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

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

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

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

Virulence assays of the wild-type and <i>rpfG-, hgdA- and hgdC-</i>related single, double and triple mutant strains on rice cv. Jingang 30.

<p>(A) The length of disease lesions was measured at 14 days after pressure inoculation of the wild-type (WT), <i>ΔrpfG</i>, complemented <i>ΔrpfG (rpfG)</i>, <i>ΔhgdA</i>, <i>ΔhgdA/hgdC</i>, <i>ΔhgdA/rpfG</i>, <i>ΔrpfG/hgdC</i>, <i>ΔhgdA/rpfG/hgdC</i> strains, respectively. Ten to 15 leaves were scored for each strain; means ± standard error (SE) are shown. (B) <i>In planta</i> bacterial populations of <i>Xoc</i> RS105, <i>ΔrpfG</i> and <i>ΔrpfG(rpfG)</i> at the specific time points after inoculation. Data are presented as means ± SE.</p

Bacterial strains and plasmids.

<p>Bacterial strains and plasmids.</p

Effects of mutations of genes encoding several putative glycosyltransferases on biofilm formation and EPS production in <i>Xoc</i> wild-type and <i>ΔrpfG</i> mutant backgrounds.

<p>(A) Elevated biofilm formation in <i>Xoc ΔrpfG</i> was reduced to the wild-type level when <i>xagA</i>, <i>xagB</i> and <i>gumD</i>, but not <i>pgaA</i> or <i>pgaC</i> was deleted singly in the <i>ΔrpfG</i> genotype. The full length <i>xagA</i> and <i>gumD</i> genes restored the ability of biofilm formation in <i>ΔrpfG/xagA</i> and <i>ΔrpfG/gumD</i> mutants, respectively. (B) The ability of <i>ΔgumD</i> to form biofilm was greatly attenuated in the wild-type background, but was restored by complementation. Biofilm formation in the <i>ΔpgaA</i>, <i>ΔpgaC, ΔxagA</i> and <i>ΔxagB</i> single mutants was not altered compared to the wild-type strain. (C) EPS production was significantly reduced in the <i>ΔgumD</i> mutant, but not in <i>ΔpgaA</i>, <i>ΔpgaC, ΔxagA</i> and <i>ΔxagB</i> mutants compared to the wild-type. These experiments were repeated at least three times with similar results.</p

Mutation of H231 and D232 residues in the HD-GYP domain of <i>Xoc</i> RpfG disrupts the PDE activity and regulatory function.

<p>(A) Purified RpfG_AA-GYP completely lost its PDE activity detected by colorimetric assays. (B–D) RpfG_AA-GYP lost the ability to restore EPS production (B), biofilm formation (C) and virulence to rice (D) of <i>ΔrpfG</i> to the wild-type level. WT: wild-type, EV: empty vector.</p