Yield (max OD₆₀₀) of <i>Pseudomonas fluorescens</i> F113 grown under anaerobic conditions in the presence of different concentrations of final electron acceptors (nitrate and nitrite) in SA and LB media.

<p>Yield (max OD₆₀₀) of <i>Pseudomonas fluorescens</i> F113 grown under anaerobic conditions in the presence of different concentrations of final electron acceptors (nitrate and nitrite) in SA and LB media.</p

Motility regulation under denitrification conditions.

<p>Swimming motility phenotype of <i>P</i>. <i>fluorescens</i> F113 and isogenic mutants affected in swimming motility. Experiments were done on SA (0.3% agar) under anaerobic (denitrification) conditions. Haloes were measured 48 h after inoculation. Experiments were performed three times in triplicate. Statistically significant results are shown as: * (p<0.1); ** (p<0.05); *** (p<0.01).</p

The three chemotaxis systems are important for rhizosphere colonization.

<p>Competitive colonization root-tip assay of <i>P</i>. <i>fluorescens</i> F113 and isogenic mutants affected in either of the <i>cheA</i> genes. Equal amounts (10⁸ cells) of F113 and its competitors were inoculated per plant. F113 was also compared with an isogenic strain tagged with a kanamycin resistance gene as a control. Experiments were done in triplicate with 20 plants in each experiment. Different letters indicate statistically significant differences (p<0.05).</p

Growth of <i>P</i>. <i>fluorescens</i> F113 under denitrification conditions.

<p>Growth curves of F113 with different final electron acceptors. Nitrate concentration was 40mM, nitrite concentration was 10mM. Anaerobic conditions were obtained by flushing inoculated medium with argon. For anaerobic conditions, air-tight tubes were used. F113 was unable to grow anaerobically in LB or SA media without nitrate or nitrite. Experiments were done in triplicate (A) Growth curves of F113 in LB and SA media supplemented with nitrate (B) Growth curves of F113 in LB medium (aerobic), LB supplemented with nitrate and LB supplemented with nitrite.</p

SadB and GacAS regulate <i>algU</i> expression.

<p>(<b>A</b>) Analysis of the swimming motility of F113 wild-type, mutants in the Gac-SadB-AlgU cascade, and <i>amrZ</i> complementation (p<i>amrZ</i>). Different letters indicate significant statistical differences (p<0.05). At 18 h, F113 wild-type strain swimming halo diameter is 11±0.55 (mm). (<b>B</b>) qRT-PCR expression analysis of <i>algU</i> (black) and <i>amrZ</i> (grey) genes (primers qalgUF-R and qamrZF-R, respectively) in F113 wild-type, <i>gacS</i><b><i>⁻</i></b>, <i>sadB</i><b><i>⁻</i></b>, and double mutant <i>gacS-sadB</i>. <i>16S</i> gene expression (primers 16SF-R) was used for normalization. Different letters indicate significant statistical differences (p<0.05).</p

Hypothetical model for the environmental regulation of flagellar synthesis in <i>P. fluorescens</i> F113 through the Gac-SadB cascades.

<p>Arrows indicate positive control and perpendicular lines negative control.</p

Gac-mediated downregulation of <i>fleQ</i> expression is independent of Vfr but dependent on AmrZ and AlgU.

<p>(<b>A</b>) Analysis of the swimming motility of F113 wild-type, mutants in the Gac-AlgU cascade, and complemented <i>amrZ</i> (p<i>amrZ</i>). Different letters indicate significant statistical difference (p<0.05). At 18 h, F113 wild-type strain swimming halo diameter is 11±0.55 (mm). (<b>B</b>) Western blot analysis of extracellular proteins from F113 wild-type strain and its isogenic mutants <i>amrZ</i><b><i>⁻</i></b> and <i>algU</i><b><i>⁻</i></b>, reacted with an anti-flagellin antiserum. Loading control corresponds to a Coomassie-stained gel portion. (<b>C</b>) qRT-PCR expression analysis of the <i>fleQ</i> gene (primers qfleQF-R) in F113 wild-type, <i>amrZ</i><b><i>⁻</i></b> and <i>algU</i><b><i>⁻</i></b>. <i>16S</i> gene expression (primers 16SF-R) was used for normalization. To control for DNA contamination, PCR of RNA was performed using the same primer pairs. Different letters indicate significant statistical differences (p<0.05).</p

Negative regulation of motility by the Gac system acts through downregulation of the <i>fleQ</i> gene transcription during exponential phase.

