Hydrogen peroxide resistance of Acetobacter pasteurianus NBRC3283 and its relationship to acetic acid fermentation

The bacterium Acetobacter pasteurianus can ferment acetic acid, a process that proceeds at the risk of oxidative stress. To understand the stress response, we investigated catalase and OxyR in A. pasteurianus NBRC3283. This strain expresses only a KatE homolog as catalase, which is monofunctional and growth dependent. Disruption of the oxyR gene increased KatE activity, but both the katE and oxyR mutant strains showed greater sensitivity to hydrogen peroxide as compared to the parental strain. These mutant strains showed growth similar to the parental strain in the ethanol oxidizing phase, but their growth was delayed when cultured in the presence of acetic acid and of glycerol and during the acetic acid peroxidation phase. The results suggest that A. pasteurianus cells show different oxidative stress responses between the metabolism via the membrane oxidizing pathway and that via the general aerobic pathway during acetic acid fermentation.     

The role of oxyR and soxRS in oxidative stress survival in Shigella flexneri

Shigella flexneri, a facultative intracellular pathogen, is exposed to a variety of environments inside and outside of the human host. Some of these environments may contain significant oxidative stress. S. flexneri mutants were generated with deletions in the major oxidative stress regulators oxyR and/or soxRS to test their importance in Shigella biology. Strains that contained a deletion of oxyR had reduced growth and survival during aerobic growth, but not microaerobic growth. The mutants were also defective in surviving exposure to oxidative stress: oxyR mutants were sensitive to hydrogen peroxide, while soxRS mutants were sensitive to superoxide. Although the ΔsoxRS, ΔoxyR, and ΔoxyR/ΔsoxRS mutant Shigellae survived similarly to the parental strains within macrophages, the mutants formed plaques on Henle cell monolayers that were slightly smaller than the plaques formed by the wildtype strain.     

Cj1386 is an ankyrin-containing protein involved in heme trafficking to catalase in Campylobacter jejuni

Campylobacter jejuni, a microaerophilic bacterium, is the most frequent cause of human bacterial gastroenteritis. C. jejuni is exposed to harmful reactive oxygen species (ROS) produced during its own normal metabolic processes and during infection from the host immune system and from host intestinal microbiota. These ROS will damage DNA and proteins and cause peroxidation of lipids. Consequently, identifying ROS defense mechanisms is important for understanding how Campylobacter survives this environmental stress during infection. Construction of a ΔCj1386 isogenic deletion mutant and phenotypic assays led to its discovery as a novel oxidative stress defense gene. The ΔCj1386 mutant has an increased sensitivity toward hydrogen peroxide. The Cj1386 gene is located directly downstream from katA (catalase) in the C. jejuni genome. A ΔkatAΔ Cj1386 double deletion mutant was constructed and exhibited a sensitivity to hydrogen peroxide similar to that seen in the ΔCj1386 and ΔkatA single deletion mutants. This observation suggests that Cj1386 may be involved in the same detoxification pathway as catalase. Despite identical KatA abundances, catalase activity assays showed that the ΔCj1386 mutant had a reduced catalase activity relative to that of wild-type C. jejuni. Heme quantification of KatA protein from the ΔCj1386 mutant revealed a significant decrease in heme concentration. This indicates an important role for Cj1386 in heme trafficking to KatA within C. jejuni. Interestingly, the ΔCj1386 mutant had a reduced ability to colonize the ceca of chicks and was outcompeted by the wild-type strain for colonization of the gastrointestinal tract of neonate piglets. These results indicate an important role for Cj1386 in Campylobacter colonization and pathogenesis.     

Proteomic analysis of the oxidative stress response in Candida albicans

An efficient oxidative stress response (OSR) is important for the facultative pathogenic yeast Candida albicans to survive within the human host. We used a large scale 2-D protein gel electrophoresis approach to analyze the stress response mechanisms of C. albicans after treatment with hydrogen peroxide and the thiol oxidizing agent, diamide. Quantitation of in vivo protein synthesis after pulse labeling of the proteins with radioactive L-[35S]-methionine resulted in characteristic proteome signatures for hydrogen peroxide and diamide with significant overlap of 21 up-regulated proteins for both stressors. Among the induced proteins were enzymes with known antioxidant functions like catalase or thioredoxin reductase and a set of oxidoreductases. 2-D gel analysis of mutants in the CAP1 gene revealed that the synthesis of 12 proteins is controlled by the oxidative stress regulator Cap1p. Stressing its importance for the C. albicans OSR, all 12 proteins were also induced after oxidative challenge by hydrogen peroxide or diamide.     

