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

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

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

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

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