Predicted domains of <i>B</i>. <i>dolosa</i> strain AU0158 FixL and FixJ.

<p>Domains predicted by SMART [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006116#ppat.1006116.ref025" target="_blank">25</a>]. PAS domains (named after 3 proteins in which they occur, namely, Per, Arnt, and Sim) are seen commonly in signaling proteins where they function as signal sensor and often include a cofactor such as heme. Domain abbreviations: TM- transmembrane, PAC- Motif C-terminal to PAS motif, HisKA-histidine kinase, HATPase- histidine kinase-associated ATPase, REC-CheY homologous receiver domain, and HTH LuxR-helix-turn-helix-Lux regulon (DNA binding domain).</p

The <i>fixLJ</i> pathway is a global regulator of gene expression.

<p>Volcano plot depicting the differential regulation of genes in the <i>fixLJ</i> deletion mutant relative to the wild-type strain AU0158 measured by RNA-seq. Green dots signify genes with expression 2-fold higher in the <i>fixLJ</i> deletion mutant relative to strain AU0158 with a q < 0.05. Red dots signify genes with expression 2-fold lower in the <i>fixLJ</i> deletion mutant relative to strain AU0158 with a q < 0.05.</p

The <i>B</i>. <i>dolosa fixLJ</i> deletion mutant is less invasive of epithelial and macrophage-like cells.

<p>A549 cells (A) or THP-1 cells treated with 200 nM PMA for 3 days (B) in 24-well plates were infected with ~2x10⁶ CFU/well (MOI of ~10:1) of strain AU0158, its <i>fixLJ</i> deletion mutant, or its <i>fliC</i> deletion mutant for 2 hours, after which the percent of internalized bacterial relative to the total bacterial growth was determined by killing extracellular bacteria with kanamycin (1 mg/mL). Means from 2–3 separate experiments with three replicates per experiment are plotted with error bars representing one standard deviation. *<i>P</i><0.05 by 1-way ANOVA with Tukey’s multiple comparison test. (C) THP-1-derived macrophages were infected with ~2x10⁶ CFU/well of <i>B</i>. <i>dolosa</i> for 2 hours, after which the extracellular bacteria were killed by treatment with kanamycin (1 mg/mL) and number of intracellular bacteria were determined after a 24-hour incubation. *<i>P</i><0.05 by t test. (D) THP-1-derived macrophages were infected with ~2x10⁶ CFU/well of <i>B</i>. <i>dolosa</i> for varying amounts of time (15 min-2 hours) after which the number of internalized bacteria was determined by killing extracellular bacteria with kanamycin (1 mg/mL). *<i>P</i><0.05 by t test compared to the <i>fixLJ</i> deletion mutant at that time point. (E) THP-1-derived macrophages were infected with ~2x10⁶ CFU/well of <i>B</i>. <i>dolosa</i> for 2 hours, after which the extracellular bacteria were treated with kanamycin (1 mg/mL) for varying amounts of time, after which the percent of internalized bacterial relative to the total bacterial growth within the initial 2-hour infection was determined. *<i>P</i><0.05 by t test compared to strain AU0158 at that time point. # <i>P</i><0.05 by 1-way ANOVA with Tukey’s multiple comparison test compared to hour-1 measurement. (C-E) Means from representative experiment repeated twice. Separate experiments with three replicates per experiment are plotted with error bars representing one standard deviation.</p

<i>Burkholderia</i> FixLJ functions as an oxygen sensor.

<p><i>B</i>. <i>dolosa</i> (strain AU0158 or its <i>fixLJ</i> deletion mutant), <i>B</i>. <i>cenocepacia</i> (strain J2315), and <i>B</i>. <i>multivorans</i> (strain ATCC23344) carrying a <i>pfixK-lacZ</i> reporter plasmid. Negative Control denotes reporter plasmid carrying <i>S</i>. <i>meliloti fixK</i> promoter sequence. EV denotes empty complementation vector. LacZ activity was quantified by Miller Units. Bars represent the means of triplicate biological replicates and error bars represent one standard deviation (representative of three independent experiments). *P<0.001 by 1-way ANOVA with Tukey’s multiple comparison test.</p

The <i>B</i>. <i>dolosa fixLJ</i> deletion mutant is cleared faster in a murine pneumonia model.

