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.
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We thank Jennifer Dale and Dawn Manias for designing the arrayed Tn library, Shalane Porter and Dinesha Walak of the University of Minnesota Genomics Center for assistance with the arrayed transposon library, Yuqing Chen for providing the tetracycline-resistant plasmid pCIE-tet, and Leanne Higgins and Todd Markowski at the University of Minnesota College of Biological Sciences Center for Mass Spectrometry and Proteomics for assistance with mass spectrometry. Barbara Murray (University of Texas, Houston) generously provided anti-EbpC and anti-Ace antibodies. The Experimental Surgery Suite at the University of Minnesota provided porcine samples, and we thank Anne-Marie Leuck for helpful discussions regarding experimental design. J.L.E.W. was supported by American Heart Association Grant #19POST34450124/J.L.E.W./2018 and NIH grant NHLBI T32 HL07741. This work was also supported by NIH grants 1R21AI20601, 5R35GM118079, and 1RO1AI122742 to G.M.D.