Involvement of <Emphasis Type="Italic">soxRS</Emphasis> Regulon in Response of <Emphasis Type="Italic">Escherichia coli</Emphasis> to Oxidative Stress Induced by Hydrogen Peroxide

The effect of hydrogen peroxide on the activity of soxRS and oxyR regulon enzymes in different strains of Escherichia coli has been studied. Treatment of bacteria with 20 μM H₂O₂ caused an increase in catalase and peroxidase activities (oxyR regulon) in all strains investigated. It is shown for the first time that oxidative stress induced by hydrogen peroxide causes in some E. coli strains a small increase in activity of superoxide dismutase and glucose-6-phosphate dehydrogenase (soxRS regulon). This effect is cancelled by chloramphenicol, an inhibitor of protein synthesis in prokaryotes. The increase in soxRS regulon enzyme activities was not found in the strain lacking the soxR gene. These results provide evidence for the involvement of the soxRS regulon in the adaptive response of E. coli to oxidative stress induced by hydrogen peroxide. __________ Translated from Biokhimiya, Vol. 70, No. 11, 2005, pp. 1506–1513. Original Russian Text Copyright ? 2005 by Semchyshyn, Bagnyukova, Lushchak.     

OxyR,an important oxidative stress regulator to phenazines production and hydrogen peroxide resistance in Pseudomonas chlororaphis GP72

Pseudomonas chlororaphis GP72 is an important plant growth-promoting rhizobacteria (PGPR) with a wide-spectrum antibiotic activity toward several soil-borne pathogens. The adaption of this strain to different environmental oxidative stress and redox phenazine pigment by the predicted regulator OxyR were investigated. The deletion of oxyR led to a significant reduction of the viability, production of three phenazine derivatives and resistance to hydrogen peroxide and paraquat on the KB agar plates. However, the mutant ΔoxyR grew better with shorter delay. In addition, the mutant ΔoxyR showed an increased resistance to hydrogen peroxide, which occurred at the concentration varying from 1.0 mM to 5.0 mM in the KB broth, as compared with the wild type. In addition, the biofilm formation ability was obviously enhanced and influenced by the different oxidants in the mutant. Quantitative RT-PCR experiments indicated that the expression of katG, ahpC, ahpD and phzE were increased in the oxyR mutant background in response to hydrogen peroxide. katG was mainly responsible for the enhanced resistance to hydrogen peroxide. The loss of oxyR is suggested to benefit the hydrogen peroxide inducible gene expression. Thus, OxyR is an important global regulator that regulates multiple pathways to enhance the survival of P. chlororaphis GP72 exposed to different oxidative stresses.     

Molecular and physiological effects of mycobacterial oxyR inactivation

The majority of slow-growing mycobacteria have a functional oxyR, the central regulator of the bacterial oxidative stress response. In contrast, this gene has been inactivated during the evolution of Mycobacterium tuberculosis. Here we inactivated the oxyR gene in Mycobacterium marinum, an organism used to model M. tuberculosis pathogenesis. Inactivation of oxyR abrogated induction of ahpC, a gene encoding alkylhydroperoxide reductase, normally activated upon peroxide challenge. The absence of oxyR also resulted in increased sensitivity to the front-line antituberculosis drug isoniazid. Inactivation of oxyR in M. marinum did not affect either virulence in a fish infection model or survival in human macrophages. Our findings demonstrate, at the genetic and molecular levels, a direct role for OxyR in ahpC regulation in response to oxidative stress. Our study also indicates that oxyR is not critical for virulence in M. marinum. However, oxyR inactivation confers increased sensitivity to isonicotinic acid hydrazide, suggesting that the natural loss of oxyR in the tubercle bacillus contributes to the unusually high sensitivity of M. tuberculosis to isoniazid.     

