Detection of superoxide dismutase (Cu–Zn) isoenzymes in leaves and pseudobulbs of Bulbophyllum morphologlorum Kraenzl orchid by comparative proteomic analysis

Typically, biological systems are protected from the toxic effect of free radicals by antioxidant defense. Extracts from orchids have been reported to show high levels of exogenous antioxidant activity including Bulbophyllum orchids but so far, there have been no reports on antioxidant enzymes. Therefore, differences in protein expression from leaves and pseudobulbs of Bulbophyllum morphologlorum Kraenzl and Dendrobium Sonia Earsakul were studied using two-dimensional gel electrophoresis and mass spectrometry (LC/MS/MS). Interestingly, the largest group of these stress response proteins were associated with antioxidant defense and temperature stress, including superoxide dismutase (Cu–Zn) and heat shock protein 70. The high expression of this antioxidant enzyme from Bulbophyllum morphologlorum Kraenzl was confirmed by activity staining on native-PAGE, and the two Cu/Zn-SODs isoenzymes were identified as Cu/Zn-SOD 1 and Cu/Zn-SOD 2 by LC/MS/MS. The results suggested that Bulbophyllum orchid can be a potential plant source for medicines and natural antioxidant supplements.     

Agrobacterium tumefaciens soxR is involved in superoxide stress protection and also directly regulates superoxide-inducible expression of itself and a target gene

Inactivation of Agrobacterium tumefaciens soxR increases sensitivity to superoxide generators. soxR expression is highly induced by superoxide stress and is autoregulated. SoxR also directly regulates the superoxide-inducible expression of atu5152. Taken together, the physiological role of soxR and the mechanism by which it regulates expression of target genes make the A. tumefaciens SoxR system different from other bacterial systems.     

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.     

Analysis of growth phase regulated KatA and CatE and their physiological roles in determining hydrogen peroxide resistance in Agrobacterium tumefaciens

Agrobacterium tumefaciens possesses two catalases, a bifunctional catalase-peroxidase, KatA and a homologue of a growth phase regulated monofunctional catalase, CatE. In stationary phase cultures and in cultures entering stationary phase, total catalase activity increased 2-fold while peroxidase activity declined. katA and catE were found to be independently regulated in a growth phase dependent manner. KatA levels were highest during exponential phase and declined as cells entered stationary phase, while CatE was detectable at early exponential phase and increased during stationary phase. Only small increases in H2O2 resistance levels were detected as cells entering stationary phase. The katA mutant was more sensitive to H2O2 than the parental strain during both exponential and stationary phase. Inactivation of catE alone did not significantly change the level of H2O2 resistance. However, the katA catE double mutant was more sensitive to H2O2 during both exponential and stationary phase than either of the single catalase mutants. The data indicated that KatA plays the primary role and CatE acts synergistically in protecting A. tumefaciens from H2O2 toxicity during all phases of growth. Catalase-peroxidase activity (KatA) was required for full H2O2 resistance. The expression patterns of the two catalases in A. tumefaciens reflect their physiological roles in the protection against H2O2 toxicity, which are different from other bacteria.     

The role of a bifunctional catalase-peroxidase KatA in protection of Agrobacterium tumefaciens from menadione toxicity

Agrobacterium tumefaciens is an aerobic plant pathogenic bacterium that is exposed to reactive oxygen species produced either as by-products of aerobic metabolism or by the defense systems of host plants. The physiological function of the bifunctional catalase-peroxidase (KatA) in the protection of A. tumefaciens from reactive oxygen species other than H(2)O(2) was evaluated in the katA mutant (PB102). Unexpectedly, PB102 was highly sensitive to the superoxide generator menadione. The expression of katA from a plasmid vector complemented the menadione-hypersensitive phenotype. A. tumefaciens possesses an additional catalase gene, a monofunctional catalase encoded by catE. Neither inactivation nor high-level expression of the catE gene altered the menadione resistance level. Moreover, heterologous expression of the catalase-peroxidase-encoding gene katG from Burkholderia pseudomallei, but not the monofunctional catalase gene katE from Xanthomonas campestris could restore normal levels of menadione resistance to PB102. A recent observation suggests that the menadione resistance phenotype involves increased activities of organic peroxide-metabolizing enzymes. Heterologous expression of X. campestris alkyl hydroperoxide reductase from a plasmid vector failed to complement the menadione-sensitive phenotype of PB102. The level of menadione resistance shows a direct correlation with the level of peroxidase activity of KatA. This is a novel role for KatA and suggests that resistance to menadione toxicity is mediated by a new, and as yet unknown, mechanism in A. tumefaciens.