K-116. Functional Genomic Screening for Biofilm Mutants by Colony Morphology in Variovorax paradoxus EPS

M. J. Pehl, K. Kong, P. M. Orwin;
California State Univ., San Bernardino, CA.

Variovorax paradoxus is a common soil microorganism that plays an important role within the soil microcommunity. Variovorax paradoxus strain EPS was isolated from the soil behind Cal State San Bernardino, and displays a mucoid phenotype on several common laboratory media. Using transposon mutagenesis to interrupt genes, a mutant library was created and screened for altered colony morphology on 0.5 and 2.5 g/L YE agar. A total of about 15000 mutants were screened using this technique. Mutants with altered morphology were subsequently assayed for altered biofilm phenotype using 96-well static biofilm assay. The interrupted genes from mutants with significantly altered biofilm levels, were cloned using the E. coli origin of replication present in Tn5. Partial sequencing of the interrupted genes in these mutants revealed a variety of likely homologues to known genes involved in biofilm formation. Interruptions of the pilY1 fimbrial tip adhesin, exopolysaccharide production and transport genes, a putative sensor histidine kinase, and various glycoprotein modification genes were identified in both screened populations. Several independent insertions into a Group 1 glycosyl transferase gene as well as the pilY1 gene lead us to hypothesize that these gene products play important roles in V paradoxus biofilm development. Our results suggest that there are many links between V. paradoxus colony morphology and biofilm physiology, similar to results in other microbes. We also observe that many genes are involved in a central biofilm formation apparatus, regardless of nutrient conditions.