Ralstonia solanacearum extracellular polysaccharide is a specific elicitor of defense responses in wilt-resistant tomato plants

Ralstonia solanacearum, which causes bacterial wilt of diverse plants, produces copious extracellular polysaccharide (EPS), a major virulence factor. The function of EPS in wilt disease is uncertain. Leading hypotheses are that EPS physically obstructs plant water transport, or that EPS cloaks the bacterium from host plant recognition and subsequent defense. Tomato plants infected with R. solanacearum race 3 biovar 2 strain UW551 and tropical strain GMI1000 upregulated genes in both the ethylene (ET) and salicylic acid (SA) defense signal transduction pathways. The horizontally wilt-resistant tomato line Hawaii7996 activated expression of these defense genes faster and to a greater degree in response to R. solanacearum infection than did susceptible cultivar Bonny Best. However, EPS played different roles in resistant and susceptible host responses to R. solanacearum. In susceptible plants the wild-type and eps(-) mutant strains induced generally similar defense responses. But in resistant Hawaii7996 tomato plants, the wild-type pathogens induced significantly greater defense responses than the eps(-) mutants, suggesting that the resistant host recognizes R. solanacearum EPS. Consistent with this idea, purified EPS triggered significant SA pathway defense gene expression in resistant, but not in susceptible, tomato plants. In addition, the eps(-) mutant triggered noticeably less production of defense-associated reactive oxygen species in resistant tomato stems and leaves, despite attaining similar cell densities in planta. Collectively, these data suggest that bacterial wilt-resistant plants can specifically recognize EPS from R. solanacearum.     

Increasing the oxidative stress response allows Escherichia coli to overcome inhibitory effects of condensed tannins

Tannins are plant-derived polyphenols with antimicrobial effects. The mechanism of tannin toxicity towards Escherichia coli was determined by using an extract from Acacia mearnsii (Black wattle) as a source of condensed tannins (proanthocyanidins). E. coli growth was inhibited by tannins only when tannins were exposed to oxygen. Tannins auto-oxidize, and substantial hydrogen peroxide was generated when they were added to aerobic media. The addition of exogenous catalase permitted growth in tannin medium. E. coli mutants that lacked HPI, the major catalase, were especially sensitive to tannins, while oxyR mutants that constitutively overexpress antioxidant enzymes were resistant. A tannin-resistant mutant was isolated in which a promoter-region point mutation increased the level of HPI by 10-fold. Our results indicate that wattle condensed tannins are toxic to E. coli in aerobic medium primarily because they generate H(2)O(2). The oxidative stress response helps E. coli strains to overcome their inhibitory effect.     

Effect of a waaL mutation on lipopolysaccharide composition, oxidative stress survival, and virulence in Erwinia amylovora

Erwinia amylovora , the causal agent of fire blight, is an enterobacterial pathogen of Rosaceous plants including apple and pear. We have been studying the response of E. amylovora to oxidative stress because, during infection, the bacterium elicits an oxidative burst response in host plants. During the screening of a transposon mutant library for hydrogen peroxide sensitivity, we identified a mutant carrying an insertion in waaL , a gene involved in lipopolysaccharide biosynthesis, that was more sensitive to hydrogen peroxide than the parental wild-type strain. We also confirmed that a waaL mutant of Pseudomonas aeruginosa exhibited an increased sensitivity to hydrogen peroxide compared with the wild-type strain. The E. amylovora waaL mutant was also reduced in virulence, showed a decrease in twitching motility, and was more sensitive to polymyxin B than the wild type. Each of these phenotypes was complemented by the cloned waaL gene. Our results highlight the importance of the lipopolysaccharide layer to virulence in E. amylovora and the unexpected finding of an additional function of lipopolysaccharide in protection from oxidative stress in E. amylovora and P. aeruginosa .     

Chemotaxis is required for virulence and competitive fitness of the bacterial wilt pathogen Ralstonia solanacearum

Ralstonia solanacearum, a soilborne plant pathogen of considerable economic importance, invades host plant roots from the soil. Qualitative and quantitative chemotaxis assays revealed that this bacterium is specifically attracted to diverse amino acids and organic acids, and especially to root exudates from the host plant tomato. Exudates from rice, a nonhost plant, were less attractive. Eight different strains from this heterogeneous species complex varied significantly in their attraction to a panel of carbohydrate stimuli, raising the possibility that chemotactic responses may be differentially selected traits that confer adaptation to various hosts or ecological conditions. Previous studies found that an aflagellate mutant lacking swimming motility is significantly reduced in virulence, but the role of directed motility mediated by the chemotaxis system was not known. Two site-directed R. solanacearum mutants lacking either CheA or CheW, which are core chemotaxis signal transduction proteins, were completely nonchemotactic but retained normal swimming motility. In biologically realistic soil soak virulence assays on tomato plants, both nonchemotactic mutants had significantly reduced virulence indistinguishable from that of a nonmotile mutant, demonstrating that directed motility, not simply random motion, is required for full virulence. In contrast, nontactic strains were as virulent as the wild-type strain was when bacteria were introduced directly into the plant stem through a cut petiole, indicating that taxis makes its contribution to virulence in the early stages of host invasion and colonization. When inoculated individually by soaking the soil, both nontactic mutants reached the same population sizes as the wild type did in the stems of tomato plants just beginning to wilt. However, when tomato plants were coinoculated with a 1:1 mixture of a nontactic mutant and its wild-type parent, the wild-type strain outcompeted both nontactic mutants by 100-fold. Together, these results indicate that chemotaxis is an important trait for virulence and pathogenic fitness in this plant pathogen.     

