Transcriptome changes in response to ParB overproduction.

<p>(<b>A</b>) Statistics of loci with significant expression change (FC<-2 or >2, <i>p</i>-value <0.05). RNA was isolated from PAO1161 cultures grown in L broth without Ara (WT), PAO1161 (pKGB8 <i>araBAD</i>p) cultures grown under selection in L broth with 0.02% Ara (empty vector control, EV), PAO1161 (pKGB9 <i>araBAD</i>p-<i>parB</i>) cultures grown under selection in L broth without Ara (mild ParB excess, ParB+) or with 0.02% Ara (higher ParB excess, ParB<i>+++</i>). (<b>B</b>) Venn diagram for sets of loci with significant expression change between EV <i>vs</i> WT, ParB+ <i>vs</i> EV and ParB+++ <i>vs</i> EV. (<b>C</b>) Classification of loci with altered expression according to PseudoCAP categories [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0181726#pone.0181726.ref057" target="_blank">57</a>]. When a gene was assigned to multiple categories, one category was arbitrarily selected (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0181726#pone.0181726.s006" target="_blank">S2 Table</a>). The PseudoCAP categories were grouped into six classes as marked. White and black bars correspond to the numbers of respectively, upregulated and downregulated genes in a particular category.</p

Validation of the microarray data by RT-qPCR.

<p>Validation of the microarray data by RT-qPCR.</p

Plasmids used in this study.

<p>Plasmids used in this study.</p

Bacterial strains used in this study.

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

Selected genes whose expression was identified as ParB regulated.

<p>Selected genes whose expression was identified as ParB regulated.</p

Influence of ParB level on expression of genes adjacent to <i>parS6</i>.

<p>(<b>A</b>) Mean level of expression of <i>PA0488</i>–<i>PA0498</i> genes in ParB+ and ParB+++ cells relative to EV cells (microarray data). Filled markers indicate statistically different expression relative to EV (<i>p</i>-value < 0.05 in ANOVA test). Operons (according to the DOOR 2.0 database [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0181726#pone.0181726.ref073" target="_blank">73</a>]) are labelled with different shades of grey. (<b>B</b>) RT-qPCR analysis of expression of <i>PA0492</i>, <i>PA0493</i> and <i>PA0494</i> genes in <i>parB</i>_null and <i>parS</i>_null strains relative to WT cells. Data represent mean ±SD from three biological replicates. The differences between strains / conditions are not statistically significant (<i>p</i>-value > 0.05 in two-sided Student’s <i>t</i>-test assuming equal variance).</p

ParB excess induces the expression of chromosomal <i>bexR</i>p-<i>lacZ</i> transcriptional fusion.

<p>PAO1161::<i>bexR</i>p-<i>lacZ</i> strain contains <i>bexR</i>p-<i>lacZ</i> transcriptional fusion inserted in the intergenic region of PAO1161 genome. White colony from L agar with X-gal was inoculated and used as a recipient in conjugation with either S17-1 (pKGB8) or S17-1 (pKGB9 <i>araBAD</i>p-<i>parB</i>) donor cells. Conjugants were grown on selective L agar plates supplemented with X-gal. The photographs show representative plates of PAO1161::<i>bexR</i>p-<i>lacZ</i> with both plasmids.</p

Effects of ParB excess in <i>P</i>. <i>aeruginosa</i>.

<p>(<b>A</b>) Growth of <i>P</i>. <i>aeruginosa</i> PAO1161 (pKGB8 <i>araBAD</i>p) and PAO1161 (pKGB9 <i>araBAD</i>p-<i>parB</i>) strains in L broth with different arabinose concentrations. Data represent mean OD₆₀₀. (<b>B</b>) Western blot analysis of ParB levels in the tested strains. Each lane contains extract from 10⁹ cells. Blots were subjected to immunodetection using primary anti-ParB antibodies. Representative blot is shown. Signals on the blots were quantified. Data represent mean ParB level ±SD relatively to the control strain PAO1161 (pKGB8). Purified His₆-ParB was used to generate standard curves. M–molecular weight marker. <b>(C</b>) Biofilm formation in the static cultures of PAO1161, PAO1161 (pKGB8) and PAO1161 (pKGB9). Strains were grown without or with 0.02% arabinose until OD₆₀₀ 0.5. Biofilm was stained with crystal violet and assessed by measurement of OD₅₉₀. Data represent mean OD₅₉₀/OD₆₀₀ ratio ±SD from 3 biological replicates. *—<i>p</i>-value < 0.05 in two-sided Student’s <i>t</i>-test assuming equal variance.</p

Influence of ParB on gene expression in the <i>parS1-4</i> region.

<p>(<b>A</b>) Mean level of expression of <i>dnaA</i>-<i>def</i> (<i>PA0001</i>-<i>PA0019</i>) genes in ParB+ and ParB+++ cells relative to EV as revealed by microarray analysis. Filled markers indicate statistically different expression relative to EV (<i>p</i>-value < 0.05 in ANOVA test). Arrangement of the genes in the chromosome is shown below. Operons (according to the DOOR 2.0 database [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0181726#pone.0181726.ref073" target="_blank">73</a>]) are marked in grey. (<b>B</b>) Expression of <i>PA0001</i>-<i>PA0016</i> <b>(</b><i>dnaA</i>–<i>trkA</i>) genes in ParB+ and ParB+++ cells relative to EV cells. (<b>C</b>) RT-qPCR analysis of expression of <i>dnaA</i>–<i>trkA</i> genes in <i>parB</i>_null and <i>parS</i>_null strains relative to WT cells. Cells were grown in L broth. (<b>D</b>) RT-qPCR analysis of expression of <i>dnaA</i>–<i>trkA</i> genes in <i>parS</i>_null (pKGB9 <i>araBADp</i>-<i>parB</i>) and <i>parS</i>_null (pKGB8 <i>araBAD</i>p) relative to EV [PAO1161 (pKGB8 <i>araBAD</i>p)]. Cells were grown in L broth supplemented with chloramphenicol and 0.02% arabinose. RT-qPCR data represent mean ±SD from three biological replicates. Filled symbols indicate significantly different expression (<i>p</i>-value <0.05 in two-sided Student’s <i>t</i>-test assuming equal variance) relative to the control cells labelled as blue squares. The differences in expression of genes in <i>parS</i>_null (pKGB9) strain relative to <i>parS</i>_null (pKGB8) strain are not statistically significant.</p

Influence of ParB on the activities of <i>PA0011</i> and <i>PA0013</i> promoters.

<p>(<b>A</b>) DNA sequences preceding <i>PA0011</i> (<i>PA0011</i>p) and <i>PA0013</i> (<i>PA0013</i>p) and their mutated versions, <i>PA0011</i>p_{<i>parS3</i>mut} and <i>PA0013</i>p_{<i>parS4</i>mut}, cloned upstream of promoter-less <i>lacZ</i> cassette in pPJB132 are presented. Putative promoters’ motifs, RBS sequences and start codons are indicated. Promoter -35 and -10 boxes were predicted using BPROM [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0181726#pone.0181726.ref074" target="_blank">74</a>]. β-galactosidase activity was measured in extracts from PAO1161::<i>araBAD</i>p (control strain) (<b>B</b>) and PAO1161::<i>araBAD</i>p-<i>flag</i>-<i>parB</i> (ParB-overproducing strain) (<b>C</b>) cells carrying pPJB132 derivatives as indicated. Data represent mean activity from at least three cultures ±SD. *—<i>p</i>-value < 0.05 in two-sided Student’s <i>t</i>-test assuming equal variance.</p