RESUMEN
Xanthomonas campestris pv. campestris causes black rot in cruciferous crops. Hydrogen peroxide (H(2)O(2)) production and accumulation is an important initial response in plant defense against invading microbes. The role of genes involved in the bacterial H(2)O(2) protection system in pathogenicity was evaluated. Mutants of katA (encoding a monofunctional catalase) and, to a lesser extent, katG (encoding a catalase-peroxidase) and oxyR (encoding a H(2)O(2) sensor and a transcription regulator), are hypersensitive to H(2)O(2) treatments that mimic the plant H(2)O(2) burst. These data correlate with the results of pathogenicity testing that show katA, katG, and oxyR mutants are avirulent on a compatible plant. Moreover, exposure to H(2)O(2) (1, 2, and 4 mM) highly induces the expression of genes in the OxyR regulon, including katA, katG, and ahpC. The avirulent phenotype of the oxyR mutant is partly because of its inability to mount an adaptive response upon exposure to an H(2)O(2) burst. Our data provide insights into important roles of a transcription regulator and other genes involved in peroxide stress protection in the virulence of X. campestris pv. campestris.
Asunto(s)
Peróxido de Hidrógeno/toxicidad , Estrés Oxidativo , Enfermedades de las Plantas/microbiología , Regulón , Xanthomonas campestris/patogenicidad , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Catalasa/metabolismo , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Peroxidasa/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Virulencia , Xanthomonas campestris/efectos de los fármacos , Xanthomonas campestris/genética , Xanthomonas campestris/metabolismoRESUMEN
Xanthomonas campestris pv. campestris katG encodes a catalase-peroxidase that has a role in protecting the bacterium against micromolar concentrations of H(2)O(2). A knockout mutation in katG that causes loss of catalase-peroxidase activity correlates with increased susceptibility to H(2)O(2) and a superoxide generator and is avirulent in a plant model system. katG expression is induced by oxidants in an OxyR-dependent manner.
Asunto(s)
Proteínas Bacterianas/fisiología , Catalasa/metabolismo , Peróxido de Hidrógeno/toxicidad , Peroxidasas/metabolismo , Virulencia/efectos de los fármacos , Proteínas Bacterianas/genética , Southern Blotting , Catalasa/genética , Peroxidasas/genética , Reacción en Cadena de la Polimerasa , Raphanus/microbiología , Virulencia/genética , Xanthomonas campestris/efectos de los fármacos , Xanthomonas campestris/enzimología , Xanthomonas campestris/patogenicidadRESUMEN
Xanthomonas campestris pv. campestris, a soil-borne plant-pathogenic bacterium, is exposed to multiple stresses in the environment and during interaction with a host plant. The roles of hydrogen peroxide (H(2) O(2) )-protective genes (katA, katG, and ahpC) and a peroxide sensor/transcription regulator (oxyR) in the viability of X. campestris pv. campestris at an elevated temperature were evaluated. The single katA and katG mutants showed moderate decreased survival after the heat treatment, while the double katA-katG and oxyR mutants were the most vulnerable to the heat treatment compared with a wild-type strain. However, ahpC provided no protective function against the heat treatment. Flow cytometric analysis revealed an increased accumulation of peroxide in cells treated with heat. Altogether, the data revealed a crucial role of genes in the H(2) O(2) detoxification system for protection against lethal heat shock in X. campestris pv. campestris.
Asunto(s)
Calor , Viabilidad Microbiana/efectos de los fármacos , Viabilidad Microbiana/efectos de la radiación , Estrés Oxidativo , Estrés Fisiológico , Xanthomonas campestris/enzimología , Xanthomonas campestris/fisiología , Biotransformación , Peróxido de Hidrógeno/metabolismo , Peróxido de Hidrógeno/toxicidad , Oxidantes/metabolismo , Oxidantes/toxicidad , Xanthomonas campestris/efectos de los fármacos , Xanthomonas campestris/efectos de la radiaciónRESUMEN
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.
Asunto(s)
Cobre/farmacología , Peróxido de Hidrógeno/toxicidad , Iones/farmacología , Xanthomonas campestris/efectos de los fármacos , Xanthomonas campestris/metabolismo , Proteínas Bacterianas/genética , Farmacorresistencia Bacteriana/genética , Xanthomonas campestris/genéticaRESUMEN
Transposon mini-Tn7 vectors insert into the chromosome of several Gram-negative bacteria in a site-specific manner. Here, we showed the application of mini-Tn7 as single copy site-specific integration vector system for Xanthomonas campestris pv. campestris. The transposition of the mini-Tn7 into the bacterial genome was detected at a Tn7 attachment (attTn7) site located downstream of glmS1. Furthermore, using a newly constructed vector pBBR1FLP2 containing the flipase (FLP) recombinase for site-specific excision of the sequence between the FLP recombinase target (FRT) sites, and a sacB counter selection marker, an unmarked mini-Tn7 insertion mutant was created. Mini-Tn7 insertion did not affect bacterial virulence on the tested plant. The mini-Tn7 and FLP-FRT systems also work well in Xanthomonas oryzae pv. oryzae.