The role of the natural polyamine putrescine in defense against oxidative stress in Escherichia coli

Putrescine up-regulated, in a concentration-dependent manner, the expression levels of the oxyR and katG genes of Escherichia coli cells exposed to hydrogen peroxide. Its stimulatory effect was more pronounced under conditions of strong oxidative stress. 1,4-Diamino-2-butanone, a specific inhibitor of putrescine synthesis, also inhibited oxyR expression under oxidative stress. When added to inhibited cells, putrescine relieved this inhibitory effect. Addition of putrescine to E. coli cultures exposed to oxidative stress led to increased cell survival.     

Evidence against a direct antimicrobial role of H2O2 in the infection of plants by Erwinia chrysanthemi

We have investigated the role of bacterial resistance to oxidative stress in pathogenesis. The oxyR gene from the pathogenic bacterium Erwinia chrysanthemi has been characterized. It is closely related to that found in Escherichia coli (88% overall amino acid identity). An E. chrysanthemi oxyR mutant strain was constructed by marker exchange. After induction with a sublethal dose of H2O2, this mutant was more sensitive to H2O2 and showed reduced levels of catalase and glutathione reductase activities, compared with the wild type. The oxyR mutant was unable to form individual colonies on agar plates unless catalase was added exogenously. However, it retained full virulence in potato tubers and tobacco leaves. These results suggest that the host-produced H2O2 has no direct antimicrobial effect on the interaction of E. chrysanthemi with the two plant species.     

Positive control of a regulon for defenses against oxidative stress and some heat-shock proteins in Salmonella typhimurium

S. typhimurium become resistant to killing by hydrogen peroxide and other oxidants when pretreated with nonlethal levels of hydrogen peroxide. During adaptation to hydrogen peroxide, 30 proteins are induced. Nine are constitutively overexpressed in dominant hydrogen peroxide-resistant oxyR mutants. Mutant oxyR1 is resistant to a variety of oxidizing agents and overexpresses at least five enzyme activities involved in defenses against oxidative damage. Deletions of oxyR are recessive and uninducible by hydrogen peroxide for the nine proteins overexpressed in oxyR1, demonstrating that oxyR is a positive regulatory element. The oxyR1 mutant is also more resistant than the wild-type parent to killing by heat, and it constitutively overexpresses three heat-shock proteins. The oxyR regulatory network is a previously uncharacterized global regulatory system in enteric bacteria.     

Escherichia coli response to hydrogen peroxide: a role for DNA supercoiling, topoisomerase I and Fis

Bacterial cells respond to the deleterious effects of reactive oxygen species by inducing the expression of antioxidant defence genes. Here we show that treatment with hydrogen peroxide leads to a transient decrease in DNA negative supercoiling. We also report that hydrogen peroxide activates topA P1 promoter expression. The peroxide-dependent topA P1 activation is independent of oxyR, but is mediated by Fis. This nucleoid-associated protein binds to the promoter region of topA. We also show that a fis deficient mutant strain is extremely sensitive to hydrogen peroxide. Our results suggest that topA activation by Fis is an important component of the Escherichia coli response to oxidative stress.     

Induction of the manganese-containing superoxide dismutase in Escherichia coli is independent of the oxidative stress (oxyR-controlled) regulon

The synthesis of manganese-superoxide dismutase in response to hydrogen peroxide and to paraquat was examined in strains of Escherichia coli with different mutations in the oxyR gene. Hydrogen peroxide treatment did not induce manganese-superoxide dismutase, but did induce the oxyR regulon. Paraquat induced this enzyme in a strain compromised in its ability to induce the defense response against oxidative stress (oxyR deletion) as well as in a strain that is constitutive and overexpresses the oxyR regulon. Catalase (HPI), but not manganese-superoxide dismutase, was over-expressed under anaerobic conditions in a strain harboring a constitutive oxyR mutation. The data clearly demonstrate that the induction of manganese-superoxide dismutase is independent of the oxyR-controlled regulon.