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

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

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

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

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

Identification of <i>Xanthomonas campestris</i> XC_3703 as a cyclic di-GMP- binding protein.

<p>(A) SDS-Polyacrylamide gel separation of <i>Xcc</i> proteins retained on cyclic di-GMP-coupled beads. Lanes from left: Molecular weight markers; Total crude extract of <i>Xcc</i> soluble proteins; Proteins retained by the cyclic di-GMP-coupled beads. The protein band indicated by the asterisk was identified by mass spectrometry of tryptic peptides as XC_3703. (B) Isothermal titration calorimetry analysis of binding of cyclic di-GMP and cyclic GMP by XC_3703. Lower panels show the integrated data obtained from the raw data, after subtracting the heat of dilution. Injection of cyclic di-GMP yielded an endothermic binding isotherm. Experimental data were fitted using the MicroCal ORIGIN version 7.0 software and a <i>K</i>_d for cyclic di-GMP was calculated as 2 µM. No binding of cyclic GMP to XC_3703 was detected.</p

Protein interaction, transcription and phenotypic profiling reveal an overlap in regulatory influence of XC_3703 and XC_2801.

<p>(A) Domains of XC_2801 were predicted by SMART with amino acid positions indicated. Domain abbreviations are; HTH_1: helix-turn-helix DNA binding domain; LysR_substrate: small molecule binding domain associated with LysR family transcriptional regulators. Black lines below figures represent constructs cloned into either pBT or pTRG vectors to assess potential protein-protein interactions with XC_3703 using the bacterial two-hybrid assay. (+) indicates that an interaction was detected. (B) Venn diagrams showing the overlap of genes whose expression is down-regulated or up-regulated in the <i>XC_2801</i> or <i>XC_3703</i> mutant strain. Changes in gene expression in <i>XC_2801</i> and <i>XC_3703</i> deletion mutants compared with the wild-type <i>Xcc</i> were measured by RNA-Seq. Further detail of data analysis is given in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004429#s4" target="_blank">Materials and Methods</a> section and the complete RNA Seq data sets are detailed in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004429#ppat.1004429.s010" target="_blank">Table S4</a>. (C) Virulence of different <i>Xcc</i> strains assessed after spray inoculation. The percentage of the total number of inoculated leaves that showed typical black rot disease symptoms at the leaf margin is given. Values are means and standard deviations of three replicates, each comprising 25 plants and approximately 100 leaves. Asterisks indicate values that are significantly different from the wild-type at <i>p</i><0.01 by two-tailed Student's <i>t</i>-test. Complementation of the <i>XC_2801</i> mutant by introduction of <i>XC_2801</i> cloned into pLAFR3 (indicated as XC_2801(p2801)) restored the phenotype towards wild-type. (D) Biofilm and cell adhesion of the <i>XC_2801</i> mutant to a glass surface was decreased compared to wild-type strain 8004. Complementation of the <i>XC_2801</i> mutant by introduction of <i>XC_2801</i> cloned into pLAFR3 (indicated as XC_2801(p2801)) restored the phenotype towards wild-type. Biofilm biomass was measured by crystal violet staining and expressed as a ratio of absorbance at 550/600 nm. Values given are the mean and standard deviation of triplicate measurements. Asterisks indicate values that are significantly different from the wild-type at <i>p</i><0.01 by two-tailed Student's <i>t</i>-test.</p

Transcriptome and phenotypic characterisation of an <i>XC_3703</i> deletion mutant reveals roles in biofilm formation and virulence in <i>Xcc</i>.

<p>(A) The virulence of the <i>Xcc</i> strains was tested by measurement of the lesion length after bacteria were introduced into the vascular system of Chinese Radish by leaf clipping. (i) Representative virulence assays for (from left to right) <i>Xcc</i> wild-type (8004), <i>XC_3703</i> deletion mutant and negative control (H₂O). (ii) Re-integration of the gene encoding <i>XC_3703</i> into a neutral site in the mutant chromosome (indicated as <i>XC_3703</i> (cXC_3703)) restored the reduced virulence of the mutant towards wild-type. Values given are the mean and standard deviation of triplicate measurements each comprising of 30 leaves. Asterisks indicate values that are significantly different from the wild-type (p<0.01, two-tailed Student's <i>t</i>-test). (B) Deletion of <i>XC_3703</i> also led to decreased biofilm and cell adhesion on a glass surface when assessed by crystal violet staining. Biofilm biomass is measured as a ratio of absorbance at 550 and 600 nm. Re-integration of the gene encoding <i>XC_3703</i> into the chromosome (indicated as <i>XC_3703</i> (c3703)) restores the phenotypes towards wild-type. Values given are the mean and standard deviation of triplicate measurements. Asterisks indicate values that are significantly different from the wild-type (p<0.01, two-tailed Student's <i>t</i>-test). (C) Differential expression of selected genes implicated in virulence or biofilm formation in the <i>XC_3703</i> deletion mutant and wild-type as determined by RNA-Seq (dark grey) and qRT–PCR (light grey). Mutation of <i>XC_3703</i> affected transcript levels of <i>XC_0026</i> (cellulase), <i>XC_0027</i> (endoglucanase), <i>XC_1058</i> (pilin), <i>XC_1165</i> (TonB receptor) <i>XC_1732</i> (glycosyltransferase), <i>XC_2013</i> (MASE1 domain-containing protein), <i>XC_2724</i> (methyltransferase), and <i>XC_3591</i> (pectate lyase). The qRT–PCR data were normalised to 16S rRNA and is presented as the fold change with respect to the wild-type for each gene. Data (means ± standard deviation) are representative of four independent biological experiments. The complete RNA-Seq data set is detailed in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004429#ppat.1004429.s010" target="_blank">Table S4</a>.</p

XC_2801 binds to specific promoters and only in the presence of XC_3703.

<p>(A) Promoter-fusion analysis of the upstream regions <i>flhB</i>, <i>aaeB</i>, <i>flgG</i> and <i>fliL</i> revealed significantly decrease in promoter activity in the <i>XC_2801</i> mutant strain. Values given are the mean and standard deviation of triplicate measurements. Values for promoter activity in the <i>XC_2801</i> mutant strain are significantly different from the wild-type at <i>p</i><0.01 by two-tailed Student's <i>t</i>-test. The decrease in promoter activity was consistently observed in three independent experiments each consisting of three biological replicates. (B) Binding of XC_2801 to the promoter regions of <i>flhB</i>, <i>aaeB</i>, <i>flgG</i> and <i>fliL</i> in the absence (i) or presence (ii) of XC_3703 assessed by the use of electromobility shift assay (EMSA). Each lane contained 1.5 nM DIG-labelled Probe DNA, and in addition nanomolar concentrations of purified His6-tag XC_2801 protein as indicated below each well. (C) Bioinformatics reveals a putative LysR family regulator-binding site (<u>T</u>CCCGAATCCCCG<u>A</u>) located 80-67 nucleotides upstream of the predicted translational start site of <i>flhB</i>. (D) Although a shift was observed with the <i>flhB</i> upstream (promoter) region, no shift was observed with an overlapping DNA fragment that lacks the putative XC_2801 binding site (indicated as truncated <i>flhB</i>). The nanomolar concentration of His-tagged XC_2801 used in each assay is indicated below each well. DIG-labelled DNA at 1.5 nM was used.</p