KatG, the bifunctional catalase of xanthomonas citri subsp. citri, responds to hydrogen peroxide and contributes to epiphytic survival on citrus leaves

Xanthomonas citri subsp. citri (Xcc) is the bacterium responsible for citrus canker. This bacterium is exposed to reactive oxygen species (ROS) at different points during its life cycle, including those normally produced by aerobic respiration or upon exposition to ultraviolet (UV) radiation. Moreover, ROS are key components of the host immune response. Among enzymatic ROS-detoxifying mechanisms, catalases eliminate H2O2, avoiding the potential damage caused by this specie. Xcc genome includes four catalase genes. In this work, we studied the physiological role of KatG, the only bifunctional catalase of Xcc, through the construction and characterization of a modified strain (XcckatG), carrying an insertional mutation in the katG gene. First, we evaluated the involvement of KatG in the bacterial adaptive response to H2O2. XcckatG cultures exhibited lower catalase activity than those of the wild-type strain, and this activity was not induced upon treatment with sub-lethal doses of H2O2. Moreover, the KatG-deficient mutant exhibited decreased tolerance to H2O2 toxicity compared to wild-type cells and accumulated high intracellular levels of peroxides upon exposure to sub-lethal concentrations of H2O2. To further study the role of KatG in Xcc physiology, we evaluated bacterial survival upon exposure to UV-A or UV-B radiation. In both conditions, XcckatG showed a high mortality in comparison to Xcc wild-type. Finally, we studied the development of bacterial biofilms. While structured biofilms were observed for the Xcc wild-type, the development of these structures was impaired for XcckatG. Based on these results, we demonstrated that KatG is responsible for Xcc adaptive response to H2O2 and a key component of the bacterial response to oxidative stress. Moreover, this enzyme plays an important role during Xcc epiphytic survival, being essential for biofilm formation and UV resistance.Para citar este articulo: Tondo ML, Delprato ML, Kraiselburd I, Fernández Zenoff MV, Farías ME, Orellano EG (2016) KatG, the Bifunctional Catalase of Xanthomonas citri subsp. citri, Responds to Hydrogen Peroxide and Contributes to Epiphytic Survival on Citrus Leaves. PLoS ONE 11(3): e0151657. doi:10.1371/journal.pone.0151657Fil: Tondo, María Laura. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario (IBR -CONICET); Argentina.Fil: Delprato, María Laura. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario (IBR -CONICET); Argentina.Fil: Kraiselburd, Ivana. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario (IBR -CONICET); Argentina.Fil: Fernández Zenoff, María Verónica. Planta Piloto de Procesos Industriales Microbiológicos (PROIMI -CONICET); Argentina.Fil: Farías, María Eugenia. Planta Piloto de Procesos Industriales Microbiológicos (PROIMI -CONICET); Argentina.Fil: Orellano, Elena G. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario (IBR -CONICET); Argentina

Native Killer Yeasts as Biocontrol Agents of Postharvest Fungal Diseases in Lemons.

Economic losses caused by postharvest diseases represent one of the main problems of the citrus industry worldwide. The major diseases affecting citrus are the "green mold" and "blue mold", caused by Penicillium digitatum and P. italicum, respectively. To control them, synthetic fungicides are the most commonly used method. However, often the emergence of resistant strains occurs and their use is becoming more restricted because of toxic effects and environmental pollution they generate, combined with trade barriers to international markets. The aim of this work was to isolate indigenous killer yeasts with antagonistic activity against fungal postharvest diseases in lemons, and to determine their control efficiency in in vitro and in vivo assays. Among 437 yeast isolates, 8.5% show to have a killer phenotype. According to molecular identification, based on the 26S rDNA D1/D2 domain sequences analysis, strains were identified belonging to the genera Saccharomyces, Wickerhamomyces, Kazachstania, Pichia, Candida and Clavispora. Killers were challenged with pathogenic molds and strains that caused the maximum in vitro inhibition of P. digitatum were selected for in vivo assays. Two strains of Pichia and one strain of Wickerhamomyces depicted a significant protection (p <0.05) from decay by P. digitatum in assays using wounded lemons. Thus, the native killer yeasts studied in this work showed to be an effective alternative for the biocontrol of postharvest fungal infections of lemons and could be promising agents for the development of commercial products for the biological control industry

Catalase activity pattern in the Xcc<i>katG</i> mutant.

