Domain organizations of PA2571, PA2572 and PA2573.

<p>Domains were predicted by SMART with amino acid positions indicated. Domain abbreviations are as follows; HisKA (Histidine Kinase A phosphoacceptor domain), HATPase_c (Histidine kinase-like ATPase), REC (Receiver domain), HD (superfamily with predicted or known phosphohydrolase activity), HAMP (Histidine kinase, Adenylyl cyclase, Methyl binding protein, Phosphatase domain), and MA (Methyl-accepting chemotaxis-like domain). Vertical bars represent predicted transmembrane domains (SOUSI). Black lines below figures represent constructs cloned into either pBT or pTRG vectors to assess potential protein-protein interactions using the bacterial two-hybrid assay.</p

Production of virulence factors.

<p>Effects of mutations of <i>PA2572</i> and <i>PA2573</i> on production of pyoverdine (A) and pyocyanin (B) in <i>P. aeruginosa</i>. Complemented strains (indicated by pPA2572 and pPA2573) had wild-type levels of the factors. Phenotypic effects of mutations were complemented with reintroduction of genes into mutant strains. Error bars represent the mean ± standard deviation of three independent experiments in triplicate.</p

Venn diagrams representing unique and overlapping gene regulation in PA2572 and PA2573 mutants.

<p>Mutant strains were compared to the parental PAO1 strain during exponential growth phase (0.6–0.7 OD₆₀₀) in LB media. Genes were considered to have significant alteration in expression based on two-fold changes compared with wild-type. The numbers of genes regulated in an upward direction (A) and a downward direction (B) are shown.</p

Bacterial two-hybrid analysis investigating potential protein-protein interactions.

<p>(+) indicates a positive protein-protein interaction based on growth of colonies on Selective Screening Media (SSM) (M9+ His-dropout media containing 5 mM 3-AT). (−) indicates a negative interaction based on lack of growth on SSM. (-TM) represents a PA2573 construct without the N-terminal transmembrane domains. Results were observations from three independent experiments.</p

Motility of strains.

<p>Motility phenotypes of <i>P. aeruginosa</i> for swimming (0.3% agar) and swarming (0.5% Eiken agar) after 24 hours at 37°C. Mutant strains (indicated by PA2572 and PA2573) have reduced swimming and swarming, whereas complemented strains (indicated by pPA2572 and pPA2573) had wild-type levels of motility. Each plate is representative of the motility phenotype observed for strains in triplicate in three independent experiments.</p

Validation of transcriptome data using quantitative and semi-quantitative real-time PCR.

<p>Abbreviations and symbols: NS (Not significant, fold change was less than 2.0), RND (Resistance-Nodulation-Cell Division), + (represents increased gene expression in mutant strain based on increased abundance of PCR amplicon compared to PAO1), − (represents decreased gene expression in mutant strain based on decreased abundance of PCR amplicon compared to PAO1), NC (no apparent change in abundance was observed).</p