Analysis of surface behaviors to determine efficacy of Varpa_4680 (<i>wbu</i>B) and Varpa_5900 (<i>pil</i>Y1) complementation.

<p>Biofilm levels after 24 h incubation and swarm diameters at 48 h for wt and mutant strains expressing either <i>wbu</i>B (A, B) or <i>pil</i>Y1 (C, D) <i>in trans</i> compared to vector controls. 86 = Varpa_4680::Tn5, 223 = Varpa_5900::Tn5. Error was computed as +/−SEM. All p-values were calculated using the student's unpaired T-test. For all panels a = p<0.01 compared to wt+vec, b = p<0.01 compared to 86+vec (A,B) or 223+vec (C,D).</p

Expression of <i>wbu</i>B (grey bars) and <i>pil</i>Y1(black bars) assessed directly by qPCR.

<p>Planktonic cells, biofilm cells, and plate cultures from 0.5% agarose solidified YE plates or FW swarming plates were harvested at 48 h of growth and compared to aerated liquid culture in log phase (19 h) or stationary phase (26 h). RNA levels were determined in comparison to a luciferase spike added to each sample, and fold expression relative to log phase was assessed using the Pfaffl method.</p

Mutants from Colony Morphology Screens.

<p>Mutants from Colony Morphology Screens.</p

Swarms of <i>V. paradoxus</i> EPS imaged using a stereomicroscope.

<p>(A) wt+vec, (B) wt+<i>pil</i>Y1, (C) mut223+vec, (D) mut223+<i>pil</i>Y1.</p

Phylogenetic and nucleotide composition suggests Varpa_4680 entered the <i>V. paradoxus</i> EPS genome by horizontal transfer.

<p>A) Phylogenetic tree based on WbuB amino acid sequences created using ClustalW multiple sequence alignment and subsequent Bayesian inference of phylogeny(Mr. Bayes). B) Phylogenetic tree using the 16s rDNA sequences from the same set of organisms as in (A), using Bayesian inference of phylogeny based on the HKY85 nucleotide substitution model. For both A) and B) Vp EPS = <i>Variovorax paradoxus</i> EPS, Pn = <i>Polaromonas naphthlenivorans</i> CJ2, Bp = <i>Bordatella pertussis</i> Tohama 1, Pao1 = <i>Pseudomonas aeruginosa</i> PAO1, Cv = <i>Chromobacterium violaceum</i> ATCC 12472, Sd = <i>Sulfurimonas denitrificans</i> DSM 1251 C) G/C analysis of 35 kb region of the <i>V. paradoxus</i> EPS genome including <i>wbu</i>B. Total region spans orfs Varpa_4661-4691. Low G/C region from 18.4 kb to 27.1 kb spans orfs Varpa_4679-4684.</p

Swarm edges at 24 h on 0.5% FW plates+25 µg/ml Km were photographed using a 10× phase contrast lens (scale bar = 10 microns).

<p>A) wt+vec, B) wt+<i>wbu</i>B, C) mut86+vec, D) mut86+<i>wbu</i>B.</p

Strains, Vectors, Primers.

<p>Strains, Vectors, Primers.</p

Analysis of surface behaviors to examine complementation of Varpa_4664 (<i>shk</i>S).

<p>Biofilm levels after 24 h incubation (A) and swarm diameters (B) at 48 h for wt and mut99 expressing either <i>shk</i>S or <i>shk</i>R <i>in trans</i> compared to vector controls. Error was computed as +/−SEM. P-values were calculated using the student's unpaired T-test. For both panels, a = p<0.01 compared to wt+vec, b = p<0.01 compared to mut99+vec.</p