and the sugar moieties producing single- and double-strand breaks in the backbone, adducts of base and sugar groups, and cross-links to other molecules, lesions that block replication [41, 42]. The spectrum of adducts in oxidized DNA in vitro and in vivo includes more than 20 known products, including damage to all four bases and thymine-tyrosine cross-links [14]. The oxidation of proteins, Elisa Cabiscol Jordi Tamarit Joaquim Ros Department of Basic Medical Sciences, Faculty of Medicine, University of Lleida, Spain Received 23 September 1999 Accepted 27 December 1999 Correspondence to: Joaquim Ros. Departament de Ci��ncies M��diques B��siques. Facultat de Medicina. Rovira Roure, 44. 25198 Lleida. Spain. Tel.: +34-973702407. Fax: +34-973702426. E-mail: joaquim.ros@cmb.udl.es REVIEW ARTICLE INTERNATL MICROBIOL (2000) 3:3���8 �� Springer-Verlag Ib��rica 2000 Oxidative stress in bacteria and protein damage by reactive oxygen species Summary The advent of O2 in the atmosphere was among the first major pollution events occurred on earth. The reaction between ferrous iron, very abundant in the reductive early atmosphere, and oxygen results in the formation of harmful superoxide and hydroxyl radicals, which affect all macromolecules (DNA, lipids and proteins). Living organisms have to build up mechanisms to protect themselves against oxidative stress, with enzymes such as catalase and superoxide dismutase, small proteins like thioredoxin and glutaredoxin, and molecules such as glutathione. Bacterial genetic responses to oxidative stress are controlled by two major transcriptional regulators (OxyR and SoxRS). This paper reviews major key points in the generation of reactive oxygen species in bacteria, defense mechanisms and genetic responses to oxidative stress. Special attention is paid to the oxidative damage to proteins. Key words Oxygen �� Oxidative stress �� Reactive oxygen species �� Protein oxidation �� Carbonyl groups

which has been traditionally less well characterized, is one of the aims of this review. Several classes of damage are documented [21, 44], including oxidation of sulfhydryl groups, reduction of disulfides, oxidative adduction of amino acid residues close to metal-binding sites via metal-catalyzed oxidation, reaction with aldehydes, modification of prosthetic groups or metal clusters, protein-protein cross-linking and peptide fragmentation. All these modifications are deleterious to the cell, since they lead to a loss of function of membranes and proteins, and block DNA replication or cause mutations. Defense mechanisms The appearance of oxygen in the atmosphere led to the development of defense mechanisms that either kept the concentration of the O2-derived radicals at acceptable levels or repaired oxidative damages. Iron plays a significant role in biology (transport, storage and activation of mole- cular oxygen, reduction of ribonucleotides, activation and decomposition of peroxides, and electron transport) and Fe2+ is required for the growth of almost all living cells. Due to its potential damaging effects, in bacteria, iron solubilization and metabolism is strictly regulated at two levels: (i) the entrance to the cell by specific membrane-bound receptors, and (ii) inside the cell, by two proteins, bacterioferritin and ferritin, very similar to the eukaryotic ferritin, but presenting ferroxidase activity. Some molecules are constitutively present and help to maintain an intracellular reducing environment or to scavenge chemically reactive oxygen. Among these molecules are nonenzymatic antioxidants such as NADPH and NADH pools, ��-carotene, ascorbic acid, ��-tocopherol, and glutathione (GSH). GSH, present at high concentrations, maintain a strong reducing environment in the cell, and its reduced form is maintained by glutathione reductase using NADPH as a source of reducing power. In addition, specific enzymes decrease the steady-state levels of reactive oxygen. Two superoxide dismutases (SOD), which convert O2��� to H2O2 and O2, have been described in Escherichia coli: an iron-containing enzyme, whose expression is modulated by intracellular iron levels [38], and a manganese- containing SOD, the predominant enzyme during aerobic growth, whose expression is transcriptionally regulated by at least six control systems [6]. A third SOD activity with properties like eukaryotic CuZn-SOD has been found in the E. coli periplasmic space [2]. In E. coli, H2O2 is removed by two catalases (yielding H2O and O2): hydroperoxidase I (HPI),which is present during aerobic growth and transcriptionally controlled at different levels [20], and hydroperoxidase II (HPII), which is induced during stationary phase [49]. Glutathione peroxidase and DT-diaphorase are also scavenging enzymes. Secondary defenses include DNA-repair systems and proteolytic and lipolytic enzymes. DNA repair enzymes [reviewed in ref. 13] include endonuclease IV, which is induced by oxidative stress, and exonuclease III, which is induced in the stationary phase and in starving cells. Both enzymes act on duplex DNA cleaning up DNA 3' termini. Prokaryotic cells contain catalysts able to repair directly some covalent modifications to the primary structure of proteins. One of the most frequent modifications is the reduction of oxidized disulfide bonds: (i) thioredoxin reductase transfers electrons from NADPH to thioredoxin via a flavin carrier, (ii) glutaredoxin is also able to reduce disulfide bonds, but using GSH as an electron donor and, (iii) protein disulfide isomerase facilitates disulfide exchange reactions with large inactive protein substrates, besides having chaperone activity. Oxidation of methionine to methionine sulfoxide can be repaired by methionine sulfoxide reductase. Recent experimental data described that surface-exposed methionine residues surrounding the entrance to the active site are preferentially oxidized without loss of catalytic activity, and suggested that methionine residues could function as a ���last-chance��� antioxidant defense system for proteins [31]. Genetic responses Genetic responses to oxidative stress occur in bacteria [extensively reviewed in ref. 19 and 26], yeast, mammalian cell lines and, in general, in all aerobic organisms. E. coli cells possess a specific defense against peroxides, mediated by the transcriptional activator OxyR, and another against superoxide, controlled by the two-stage SoxRS system. The SoxRS regulon contains at least ten genes, including those encoding the Mn- SOD, endonuclease IV, glucose-6-P DH, a fumarase, aconitase, ferredoxin reductase and micF RNA, which affects the expression of a major outer membrane protein. The oxyR gene controls, among others, the genes encoding the HPI catalase, glutaredoxin, glutathione reductase, NADPH-dependent alkyl hydroperoxide reductase, and a protective DNA-binding protein (Dps). The activation of these responses greatly increases cellular resistance to oxidative agents. Both OxyR and SoxR are present, but inactivated in ���unstressed��� cells. It was proposed that the activation of SoxR protein could result from reversible one-electron oxidation of its iron-sulfur centers [27]. More recently, it was reported that OxyR is reversibly activated by the formation of an intramolecular disulfide bond, resulting from the altered redox state of the cytosol. OxyR activation is reversed by cellular disulfide-reducing machinery, with particular dependence on glutaredoxin. The gene encoding glutaredoxin is regulated by OxyR, thus providing a mechanism for autoregulation [1, 50]. Using the formation and reduction of a disulfide bond as an ���on-off��� switch allows for rapid response to oxidative conditions (Fig. 1). The response against H2O2 does not finish with the activation of OxyR. OxyR defective mutants are able to induce around 20���30 proteins in response to H2O2. These mutants are, however, hypersensitive to H2O2 and have higher mutation rates even 4 INTERNATL MICROBIOL Vol. 3, 2000 Cabiscol et al.