Exploiting biofilm phenotypes for functional characterization of hypothetical genes in Enterococcus faecalis

Enterococcus faecalis is a commensal organism as well as an important nosocomial pathogen, and its infections are typically linked to biofilm formation. Nearly 25% of the E. faecalis OG1RF genome encodes hypothetical genes or genes of unknown function. Elucidating their function and how these gene products influence biofilm formation is critical for understanding E. faecalis biology. To identify uncharacterized early biofilm determinants, we performed a genetic screen using an arrayed transposon (Tn) library containing ~2000 mutants in hypothetical genes/intergenic regions and identified eight uncharacterized predicted protein-coding genes required for biofilm formation. We demonstrate that OG1RF_10435 encodes a phosphatase that modulates global protein expression and arginine catabolism and propose renaming this gene bph (biofilm phosphatase). We present a workflow for combining phenotype-driven experimental and computational evaluation of hypothetical gene products in E. faecalis, which can be used to study hypothetical genes required for biofilm formation and other phenotypes of diverse bacteria.