Challenging Xanthomonas campestris with low levels of arsenic mediates cross-protection against oxidant killing

Xanthomonas encounters highly toxic reactive oxygen species (ROS) from many sources, such as those generated by plants against invading bacteria, other soil bacteria and from aerobic respiration. Thus, conditions that alter intracellular ROS levels such as exposure to toxic metalloids would have profound effects on bacterial physiology. Here, we report that exposure of Xanthomonas campestris pv. phaseoli (Xp) to low levels of arsenic induces physiological cross-protection against killing by H(2)O(2) and organic hydroperoxide but not a superoxide generator. Cross-protection against H(2)O(2) and organic hydroperoxide toxicity was due to increased expression of genes encoding major peroxide-metabolizing enzymes such as alkyl hydroperoxide reductase (AhpC), catalase (KatA) and organic hydroperoxide resistance protein (Ohr). Arsenic-induced protection against H(2)O(2) and organic hydroperoxide requires the peroxide stress response regulators, OxyR and OhrR, respectively. Moreover, analyses of double mutants of the major H(2)O(2) and organic hyproperoxide-scavenging enzymes, Xp ahpC katA and Xp ahpC ohr, respectively, suggested the existence of unidentified OxyR- and OhrR-regulated genes that are involved in arsenic-induced resistance to H(2)O(2) and organic hyproperoxide killing in Xp. These arsenic-induced physiological alterations could play an important role in bacterial survival both in the soil environment and during plant-pathogen interactions.     

Exposure of phytopathogenic Xanthomonas spp. to lethal concentrations of multiple oxidants affects bacterial survival in a complex manner

During plant-microbe interactions and in the environment, Xanthomonas campestris pv. phaseoli is likely to be exposed to high concentrations of multiple oxidants. Here, we show that simultaneous exposures of the bacteria to multiple oxidants affects cell survival in a complex manner. A superoxide generator (menadione) enhanced the lethal effect of an organic peroxide (tert-butyl hydroperoxide) by 1, 000-fold; conversely, treatment of cells with menadione plus H(2)O(2) resulted in 100-fold protection compared to that for cells treated with the individual oxidants. Treatment of X. campestris with a combination of H(2)O(2) and tert-butyl hydroperoxide elicited no additive or protective effect. High levels of catalase alone are sufficient to protect cells against the lethal effect of menadione plus H(2)O(2) and tert-butyl hydroperoxide plus H(2)O(2). These data suggest that H(2)O(2) is the lethal agent responsible for killing the bacteria as a result of these treatments. However, increased expression of individual genes for peroxide (alkyl hydroperoxide reductase, catalase)- and superoxide (superoxide dismutase)-scavenging enzymes or concerted induction of oxidative stress-protective genes by menadione gave no protection against killing by a combination of menadione plus tert-butyl hydroperoxide. However, X. campestris cells in the stationary phase and a spontaneous H(2)O(2)-resistant mutant (X. campestris pv. phaseoli HR) were more resistant to killing by menadione plus tert-butyl hydroperoxide. These findings give new insight into oxidant killing of Xanthomonas spp. that could be generally applied to other bacteria.     

Copper ions potentiate organic hydroperoxide and hydrogen peroxide toxicity through different mechanisms in Xanthomonas campestris pv. campestris

Copper (Cu)-based biocides are important chemical controls for both fungal and bacterial diseases in crop fields. Here, we showed that Cu ions at a concentration of 100 μM enhanced t-butyl hydroperoxide (tBOOH) and hydrogen peroxide (H(2) O(2) ) killing of Xanthomonas campestris pv. campestris through different mechanisms. The addition of an antilipid peroxidation agent (α-tocopherol) and hydroxyl radical scavengers (glycerol and dimethyl sulphoxide) partially protected the bacteria from the Cu-enhanced tBOOH and H(2) O(2) killing, respectively. Inactivation of the alkyl hydroperoxide reductase gene rendered the mutant vulnerable to lethal doses of copper sulphate, which could be alleviated by the addition of an H(2) O(2) scavenger (pyruvate) and α-tocopherol. Taken together, the data suggest that Cu ions influence the killing effect of tBOOH through the stimulation of lipid peroxidation, while hydroxyl radical production is the underlying mechanism responsible for the Cu-ion-enhanced H(2) O(2) killing effects.     

A Xanthomonas alkyl hydroperoxide reductase subunit C (ahpC) mutant showed an altered peroxide stress response and complex regulation of the compensatory response of peroxide detoxification enzymes

Alkyl hydroperoxide reductase subunit C (AhpC) is the catalytic subunit responsible for alkyl peroxide metabolism. A Xanthomonas ahpC mutant was constructed. The mutant had increased sensitivity to organic peroxide killing, but was unexpectedly hyperresistant to H(2)O(2) killing. Analysis of peroxide detoxification enzymes in this mutant revealed differential alteration in catalase activities in that its bifunctional catalase-peroxidase enzyme and major monofunctional catalase (Kat1) increased severalfold, while levels of its third growth-phase-regulated catalase (KatE) did not change. The increase in catalase activities was a compensatory response to lack of AhpC, and the phenotype was complemented by expression of a functional ahpC gene. Regulation of the catalase compensatory response was complex. The Kat1 compensatory response increase in activity was mediated by OxyR, since it was abolished in an oxyR mutant. In contrast, the compensatory response increase in activity for the bifunctional catalase-peroxidase enzyme was mediated by an unknown regulator, independent of OxyR. Moreover, the mutation in ahpC appeared to convert OxyR from a reduced form to an oxidized form that activated genes in the OxyR regulon in uninduced cells. This complex regulation of the peroxide stress response in Xanthomonas differed from that in other bacteria.     

