Complex Interplay between FleQ, Cyclic Diguanylate and Multiple σ Factors Coordinately Regulates Flagellar Motility and Biofilm Development in <i>Pseudomonas putida</i>

<div><p>Most bacteria alternate between a free living planktonic lifestyle and the formation of structured surface-associated communities named biofilms. The transition between these two lifestyles requires a precise and timely regulation of the factors involved in each of the stages that has been likened to a developmental process. Here we characterize the involvement of the transcriptional regulator FleQ and the second messenger cyclic diguanylate in the coordinate regulation of multiple functions related to motility and surface colonization in <i>Pseudomonas putida</i>. Disruption of <i>fleQ</i> caused strong defects in flagellar motility, biofilm formation and surface attachment, and the ability of this mutation to suppress multiple biofilm-related phenotypes associated to cyclic diguanylate overproduction suggests that FleQ mediates cyclic diguanylate signaling critical to biofilm growth. We have constructed a library containing 94 promoters potentially involved in motility and biofilm development fused to <i>gfp</i> and <i>lacZ</i>, screened this library for FleQ and cyclic diguanylate regulation, and assessed the involvement of alternative σ factors σ^N and FliA in the transcription of FleQ-regulated promoters. Our results suggest a dual mode of action for FleQ. Low cyclic diguanylate levels favor FleQ interaction with σ^N-dependent promoters to activate the flagellar cascade, encompassing the flagellar cluster and additional genes involved in cyclic diguanylate metabolism, signal transduction and gene regulation. On the other hand, characterization of the FleQ-regulated σ^N- and FliA-independent P<i>lapA</i> and P<i>bcsD</i> promoters revealed two disparate regulatory mechanisms leading to a similar outcome: the synthesis of biofilm matrix components in response to increased cyclic diguanylate levels.</p></div

Assessment of combined FleQ and c-di-GMP regulation at selected promoters.

<p>β-galactosidase assays of the P<i>pp2357</i> (<b>A</b>), P<i>bifA</i> (<b>B</b>), P<i>bcsD</i> (<b>C</b>) and P<i>lapA</i> (<b>D</b>) promoter fusions in the wild-type KT2442 and <i>fleQ</i> (MRB35) strain bearing the YhjH-producing plasmid pMRB89 (low c-di-GMP) or the YedQ-producing plasmid pYedQ (high c-di-GMP). Bars represent the averages and standard deviations of three independent assays. Stars designate p-values for the Student's <i>t</i>-test for unpaired samples not assuming equal variance. *: p<0.05, **: p<0.01; ***: p<0.001.</p

Pellicle, aggregation and CR adsorption phenotypes of Δ<i>bifA</i> and <i>fleQ</i> mutants.

<p><b>A.</b> Photographs of planktonic cultures of the wild-type (KT2442), Δ<i>bifA</i> (MRB32), <i>fleQ</i> (MRB35) and Δ<i>bifA fleQ</i> (MRB50) strains showing pellicle formation and culture clarification due to aggregation. <b>B.</b> CR adsorption of the wild-type (KT2442), Δ<i>bifA</i> (MRB32), <i>fleQ</i> (MRB35) and Δ<i>bifA fleQ</i> (MRB50) strains. Data are normalized to the wild-type, set to 100%. Bars represent the averages and standard deviations of at least three independent assays.</p

Expression of the selected promoters in different <i>P</i>. <i>putida</i> backgrounds.

<p>β-galactosidase assays of the selected promoter fusions in wild-type (KT2442) and <i>fleQ</i> (MRB35) backgrounds (<b>A</b>), a <i>lapA</i>^<i>-</i> mutant (MRB34) bearing pYedQ (high c-di-GMP) or pMRB89 (low c-di-GMP) (<b>B</b>), wild-type (KT2440) and <i>rpoN</i>^<i>-</i> (KT2440<i>rpoN</i>) backgrounds (<b>C</b>), and wild-type (KT2440) and <i>fliA</i> (KT2440<i>fliA</i>::<i>aphA-3</i>) backgrounds (<b>D</b>) Bars represent the averages and standard deviations of at least three independent assays. Stars designate p-values for the Student's <i>t</i>-test for unpaired samples not assuming equal variance. *: p<0.05; **: p<0.01; ***:p<0.005.</p

Adhesion assays of KT2442 derivatives.

<p>Phase-contrast micrographs of the wild-type strain KT2442 (top) and its <i>fleQ and fliG</i> mutant derivatives MRB35 (center) and MRB47 (bottom). Two frames of the same field were taken in a 1-minute interval (left and center). The right panel shows an overlay of the two images of each strain digitally colored red (t = 0) or green (t = 1 min).</p

Screenings of the ordered promoter library for FleQ and c-di-GMP regulation.

<p>Diagonal plots showing expression of each promoter fusion in the <i>fleQ</i> (MRB35) vs. wild-type (KT2442) backgrounds (<b>A</b>), or in the presence of low vs. high levels of c-di-GMP (<b>B</b>). Red and blue lines represent 2-fold differential expression. Data points showing over 2-fold differential expression are shown in dark red or dark blue. Data points showing over differential expression greater than 1.5-fold but lower than 2-fold are shown in orange or cyan. Data from the experimental controls (KT2442 and KT2442 bearing the empty vector pMRB1) are shown in yellow and pink,respectively. Promoters selected for confirmation are indicated in each plot. The original data for these plots are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163142#pone.0163142.s007" target="_blank">S2 Table</a>.</p

Dilution series-based growth curves of KT2442 derivatives.

<p>Planktonic (left axes, open symbols) or biofilm growth (right axes, closed symbols) is plotted against the initial A₆₀₀ of each dilution. Circles represent the wild-type KT2442 strain and squares represent the <i>fleQ</i> mutant MRB35 (<b>A</b>), the <i>fliG</i> mutant MRB47 (<b>B</b>), or the Δ<i>bifA fleQ</i> mutant MRB50 (<b>C</b>). Plots display one representative experiment of at least three biological replicates. Error bars represent the standard deviation of the six technical replicates.</p

Gel mobility shift assays of FleQ on the P<i>lapA</i> and P<i>bcsD</i> promoters.

<p><b>Top:</b> Cartoon of the probes used for each of the promoter regions, indicating the restriction enzymes used for cleavage, the sizes (in bp) of the resulting fragments, and the location of the predicted FleQ binding motifs (open boxes). Drawn to scale. <b>Bottom:</b> Ethidium bromide-stained PAGE showing the results of a typical gel mobility shift assay. Each promoter region was challenged with 0, 0.45 and 4.5 μM FleQ.</p

Bacterial strains and plasmids used in this work.

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