<i>PA2214-15</i> is under the direct transcriptional control of the PA2206 LysR regulator.

<p>(<b>A</b>) Comparative genomic analysis of the metabolic-centric <i>PA2206</i> region in <i>P. aeruginosa</i> PAO1, <i>P. fluorescens</i> Pf-5, and <i>P. fluorescens</i> Pf-O1 based on the Pseudomonas Genome Database. Single genes and operons are denoted by colour and pattern fill while homology is denoted by connecting shaded regions. <i>P. fluorescens</i> encodes a <i>PA2206</i> homologue, which is adjacent to truncated genes corresponding to fragments of <i>PA2207</i> and <i>PA2212</i>, both of which are downstream of a conserved <i>PA2214-2216</i> homologous operon. TTT denotes a tripartite tricarboxylate transport system, FAH denotes a putative fumarylacetoacetate hydrolase activity, while Aldedh denotes a putative aldehyde dehydrogenase activity. (<b>B</b>) Promoter-fusion analysis of the <i>PA2214</i> upstream region revealed significantly increased promoter activity in the <i>PA2206^C</i> strain relative to <i>PA2206⁻</i>. The increase in promoter activity was consistently observed in three independent experiments consisting of three biological replicates (** p-value<0.01 by student's ttest). (<b>C</b>) <i>PA2214-lacZ</i> promoter fusion analysis performed in <i>E. coli</i> harbouring a <i>PA2206</i>-pBBR1MCS5 construct compared to vector control revealed direct regulation. Promoter activity was below baseline in the vector control (compared to empty pBBR1MCS5 plasmid), while an average of 6 Miller Units was consistently detected in the overexpressing strain (** p-value of <0.01 by student's ttest).</p

Quantitative Real Time PCR analysis confirms the influence of PA2206 on gene expression linked to the oxidative stress response.

<p>Bacterial cultures were grown to exponential phase, and aliquots were then either untreated or exposed to a 1 mM concentration of H₂O₂ for 10 mins. Expression of <i>PA3032</i>, <i>PA2821</i>, <i>PA0729.1</i> and <i>PA5129</i> was measured as fold change relative to the <i>proC</i> housekeeping gene. Mean values are represented ± standard error. Consistent with the array data, expression of <i>PA3032</i> and <i>PA2821</i> was increased in the <i>PA2206⁻</i> mutant (white bar) compared to <i>PA2206^C</i> (striped bar) and the wild-type (black bar) strain. Similarly, expression of <i>PA0729.1</i> and <i>PA5129</i> was approximately 3-fold less in the <i>PA2206⁻</i> mutant strain (p-value of <0.05 by student's ttest).</p

PA2206 has a global regulatory role in <i>P. aeruginosa</i>.

<p>(<b>A</b>) BioPerl analysis revealed the presence of the putative PA2206 consensus sequence in the promoters of the <i>pvdS</i> and <i>PA4881</i> genes. The arrangement and sequence similarity of the consensus sequence was similar to that of the <i>pcaQ</i> LTTR from <i>S. meliloti</i>. (<b>B</b>) (i) EMSA analysis of the <i>pvdS</i> promoter region with the PA2206 protein revealed an interaction at nanomolar concentrations. Concentrations as low as 800 nM were able to shift the <i>pvdS</i> promoter. (ii) The absence of binding to the <i>PA0982</i> promoter probe indicates the requirement for specificity in the predicted consensus sequence. (iii) No interaction was observed between PA2206 and a truncated <i>pvdS</i> promoter fragment located downstream of the putative LysR box suggesting that this sequence alone is sufficient for the protein-DNA interaction to occur.</p

Bacterial strains and plasmids used in this study.

<p>Bacterial strains and plasmids used in this study.</p

PA2206 binds a LysR-box overlapping the predicted −35 box in the <i>PA2214</i> promoter.

