Characterization of Xanthomonas oryzae pv. oryzae recX, a gene that is required for high-level expression of recA

A universally primed (UP)-PCR cross hybridization assay was developed for rapid identification of isolates of Rhizoctonia solani into the correct anastomosis group (AG). Twenty-one AG tester isolates belonging to 11 AGs of R. solani were amplified with a single UP primer which generated multiple PCR fragments for each isolate. The amplified products were spotted onto a filter, immobilized and used for cross hybridization against amplification products from the different isolates. Isolates within AG subgroups cross hybridize strongly, whereas between different AGs little or no cross hybridization occurs. Sixteen Rhizoctonia isolates from diseased sugar beets and potatoes were identified using the assay. The results were supported by restriction fragment length polymorphism analysis of the ITS1-5.8S-ITS2 region of the nuclear encoded ribosomal DNA. Through standardization and use of quick non-radioactive labeling techniques, the UP-PCR cross hybridization assay has potential for routine use by modern DNA chip technology.     

Atypical Adaptive and Cross-Protective Responses Against Peroxide Killing in a Bacterial Plant Pathogen, <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>

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 compounds ability to highly induce the peroxide scavenging enzyme, catalase. The levels of catalase directly correlated with the bacteriums 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. Received: 12 November 2002 / Accepted: 13 December 2002     

Multiple superoxide dismutases in Agrobacterium tumefaciens: functional analysis, gene regulation, and influence on tumorigenesis

Agrobacterium tumefaciens possesses three iron-containing superoxide dismutases (FeSods) encoded by distinct genes with differential expression patterns. SodBI and SodBII are cytoplasmic isozymes, while SodBIII is a periplasmic isozyme. sodBI is expressed at a high levels throughout all growth phases. sodBII expression is highly induced upon exposure to superoxide anions in a SoxR-dependent manner. sodBIII is expressed only during stationary phase. Analysis of the physiological function of sods reveals that the inactivation of sodBI markedly reduced levels of resistance to a superoxide generator, menadione. A mutant lacking all three Sod enzymes is the most sensitive to menadione treatment, indicating that all sods contribute at various levels towards the overall menadione resistance level. Sods also have important roles in A. tumefaciens virulence toward a host plant. A sodBI but not a sodBII or sodBIII mutant showed marked reduction in its ability to induce tumors on tobacco leaf discs, while the triple sod null mutant is avirulent.     

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 H₂O₂ resulted in 100-fold protection compared to that for cells treated with the individual oxidants. Treatment of X. campestris with a combination of H₂O₂ 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 H₂O₂ and tert-butyl hydroperoxide plus H₂O₂. These data suggest that H₂O₂ 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 H₂O₂-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.