A suppressor of the menadione-hypersensitive phenotype of a Xanthomonas campestris pv. phaseoli oxyR mutant reveals a novel mechanism of toxicity and the protective role of alkyl hydroperoxide reductase

We isolated menadione-resistant mutants of Xanthomonas campestris pv. phaseoli oxyR (oxyR(Xp)). The oxyRR2(Xp) mutant was hyperresistant to the superoxide generators menadione and plumbagin and was moderately resistant to H(2)O(2) and tert-butyl hydroperoxide. Analysis of enzymes involved in oxidative-stress protection in the oxyRR2(Xp) mutant revealed a >10-fold increase in AhpC and AhpF levels, while the levels of superoxide dismutase (SOD), catalase, and the organic hydroperoxide resistance protein (Ohr) were not significantly altered. Inactivation of ahpC in the oxyRR2(Xp) mutant resulted in increased sensitivity to menadione killing. Moreover, high levels of expression of cloned ahpC and ahpF in the oxyR(Xp) mutant complemented the menadione hypersensitivity phenotype. High levels of other oxidant-scavenging enzymes such as catalase and SOD did not protect the cells from menadione toxicity. These data strongly suggest that the toxicity of superoxide generators could be mediated via organic peroxide production and that alkyl hydroperoxide reductase has an important novel function in the protection against the toxicity of these compounds in X. campestris.     

Evaluation of the roles that alkyl hydroperoxide reductase and Ohr play in organic peroxide-induced gene expression and protection against organic peroxides in Xanthomonas campestris

Alkyl hydroperoxide reductase (ahpC) and organic hydroperoxide resistance (ohr) are distinct genes, structurally and regulatory, but have similar physiological functions. In Xanthomonas campestris pv. phaseoli inactivation of either gene results in increased sensitivity to killing with organic peroxides. An ahpC1-ohr double mutant was highly sensitive to both growth inhibition and killing treatment with organic peroxides. High level expression of ahpC or ohr only partially complemented the phenotype of the double mutant, suggesting that these genes function synergistically, but through different pathways, to protect Xanthomonas from organic peroxide toxicity. Functional analyses of Ohr and AhpC abilities to degrade organic hydroperoxides revealed that both Ohr and AhpC could degrade tert-butyl hydroperoxide (tBOOH) while the former was more efficient at degrading cumene hydroperoxide (CuOOH). Expression analysis of these genes in the mutants showed no compensatory alterations in the levels of AhpC or Ohr. However, CuOOH induced expression of these genes in the mutants was affected. CuOOH induced ahpC expression was higher in the ohr mutant than in the parental strain; in contrast, the ahpC mutation has no effect on the level of induced ohr expression. These analyses reveal complex physiological roles and expression patterns of seemingly functionally similar genes.     

Atypical adaptive and cross-protective responses against peroxide killing in a bacterial plant pathogen, Agrobacterium tumefaciens

Physiological adaptive and cross-protection responses to oxidants were investigated in Agrobacterium tumefaciens. Exposure of A. tumefaciens to sublethal concentrations of H2O2 induced adaptive protection to lethal concentrations of H2O2. Similar treatments with organic peroxide and menadione did not produce adaptive protection to subsequent exposure to lethal concentrations of these oxidants. Pretreatment of A. tumefaciens with an inducing concentration of menadione conferred cross-protection against H2O2, but not to tert-butyl hydroperoxide (tBOOH), killing. The menadione induced cross-protection to H2O2 was due to the compound's ability to highly induce the peroxide scavenging enzyme, catalase. The levels of catalase directly correlated with the bacterium's ability to survive H2O2 treatment. Some aspects of the oxidative stress response of A. tumefaciens differ from other bacteria, and these differences may be important in plant/microbe interactions.     

Involvement of ethylene and lipid signalling in cadmium-induced programmed cell death in tomato suspension cells.

