Balance between Endogenous Superoxide Stress and Antioxidant Defenses

Cells devoid of cytosolic superoxide dismutase (SOD) suffer enzyme inactivation, growth deficiencies, and DNA damage. It has been proposed that the scant superoxide (O₂⁻) generated by aerobic metabolism harms even cells that contain abundant SOD. However, this idea has been difficult to test. To determine the amount of O₂⁻ that is needed to cause these defects, we modulated the O₂⁻ concentration inside Escherichia coli by controlling the expression of SOD. An increase in O₂⁻ of more than twofold above wild-type levels substantially diminished the activity of labile dehydratases, an increase in O₂⁻ of any more than fourfold measurably impaired growth, and a fivefold increase in O₂⁻ sensitized cells to DNA damage. These results indicate that E. coli constitutively synthesizes just enough SOD to defend biomolecules against endogenous O₂⁻ so that modest increases in O₂⁻ concentration diminish cell fitness. This conclusion is in excellent agreement with quantitative predictions based upon previously determined rates of intracellular O₂⁻ production, O₂⁻ dismutation, dehydratase inactivation, and enzyme repair. The vulnerability of bacteria to increased intracellular O₂⁻ explains the widespread use of superoxide-producing drugs as bactericidal weapons in nature. E. coli responds to such drugs by inducing the SoxRS regulon, which positively regulates synthesis of SOD and other defensive proteins. However, even toxic amounts of endogenous O₂⁻ did not activate SoxR, and SoxR activation by paraquat was not at all inhibited by excess SOD. Therefore, in responding to redox-cycling drugs, SoxR senses some signal other than O₂⁻.     

Comparative study of SoxR activation by redox‐active compounds

SoxR from Escherichia coli and related enterobacteria is activated by a broad range of redox‐active compounds through oxidation or nitrosylation of its [2Fe–2S] cluster. Activated SoxR then induces SoxS, which subsequently activates more than 100 genes in response. In contrast, non‐enteric SoxRs directly activate their target genes in response to redox‐active compounds that include endogenously produced metabolites. We compared the responsiveness of SoxRs from Streptomyces coelicolor (ScSoxR), Pseudomonas aeruginosa (PaSoxR) and E. coli (EcSoxR), all expressed in S. coelicolor, towards natural or synthetic redox‐active compounds. EcSoxR responded to all compounds examined, whereas ScSoxR was insensitive to oxidants such as paraquat (Eh ?440 mV) and menadione sodium bisulphite (Eh ?45 mV) and to NO generators. PaSoxR was insensitive only to some NO generators. Whole‐cell EPR analysis of SoxRs expressed in E. coli revealed that the [2Fe–2S]¹⁺ of ScSoxR was not oxidizable by paraquat, differing from EcSoxR and PaSoxR. The mid‐point redox potential of purified ScSoxR was determined to be ?185 ± 10 mV, higher by ～ 100 mV than those of EcSoxR and PaSoxR, supporting its limited response to paraquat. The overall sensitivity profile indicates that both redox potential and kinetic reactivity determine the differential responses of SoxRs towards various oxidants.     

Antimicrobial cationic surfactant, cetyltrimethylammonium bromide, induces superoxide stress in Escherichia coli cells

Aims: To clarify whether an antibacterial surfactant, cetyltrimethylammonium bromide (CTAB), induces superoxide stress in bacteria, we investigated the generation of superoxide and hydrogen peroxide and expression of soxR, soxS and soxRS regulon genes in Escherichia coli cells with the treatment of CTAB. Methods and Results: In situ oxidative stress analyses with BES fluorescent probes revealed that generation of both superoxide and hydrogen peroxide were significantly increased with the CTAB treatment at a sublethal concentration in wild‐type strain OW6, compared with the CTAB‐resistant strain OW66. The activity of manganese–superoxide dismutase (Mn–SOD), a member of the soxRS regulon proteins, was decreased by the CTAB treatment only in strain OW6. Furthermore, quantitative real‐time PCR analyses revealed that expression of the soxRS regulon genes was not upregulated, although soxS was upregulated by the CTAB treatment in strain OW6. Conclusions: Cetyltrimethylammonium bromide treatment led E. coli cells to a generation state of superoxide and hydrogen peroxide. It was also suggested that superoxide generation was caused by inhibiting SoxS function and decreasing Mn–SOD activity. Significance and Impact of the Study: It was revealed that excess superoxide generation in bacterial cells play a key action of antibacterial surfactants.     