<p>(A-D) C57BL/6 mice were intranasally challenged with ~4x10⁸ CFU/mouse of <i>B</i>. <i>dolosa</i> strain AU0158 or its <i>fixLJ</i> deletion mutant. Bacterial loads were measured at the following sites and time points: (A) Lungs, 1 day after infection; (B) Lungs, 7 days after infection; (C) Spleen, 1 day after infection; (D) Spleen, 7 days after infection. Two <i>fixLJ</i> deletion-infected mice had undetectable bacterial levels in the spleen at day 7 (panel D, shown as 10² CFU/gm). Data is representative from 2 separate experiments with 7–8 mice per group. (E-F) C57BL/6 mice were intranasally challenged with <i>B</i>. <i>dolosa</i> AU0158 <i>ΔfixLJ</i> + <i>fixLJ</i> (6.7x10⁸ CFU/mouse) or <i>B</i>. <i>dolosa</i> AU0158 <i>ΔfixLJ</i> + empty vector (EV) (7.4x10⁸ CFU/mouse). Bacterial loads were measured 7 days after infection in the lungs (E) and spleen (F). Data are derived from one experiment done with 7–8 mice per group. Each point represents one mouse, and bars represent medians. *P<0.05 by Mann Whitney U test.</p

The <i>B</i>. <i>dolosa fixLJ</i> deletion mutant is less motile.

<p>(A) <i>B</i>. <i>dolosa</i> strain AU0158, its <i>fixLJ</i> deletion mutant, and the <i>fixLJ</i> deletion mutant complemented with <i>fixLJ</i> under the control of its own promoter or empty vector (EV) were plated on low-density (0.3%) LB agar and swimming distance was measured after incubation for 48 hours. *<i>P</i><0.05 by 1-way ANOVA with Tukey’s multiple comparison test. (B) The <i>B</i>. <i>dolosa fixLJ</i> -deletion mutant complemented with <i>fixLJ</i> under the control of a rhamnose-inducible promoter or empty vector grown in the presence of glucose (0.4%, which represses <i>fixLJ</i> expression) or rhamnose (0.4%). *P<0.05 by 1-way ANOVA with Tukey’s multiple comparison test relative to the complemented strain (Δ<i>fixLJ</i> + <i>fixLJ</i>) grown in rhamnose-containing media. For all panels, bars represent mean measurements of 3–4 replicates and error bars represent one standard deviation (representative of three independent experiments).</p

Strains used in this study.

<p>Strains used in this study.</p

<i>B</i>. <i>dolosa</i> virulence is independent of the presence of flagella.

<p>(A) <i>B</i>. <i>dolosa</i> strain AU0158 and its <i>fliC</i> deletion mutant were plated on low-density (0.3%) LB agar and swimming distance was measured after incubation for 48 hours. *P<0.05 by 1-way NOVA with Tukey’s multiple comparison test. Bars represent mean measurements of 3–4 replicates and error bars represent one standard deviation (representative of three independent experiments). (B) Overnight cultures of <i>B</i>. <i>dolosa</i> strain AU0158, the <i>fixLJ</i> deletion mutant, or the <i>fliC</i> deletion mutant were diluted 1:100 in TSB with 1% glucose, incubated for 48 hours, and then assessed for biofilm formation using crystal violet. Bars represent mean measurements of 6 replicates and error bars represent one standard deviation of the data (representative of three independent experiments). (C-D) C57BL/6 mice were intranasally challenged with 4.8 x10⁸ CFU/mouse of strain AU0158 or its <i>fliC</i> deletion mutant. Bacterial loads were measured 7 days after infection in the lungs (C) and spleen (D). Data are derived from one experiment done with 7–8 mice per group. Each point represents one mouse and bars represent medians.</p

The <i>B</i>. <i>dolosa fixLJ</i> deletion mutant produces more biofilm by crystal violet staining and has a different biofilm structure.

<p>Biofilm formation of <i>B</i>. <i>dolosa</i> AU0158 constructs on PVC plates as measured by crystal violet staining at 48 hours. (A) <i>B</i>. <i>dolosa</i> strain AU0158 produces less biofilm than its <i>fixLJ</i> deletion mutant. Strains were grown in TSB with 1% glucose at varying inocula. (B) The <i>B</i>. <i>dolosa fixLJ</i> deletion mutant complemented with <i>fixLJ</i> under the control of its own promoter produces less biofilm compared to the strain carrying an empty vector (EV). (C) A <i>B</i>. <i>dolosa fixLJ</i> deletion mutant complemented with <i>fixLJ</i> under the control of a rhamnose-inducible promoter or empty vector grown in LB in the presence of glucose (0.4%, which represses the promoter) or rhamnose (0.4%) and compared to the Δ<i>fixLJ</i> + <i>fixLJ</i> strain grown in rhamnose-containing medium. For panels A-C, bars represent mean measurements of 5–6 replicates and error bars represent one standard deviation (representative of three independent experiments). *P<0.05 compared to AU0158 by 1-way ANOVA with Tukey’s multiple comparison test. Representative mosaic images of biofilms of strain AU0158 (D) and its <i>fixLJ</i> deletion mutant (E) grown on 8-well chamber slides for 48 hours, stained with live/dead stain, and imaged by confocal microscopy. For panels D and E, live bacteria are stained green and dead are red. The inset of panel D shows Z-stack images taken at 1 μm intervals; gridlines denote 5 μm lengths. Images are representative of two independent experiments conducted with four replicates.</p

Plasmids used in this study.

<p>Plasmids used in this study.</p