Cadmium-induced cell death was studied in suspension-cultured tomato (Lycopersicon esculentum Mill.) cells (line MsK8) treated with CdSO(4). Within 24 h, cadmium treatment induced cell death in a concentration-dependent manner. Cell cultures showed recovery after 2-3 days which indicates the existence of an adaptation mechanism. Cadmium-induced cell death was alleviated by the addition of sub muM concentrations of peptide inhibitors specific to human caspases indicating that cell death proceeds through a mechanism with similarities to animal programmed cell death (PCD, apoptosis). Cadmium-induced cell death was accompanied by an increased production of hydrogen peroxide (H(2)O(2)) and simultaneous addition of antioxidants greatly reduced cell death. Inhibitors of phospholipase C (PLC) and phospholipase D (PLD) signalling pathway intermediates reduced cadmium-induced cell death. Treatment with the G-protein activator mastoparan and a cell permeable analogue of the lipid signal second messenger phosphatidic acid (PA) induced cell death. Ethylene, while not inducing cell death when applied alone, stimulated cadmium-induced cell death. Application of the ethylene biosynthesis inhibitor aminoethoxy vinylglycine (AVG) reduced cadmium-induced cell death, and this effect was alleviated by simultaneous treatment with ethylene. Together the results show that cadmium induces PCD exhibiting apoptotic-like features. The cell death process requires increased H(2)O(2) production and activation of PLC, PLD and ethylene signalling pathways.     

Mutations in oxyR resulting in peroxide resistance in Xanthomonas campestris

A spontaneous Xanthomonas campestris pv. phaseoli H(2)O(2)-resistant mutant emerged upon selection with 1 mM H(2)O(2). In this report, we show that growth of this mutant under noninducing conditions gave high levels of catalase, alkyl hydroperoxide reductase (AhpC and AhpF), and OxyR. The H(2)O(2) resistance phenotype was abolished in oxyR-minus derivatives of the mutant, suggesting that elevated levels and mutations in oxyR were responsible for the phenotype. Nucleotide sequence analysis of the oxyR mutant showed three nucleotide changes. These changes resulted in one silent mutation and two amino acid changes, one at a highly conserved location (G197 to D197) and the other at a nonconserved location (L301 to R301) in OxyR. Furthermore, these mutations in oxyR affected expression of genes in the oxyR regulon. Expression of an oxyR-regulated gene, ahpC, was used to monitor the redox state of OxyR. In the parental strain, a high level of wild-type OxyR repressed ahpC expression. By contrast, expression of oxyR5 from the X. campestris pv. phaseoli H(2)O(2)-resistant mutant and its derivative oxyR5G197D with a single-amino-acid change on expression vectors activated ahpC expression in the absence of inducer. The other single-amino-acid mutant derivative of oxyR5L301R had effects on ahpC expression similar to those of the wild-type oxyR. However, when the two single mutations were combined, as in oxyR5, these mutations had an additive effect on activation of ahpC expression.     

Induction of peroxide and superoxide protective enzymes and physiological cross-protection against peroxide killing by a superoxide generator in Vibrio harveyi

Vibrio harveyi is a causative agent of destructive luminous vibriosis in farmed black tiger prawn (Penaeus monodon). V. harveyi peroxide and superoxide stress responses toward elevated levels of a superoxide generated by menadione were investigated. Exposure of V. harveyi to sub-lethal concentrations of menadione induced high expression of genes in both the OxyR regulon (e.g., a monofunctional catalase or KatA and an alkyl hydroperoxide reductase subunit C or AhpC), and the SoxRS regulon (e.g., a superoxide dismutase (SOD) and a glucose-6-phosphate dehydrogenase). V. harveyi expressed two detectable, differentially regulated SOD isozymes, [Mn]-SOD and [Fe]-SOD. [Fe]-SOD was expressed constitutively throughout the growth phase while [Mn]-SOD was expressed at the stationary phase and could be induced by a superoxide generator. Physiologically, pre-treatment of V. harveyi with menadione induced cross-protection against subsequent exposure to killing concentrations of H(2)O(2). This induced cross-protection required newly synthesized proteins. However, the treatment did not induce significant protection against exposures to killing concentrations of menadione itself or cross-protect against an organic hydroperoxide (tert-butyl hydroperoxide). Unexpectedly, growing V. harveyi in high-salinity media induced protection against menadione killing. This protection was independent of SOD induction. Stationary-phase cells were more resistant to menadione killing than exponential-phase cells. The induction of oxidative stress protective enzymes and stress-altered physiological responses could play a role in the survival of this bacterium in the host marine crustaceans.     

Adaptive response to H(2)O(2) protects against SnCl(2) damage: the OxyR system involvement

The stannous ion, mainly the stannous chloride (SnCl(2)) salt form, is widely used as a reducing agent to label radiotracers with technetium-99m ((99m)Tc). These radiotracers can be employed as radiopharmaceuticals in nuclear medicine procedures. In this case, there is no doubt about absorption of this complex, because it is intravenously administered in humans, although biological effects of these agents have not been fully understood. In this work we used a bacterial system to study the cytotoxic potential of stannous chloride. It is known that SnCl(2) induces lesions that could be mediated by reactive oxygen species (ROS). We, thus, investigated the existence of cross-adaptive response between hydrogen peroxide (H(2)O(2)) and SnCl(2) and the role of the OxyR system known to promote cellular protection against oxidative damages. Here we describe the results obtained with prior treatment of different Escherichia coli strains with sub-lethal doses of H(2)O(2), followed by incubation with SnCl(2). Our data show that H(2)O(2) is capable of inducing cross-adaptive response against the lethality promoted by SnCl(2), suggesting the OxyR system participation through catalase, alkyl hydroperoxide reductase and superoxide dismutase enzymes