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

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

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

Electrometric record of E17R (in 20 mM Hepes, pH 7.4) with the BLM system.

<p>A. Transient currents and B. after the addition of the protonophore FCCP. Black bars indicate illumination with a 75 W XBO long-pass filtered at >495 nm. The grey bar shows the additional excitation of the M-state (>380 nm).</p

Transient absorbance changes of E17R.

<p>A. The kinetics of the absorbance changes are shown for selected wavelengths (399 nm, 517 nm and 598 nm). B. The decay-associated spectra are depicted as obtained from the global fit. The corresponding time constants are given in the figure.</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

Phylogenetic tree inferred using the Maximum likelihood method of 21 amino acid sequences of <i>Exiguobacterium</i> spp. closely related with E17R.

<p>The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1,000 replicates) are shown next to the branches. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree.</p

Multiple protein alignment of PR from <i>Exiguobacterium</i> sp. S17 (E17R), green-light absorbing proteorhodopsin from <i>Exiguobacterium sibiricum</i> (ESR) and blue-light absorbing proteorhodopsin from the uncultured gamma-proteobacterium “Hot 75m4” (BPR).

<p>Residues shared between all the protein variants are marked with asterisks. Single amino acid residue at position 106 (BPR numbering) that functions as a spectral tuning switch and accounts for most of the spectral difference between the two pigment families is highlighted in light grey. Primary proton acceptor and donor are highlighted in dark grey (D86) and with a frame (K97), respectively. The Schiff base (K232 for ESR, K226 for E17R) is indicated by a diamond. Residues differing between both green-PRs are indicated with arrows. The seven transmembrane α-helices are indicated with blue bubbles.</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

Absorption spectrum of E17R in 20 mM Hepes, 100 mM NaCl, 0.03% DDM.

<p>The inset shows the difference spectrum after bleaching with hydroxylamine. The extinction coefficient was calculated using the absorbance of the oxime product ΔA(oxime) in comparison to the bleached rhodopsin absorbance ΔA(Rh) as reference.</p