<p>(<b>A</b>) EMSA analysis of the <i>PA2214-15</i>, <i>PA2206</i> and <i>PA2216</i> promoter fragments revealed <i>PA2214-15</i> to be a direct PA2206 target. The protein concentration is marked below each lane while DNA promoter fragments were used at 15 fmoles. Three complexes were observed upon protein interaction with the <i>PA2214-15</i> (1–3) promoter fragment at low nanomolar protein concentrations, with the C2/C3 complexes predominant at higher concentrations of PA2206 protein. The <i>PA2206</i> (4–6) and <i>PA2216</i> (7–9) promoter fragments did not form a complex with the PA2206 protein. (<b>B</b>) Schematic diagram of the location and arrangement of the LysR boxes identified upstream of the predicted transcriptional start site of <i>PA2214</i>. The primer positions for each of the truncated promoter fragments are outlined below and aligned with the LysR boxes contained within each amplicon. (<b>C</b>) Mobility shift analysis of the PA2206 protein interaction with the <i>PA2214</i> promoter region. Protein was added to each reaction at 300 nM and DNA promoter fragments were used at 15 fmoles. The strong shift in lane 4 confirms that PA2206 binds to LysR box 3 overlapping the p<i>PA2214</i> predicted −35 region and that this region is sufficient for the interaction to occur. PA2206 protein did not cause a shift for any of the other truncated promoter fragments.</p

PA2206 is induced in response to H₂O₂ and is required for lethality in a zebrafish embryo model of infection.

<p>(<b>A</b>) Bacterial cultures were grown to exponential phase, and aliquots were then either untreated or exposed to 1 mM, 5 mM and 10 mM concentrations of H₂O₂. <i>PA2206</i> gene expression was measured as fold change relative to the <i>proC</i> housekeeping gene and subsequently calculated as fold change relative to the untreated sample, following 10, 20, 30, and 60 minute periods of exposure to H₂O₂. Mean values are represented ± standard error (** p-value≤0.005, *** p-value≤0.001 calculated using one-way ANOVA). (<b>B</b>) Kaplan-Meier survival curve. Twenty-six hours Zebrafish embryos were injected with ∼300 colony forming units of either mPAO1 wild-type or <i>PA2206⁻</i> mutant into their blood island. Embryos were monitored for survival daily. Mean values of three biological experiments (each 10 embryos) are shown. Statistical analysis showed that at 2 and 3 days post infection, mPAO1 wild-type killed significantly more embryos compared to the <i>PA2206⁻</i> mutant (p-value = 0.0159 using the log-rank test).</p

Susceptibility of wild-type, <i>PA2206⁻</i> and <i>PA2206</i>^C strains to oxidative stress.

<p>Mean values are represented ± standard deviation.</p

GER-derived bile may influence respiratory pathogen behaviour and biodiversity in the CF pulmonary system.

<p>Bile aspiration linked to GER is highly prevalent in CF patients and has been linked to reduced pulmonary function in these patients. As bile is shown to influence key phenotypes associated with chronic infection in <i>P. aeruginosa</i>, GER-derived bile may constitute a major host factor in chronic respiratory disease. Bile induces biofilm formation, T6SS, and QS in <i>P. aeruginosa</i>, while T3SS and swarming motility are repressed. The transcriptional and phenotypic changes are consistent with a switch towards the chronic lifestyle adopted by <i>P. aeruginosa</i> and other respiratory pathogens in the CF lung. Bile also influences biofilm formation in other respiratory pathogens prevalent in CF patients suggesting that GER-derived bile may influence biodiversity in the CF lung.</p

QS is triggered in response to bile.

<p>(<b>A</b>)(<b>i</b>) Promoter activity of the <i>pqsA-E</i> biosynthetic operon was significantly increased in the presence of 0.3% bile (p<0.005). (<b>ii</b>) TLC analysis of supernatant extracts from cells grown in the presence and absence of 0.3% bile revealed that the increased expression of the <i>pqsA-E</i> operon in the presence of bile results in increased production of the HHQ and PQS signal molecules. Synthetic HHQ and PQS were loaded as controls to facilitate identification of the signal molecules in the supernatant extract. (<b>Bi & ii</b>) Promoter activity of the <i>rhlI</i> and <i>lasI</i> AHL QS systems was also significantly increased in the presence of 0.3% bile (p<0.005). As with the PQS system, increased promoter activity was evident in early and late exponential phase growth. (<b>Biii</b>) Reversed phase TLC analysis of supernatant extracts taken from bile treated and un-treated PA14 cells grown in LB, and overlaid with soft agar containing the <i>C. violaceum</i> CV026 long chain AHL biosensor strain. Increased production of the long chain AHLs can clearly be seen in the treated sample corresponding to the position of the 3-oxo-C12-HSL standard (25 µg). All experiments were performed in triplicate and data presented is representative of at least three independent biological replicates.</p

Bacterial strains and plasmids used in this study.

*<p>Monash Unniversity, Clayton, Victoria, Australia.</p>#<p>Centre of Microbial Host Interactions, ITT Dublin, Tallaght, Dublin 24, Ireland.</p>?<p>University Medical Center, Amsterdam, The Netherlands.</p