<p>Xcc wild-type (WT), Xcc<i>katG</i> (<i>katG</i>) and cXcc<i>katG</i> (<i>ckatG</i>) strains were grown aerobically in SB medium to early exponential phase (4 h), and soluble extracts were prepared as described in the experimental section. Equal amounts of protein (25 μg) were separated by 8% (w/v) non-denaturing PAGE and stained for catalase activity [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0151657#pone.0151657.ref024" target="_blank">24</a>].</p

Effect of <i>katG</i> disruption on biofilm formation.

<p>(A) GFP-labeled Xcc strains were grown on chambered cover slides and visualized under confocal laser scanning microscopy (CLSM) after 2 days of bacterial growth. Left panels show the biofilms developed at the bottom of the chambered cover slides with a magnification of 400X and right panels show a 2X zoom of the regions marked in the previous panels. Scale bars, 50 μm. (B) Xcc strains were statically grown on glass tubes for 12 days at 28°C. Biofilm formation levels on the air-liquid interface were determined by crystal violet staining. The results show the means and standard deviations of a representative experiment with triplicate samples. The experiment was repeated three times with similar results in all cases.</p

Sensitivity of Xcc<i>katG</i> to hydrogen peroxide.

<p>(A) Hydrogen peroxide resistance of pre-adapted Xcc cells. Exponential phase cultures of Xcc wild-type and <i>katG</i> mutant were adapted with the indicated concentrations of H₂O₂ for 60 min and then exposed to 1 mM H₂O₂ for 15 min. The number of CFU was determined for each culture before and after the treatment with 1 mM H₂O₂ by plating of appropriate dilutions. The percentage of survival was calculated as the number of CFU after treatment divided by the number of CFU prior to treatment ×100. Data represent mean ± standard deviation of three independent experiments. (B) ROS accumulation upon exposure to hydrogen peroxide. Bacteria were exposed to 100 μM H₂O₂ for 1 hour, and total peroxides (-OOH) were determined in cleared extracts using the FOX II assay as described in the experimental section. Measurements were carried out in triplicate for two independent experiments, and the results are expressed as means ± standard deviations. Statistical significant differences (P < 0.05, ANOVA) between wild-type and <i>katG</i> strains are indicated by an asterisk.</p

Catalase activity of Xcc cultures in response to sub-lethal levels of hydrogen peroxide^a.

<p>Catalase activity of Xcc cultures in response to sub-lethal levels of hydrogen peroxide<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0151657#t002fn001" target="_blank">^a</a>.</p

Pathogenicity and epiphytic fitness of Xcc<i>katG</i> in orange plants.

<p>(A) Growth of Xcc strains in the apoplastic space of orange leaves. Xcc WT, Xcc<i>katG</i> and cXcc<i>katG</i> cells were inoculated at 10⁵ CFU/mL in 10 mM MgCl₂ into the intercellular spaces of fully expanded orange leaves. Bacterial populations in leaf tissues were determined by serial dilution and plating. A representative leaf 20 days after inoculation with the three strains is shown in the lower inset. Left panel, adaxial side; right panel, abaxial side. Dashed lines indicate the infiltrated area. (B) Epiphytic populations of Xcc strains on orange leaves. Bacterial cells were released from the leaf surface by sonication followed by dilution plating. Experiments were performed in triplicate; values are expressed as means ± standard deviations. Statistical significant differences (P < 0.05, ANOVA) between wild-type and <i>katG</i> strains are indicated by an asterisk.</p

Detection of catalase and peroxidase activities in Xcc cultures adapted with hydrogen peroxide.

<p>Exponential phase cultures were treated with the indicated concentrations of H₂O₂ for 60 min, and soluble extracts were prepared as described in the experimental section. Equal amounts of protein (25 μg) were separated in duplicate on 8% non-denaturing polyacrilamide gels stained for catalase (A) and peroxidase (B) activities. The position of the single catalase-peroxidase species detected is indicated by an arrow. Histograms below gels show the activity profiles obtained by densitometric quantification of the fast-migrating bands intensities. IOD, integrated optical density; A.U., arbitrary units. C, untreated control culture.</p

Degree of relative inhibition of killer yeast strains against <i>P</i>. <i>digitatum</i>, <i>P</i>. <i>italicum</i> and <i>P</i>. <i>citri</i>.

<p>Degree of relative inhibition of killer yeast strains against <i>P</i>. <i>digitatum</i>, <i>P</i>. <i>italicum</i> and <i>P</i>. <i>citri</i>.</p

Control of <i>P</i>. <i>digitatum</i> by wounds protection with killer yeasts, after 5 days of incubation.

<p>(A) Control lemons, inoculated with pathogen spore suspension. (B), (C) and (D) pretreated lemons with 27, 28 and 56 yeast strains, respectively.</p