<p>(<b>A</b>) RT-PCR expression analysis of <i>fliC</i> (primers fliCF-R), <i>fleQ</i> (primers fleQF-R), and <i>16S</i> (primers 16SF-R) genes of F113 (1), <i>gacA</i><b><i>⁻</i></b> (2), <i>gacS</i><b><i>⁻</i></b> (3), F113 p<i>rsmA</i> (4), and F113 p<i>rsmE</i> (5). (<b>B</b>) Western blot analysis of external proteins from F113 (1), <i>gacA</i><b><i>⁻</i></b> (2), <i>gacS</i><b><i>⁻</i></b> (3), F113 p<i>rsmA</i> (4), and F113 p<i>rsmE</i> (5) during exponential phase (O.D.₆₀₀ = 0.3) (a), and stationary phase (O.D.₆₀₀ = 3.5) (b), reacted with an anti-flagellin antiserum. The observed band is approximately 35 KDa and corresponds to FliC. (<b>C</b>) Percentage of flagellated cells of F113 wild-type (black bar) or <i>gacS</i><b><i>⁻</i></b> (grey bar) during exponential phase (O.D.₆₀₀ = 0.3) (a), and stationary phase (O.D.₆₀₀ = 3.5) (b). Statistical significance is shown.</p

The Gac system regulates motility through the Rsm pathway.

<p>(<b>A</b>) Analysis of the swimming motility of <i>P. fluorescens</i> F113 wild-type, F113 <i>gacA</i> mutant, F113 <i>gacS</i> mutant, F113 p<i>rsmA</i>, and F113 p<i>rsmE</i>. (<b>B</b>) Swimming motility of F113 wild-type strain and its isogenic <i>gacA</i> mutant harbouring the empty vector pVLT31 or pVLT31-<i>rsmZ</i> (p<i>rsmZ</i>). (<b>C</b>) Swimming motility of F113 wild-type strain harbouring the empty vector pVLT31, pVLT31-<i>rsmX</i> (p<i>rsmX</i>), pVLT31-<i>rsmY</i> (p<i>rsmY</i>), pVLT31-<i>rsmZ</i> (p<i>rsmZ</i>), pVLT31-<i>rsmA</i> (p<i>rsmA</i>), pVLT31-<i>rsmE</i> (p<i>rsmE</i>) or pVLT31-<i>rsmI</i> (p<i>rsmI</i>).</p

RsmA binds the <i>algUmucABD</i> polycistronic mRNA.

<p>(<b>A</b>) RT-PCR of adjacent genes in the polycistronic mRNA <i>algU</i>-<i>mucA</i>-<i>mucB</i>-<i>mucD</i>. PCR of cDNA using the primer pairs qalgUF-qmucAR (lane 1), qmucAF-qmucBR (lane 2) and qmucBF2-qmucDR (lane 3) or PCR of RNA using the same primer pairs qalgUF-mucAR (lane 4), qmucAF-qmucBR (lane 5) and qmucBF2-qmucDR (lane 6), M marker. (<b>B</b>) RNA-IP assay of F113 wild-type strain harbouring the pVLT31-rsmAHA plasmid. qRT-PCR of HA-immunoprecipitated RNA (black bar) or IgG-immunoprecipitated RNA (mock, grey bar) using the primer pairs qalgUF-R (<i>algU</i>), qmucAF-R (<i>mucA</i>), qmucBF-R (<i>mucB</i>), qmucDF-R (<i>mucD</i>), qhcnAF-R (<i>hcnA</i>) and qfliCF-R (<i>fliC</i>). The <i>fliC</i> gene was used for normalization.</p