Model of the regulatory pathway leading to swarming under phosphate depletion.

<p>Model of the regulatory pathway leading to swarming under phosphate depletion.</p

The Effect of <i>pstS</i> and <i>phoB</i> on Quorum Sensing and Swarming Motility in <i>Pseudomonas aeruginosa</i>

<div><p><i>Pseudomonas aeruginosa</i> is an opportunistic pathogen that can cause a wide range of infections and inflammations in a variety of hosts, such as chronic biofilm associated lung infections in Cystic Fibrosis patients. Phosphate, an essential nutrient, has been recognized as an important signal that affects virulence in <i>P. aeruginosa</i>. In the current study we examined the connection between phosphate regulation and surface motility in <i>P. aeruginosa</i>. We focused on two important genes, <i>pstS</i>, which is involved in phosphate uptake, and <i>phoB</i>, a central regulator that responds to phosphate starvation. We found that a mutant lacking <i>pstS</i> is constantly starved for phosphate and has a hyper swarming phenotype. Phosphate starvation also induced swarming in the wild type. The <i>phoB</i> mutant, on the other hand, did not express phosphate starvation even when phosphate was limited and showed no swarming. A double mutant lacking both genes (<i>pstS</i> and <i>phoB</i>) showed a similar phenotype to the <i>phoB</i> mutant (i.e. no swarming). This highlights the role of <i>phoB</i> in controlling swarming motility under phosphate-depleted conditions. Finally, we were able to demonstrate that PhoB controls swarming by up-regulating the Rhl quorum sensing system in <i>P. aeruginosa</i>, which resulted in hyper production of rhamonlipids: biosurfactants that are known to induce swarming motility.</p></div

<i>pstS</i> knockout results in elevated <i>rhlA</i> and <i>rhlR</i> expression.

<p>PAO1, Δ<i>pstS</i>, Δ<i>phoB</i> and Δ<i>pstS</i>Δ<i>phoB</i> were grown for 48 hours at 37°C with shaking in M9 medium_. Levels of <i>rhlA</i> and <i>rhlR</i> were measured by Real-Time PCR as described in Materials and Methods. Values were normalized to the expression of PA1769. The results shown are relative to PAO1 and represent mean+standard deviation of nine different experiments. Each experiment was performed in triplicate. Asterisks represent the significant elevation in <i>rhlA</i> and <i>rhlR</i> transcription compared to the WT (p<0.05, student’s t-test).</p

Influence of <i>pstS</i> and <i>phoB</i> deletion on swarming motility.

<p>PAO1, Δ<i>pstS</i>, Δ<i>phoB</i> and Δ<i>pstS</i>Δ<i>phoB</i> carrying an empty vector (pUCP18Ap) or a complementation plasmid (p<i>pstS</i> and p<i>phoB</i>) were grown for 24 hours at 37°C on swarming plates containing M9 (20 mM) or phosphate-depleted M9 (0.2 mM) as described in Materials and Methods.</p

Strains used in this study.

<p>AmpR -Ampicilin resistance for <i>E. coli</i> CbR – Carbenicillin resistance for <i>P. aeruginosa</i>. GmR – Gentamicin resistance.</p

Phosphate starvation promotes C4-HSL production.

<p>PAO1, Δ<i>pstS</i>, Δ<i>phoB</i> and Δ<i>pstS</i>Δ<i>phoB</i> were grown for 48 hours at 37°C with shaking in M9 medium_. C4-HSL in each strain was extracted and measured as described in Materials and Methods. β-galactosidade activity (Miller Units) represent C4-HSL levels. Results represent mean+standard deviation of three different experiments. Each experiment was performed in triplicate. Asterisks represent the significant elevation in C4-HSL production in Δ<i>pstS</i> as opposed to all other strains (p<0.05, Turkey’s post hoc test).</p

<i>rhlA</i> and <i>rhlR</i> deletion cause loss of swarming motility.

<p>A. PAO1, PAO1Δ<i>rhlA</i> and PAO1Δ<i>rhlR</i> were grown for 24 hours at 37°C on swarming plates containing M9 or phosphate-depleted M9. B. Δ<i>pstS</i>, Δ<i>pstS</i>Δ<i>rhlA</i> and Δ<i>pstS</i>Δ<i>rhlR</i> were grown for 24 hours at 37°C on swarming plates containing M9 or phosphate-depleted M9.</p

The effect of <i>pstS</i> and <i>phoB</i> deletion on phosphate starvation in <i>P.aeruginosa</i>.

<p>PAO1, Δ<i>pstS</i>, Δ<i>phoB</i> and Δ<i>pstS</i>Δ<i>phoB</i> were grown for 24 hours at 37°C on swarming plates containing M9 or phosphate-depleted M9. After 24 hours, bacteria were scraped off the plates and Alkaline Phosphatase activity was measured using p-Nitrophenyl Phosphate as described in Materials and Methods. Results were normalized to each samples’ total protein concentration using Bradford assay. Results shown represent mean+standard deviation of six different experiments. Each experiment was performed in triplicate. Asterisks represent the significant rise in AP activity compared to the WT (p<0.05, student’s t-test).</p

Additional file 3 of The role of DNA demethylation in liver to pancreas transdifferentiation

Additional file 3. Table S2. TD-prone cells compared to TD-resistant cells