Factors affecting redox potential and differential sensitivity of SoxR to redox‐active compounds

SoxR is a [2Fe‐2S]‐containing sensor‐regulator, which is activated through oxidation by redox‐active compounds (RACs). SoxRs show differential sensitivity to RACs, partly due to different redox potentials, such that Escherichia coli (Ec) SoxR with lower potential respond to broader range of RACs than Streptomyces coelicolor (Sc) SoxR. In S. coelicolor, the RACs that do not activate ScSoxR did not inhibit growth, suggesting that ScSoxR is tuned to respond to growth‐inhibitory RACs. Based on sequence comparison and mutation studies, two critical amino acids around the [2Fe‐2S] binding site were proposed as key determinants of sensitivity. ScSoxR‐like mutation (R127L/P131V) in EcSoxR changed its sensitivity profile as ScSoxR, whereas EcSoxR‐like mutation (L126R/V130P) in ScSoxR caused relaxed response. In accordance, the redox potentials of EcSoxR^R127L/P131V and ScSoxR^L126R/V130P were estimated to be ?192 ± 8 mV and ?273 ± 10 mV, respectively, approaching that of ScSoxR (?185 mV) and EcSoxR (?290 mV). Molecular dynamics simulations revealed that the R127L and P131V substitutions in EcSoxR caused more electropositive environment around [2Fe‐2S], making it harder to get oxidized. This reveals a mechanism to modulate redox‐potential in [Fe‐S]‐containing sensors by point mutations and to evolve a sensor with differential sensitivity to achieve optimal cellular physiology.     

Proposed mechanism of inactivating Escherichia coli O157:H7 by ultra‐high pressure in combination with tert‐butylhydroquinone

Aims: Investigating mechanisms of lethality enhancement when Escherichia coli O157:H7, and selected E. coli mutants, were exposed to tert‐butylhydroquinone (TBHQ) during ultra‐high pressure (UHP) treatment. Methods and Results: Escherichia coli O157:H7 EDL‐933, and 14 E. coli K12 strains with mutations in selected genes, were treated with dimethyl sulfoxide solution of TBHQ (15–30 ppm), and processed with UHP (400 MPa, 23 ± 2°C for 5 min). Treatment of wild‐type E. coli strains with UHP alone inactivated 2·4–3·7 log CFU ml^?1, whereas presence of TBHQ increased UHP lethality by 1·1–6·2 log CFU ml^?1; TBHQ without pressure was minimally lethal (0–0·6 log reduction). Response of E. coli K12 mutants to these treatments suggests that iron–sulfur cluster‐containing proteins ([Fe–S]‐proteins), particularly those related to the sulfur mobilization (SUF system), nitrate metabolism, and intracellular redox potential, are critical to the UHP–TBHQ synergy against E. coli. Mutations in genes maintaining redox homeostasis and anaerobic metabolism were associated with UHP–TBHQ resistance. Conclusions: The redox cycling activity of cellular [Fe–S]‐proteins may oxidize TBHQ, potentially leading to the generation of bactericidal reactive oxygen species. Significance and Impact of the Study: A mechanism is proposed for the enhanced lethality of UHP by TBHQ against E. coli O157:H7. The results may benefit food processors using UHP–based preservation, and biologists interested in piezophilic micro‐organisms.     

Regulators of oxidative stress response genes in Escherichia coli and their functional conservation in bacteria

Oxidative stress, through the production of reactive oxygen species, is a natural consequence of aerobic metabolism. Escherichia coli has several major regulators activated during oxidative stress, including OxyR, SoxRS, and RpoS. OxyR and SoxR undergo conformation changes when oxidized in the presence of hydrogen peroxide and superoxide radicals, respectively, and subsequently control the expression of cognate genes. In contrast, the RpoS regulon is induced by an increase in RpoS levels. Current knowledge regarding the activation and function of these regulators and their dependent genes in E. coli during oxidative stress forms the scope of this review. Despite the enormous genomic diversity of bacteria, oxidative stress response regulators in E. coli are functionally conserved in a wide range of bacterial groups, possibly reflecting positive selection of these regulators. SoxRS and RpoS homologs are present and respond to oxidative stress in Proteobacteria, and OxyR homologs are present and function in H(2)O(2) resistance in a range of bacteria, from gammaproteobacteria to Actinobacteria. Bacteria have developed complex, adapted gene regulatory responses to oxidative stress, perhaps due to the prevalence of reactive oxygen species produced endogenously through metabolism or due to the necessity of aerotolerance mechanisms in anaerobic bacteria exposed to oxygen.     

Oxidative-stress-induced production of pyocyanin by Xanthomonas campestris and its effect on the indicator target organism, Escherichia coli

Pyocyanin, a potential antimicrobial agent, was secreted by Xanthomonas campestris. Treatments with agents causing oxidative stress in the organism caused up to 4.4-fold increase in pyocyanin production. Pyocyanin added in the extracellular space did not affect growth rate of X. campestris, but decreased maximum cell concentration and specific product formation. However, the growth of Escherichia coli, the indicator target organism, was affected by pyocyanin. There was also a significant increase in the intracellular reactive oxygen species (ROS) concentration and antioxidant enzyme [catalase, superoxide dismutase (SOD)] concentrations, in the presence of pyocyanin. The intracellular ROS concentrations in E. coli formed upon exposure to pyocyanin, which is an indicator of the toxicity, was dependent on the growth phase of the organism. Studies with mutants of E. coli showed that intracellular ROS concentration was not significantly affected by the absence of the regulon OxyR, but, was significantly higher in cases when the regulon rpoS or the genes katG or katE were absent. Journal of Industrial Microbiology & Biotechnology (2000) 25, 266–272. Received 08 May 2000/ Accepted in revised form 04 August 2000