title: Genes that influence swarming motility and biofilm formation in Variovorax paradoxus EPS.
abstract: BACKGROUND
  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.
  
  
  METHODS
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  The data presented here point to complex regulation of these surface behaviors.
authors.affiliation: Department of Biology, California State University San Bernardino, San Bernardino, California, United States of America.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Pehl
authors.forename: William David
authors.initials: WD
authors.lastname: Jamieson
authors.forename: Karen
authors.initials: K
authors.lastname: Kong
authors.forename: Jessica L
authors.initials: JL
authors.lastname: Forbester
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Fredendall
authors.forename: Glenn A
authors.initials: GA
authors.lastname: Gregory
authors.forename: Jacob E
authors.initials: JE
authors.lastname: McFarland
authors.forename: Jessica M
authors.initials: JM
authors.lastname: Healy
authors.forename: Paul M
authors.initials: PM
authors.lastname: Orwin
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D018441:Biofilms / Q000254:growth & development*; D001681:Biological Assay; D001704:Biopolymers / Q000378:metabolism*; D020134:Catalytic Domain; D002462:Cell Membrane / Q000378:metabolism; D015169:Colony Count, Microbial; D042621:Comamonadaceae / Q000235:genetics* / Q000254:growth & development / Q000502:physiology*; D017124:Conserved Sequence / Q000235:genetics; D005110:Extracellular Space / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D022761:Gene Transfer, Horizontal / Q000235:genetics; D005798:Genes, Bacterial / Q000235:genetics*; D005816:Genetic Complementation Test; D005820:Genetic Testing; D008969:Molecular Sequence Data; D009068:Movement; D009154:Mutation / Q000235:genetics; D009711:Nucleotides / Q000235:genetics; D010802:Phylogeny; D016133:Polymerase Chain Reaction
chemical_list: D001426:Bacterial Proteins; D001704:Biopolymers; D009711:Nucleotides