Genes That Influence Swarming Motility and Biofilm Formation in Variovorax paradoxus EPS

Variovorax paradoxus is an aerobic soil bacterium associated with important biodegradative processes in nature. We use V. paradoxus EPS to study multicellular behaviors on surfaces.We recovered flanking sequence from 123 clones in a Tn5 mutant library, with insertions in 29 different genes, selected based on observed surface behavior phenotypes. We identified three genes, Varpa_4665, Varpa_4680, and Varpa_5900, for further examination. These genes were cloned into pBBR1MCS2 and used to complement the insertion mutants. We also analyzed expression of Varpa_4680 and Varpa_5900 under different growth conditions by qPCR.The 29 genes we identified had diverse predicted functions, many in exopolysaccharide synthesis. Varpa_4680, the most commonly recovered insertion site, encodes a putative N-acetyl-L-fucosamine transferase similar to WbuB. Expression of this gene in trans complemented the mutant fully. Several unique insertions were identified in Varpa_5900, which is one of three predicted pilY1 homologs in the EPS genome. No insertions in the two other putative pilY1 homologs present in the genome were identified. Expression of Varpa_5900 altered the structure of the wild type swarm, as did disruption of the chromosomal gene. The swarming phenotype was complemented by expression of Varpa_5900 from a plasmid, but biofilm formation was not restored. Both Varpa_4680 and Varpa_5900 transcripts were downregulated in biofilms and upregulated during swarming when compared to log phase culture. We identified a putative two component system (Varpa_4664-4665) encoding a response regulator (shkR) and a sensor histidine kinase (shkS), respectively. Biofilm formation increased and swarming was strongly delayed in the Varpa_4665 (shkS) mutant. Complementation of shkS restored the biofilm phenotype but swarming was still delayed. Expression of shkR in trans suppressed biofilm formation in either genetic background, and partially restored swarming in the mutant.The data presented here point to complex regulation of these surface behaviors

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

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

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

Mutants from Colony Morphology Screens.

<p>Mutants from Colony Morphology Screens.</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

Strains, Vectors, Primers.

<p>Strains, Vectors, Primers.</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

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