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Enterococcus faecalis is a common commensal organism and a prolific nosocomial pathogen that causes biofilm-associated infections. Numerous E. faecalis OG1RF genes required for biofilm formation have been identified, but few studies have compared genetic determinants of biofilm formation and biofilm morphology across multiple conditions. Here, we cultured transposon (Tn) libraries in CDC biofilm reactors in two different media and used Tn sequencing (TnSeq) to identify core and accessory biofilm determinants, including many genes that are poorly characterized or annotated as hypothetical. Multiple secondary assays (96-well plates, submerged Aclar discs, and MultiRep biofilm reactors) were used to validate phenotypes of new biofilm determinants. We quantified biofilm cells and used fluorescence microscopy to visualize biofilms formed by six Tn mutants identified using TnSeq and found that disrupting these genes (OG1RF_10350, prsA, tig, OG1RF_10576, OG1RF_11288, and OG1RF_11456) leads to significant time- and medium-dependent changes in biofilm architecture. Structural predictions revealed potential roles in cell wall homeostasis for OG1RF_10350 and OG1RF_11288 and signaling for OG1RF_11456. Additionally, we identified growth medium-specific hallmarks of OG1RF biofilm morphology. This study demonstrates how E. faecalis biofilm architecture is modulated by growth medium and experimental conditions and identifies multiple new genetic determinants of biofilm formation. IMPORTANCE E. faecalis is an opportunistic pathogen and a leading cause of hospital-acquired infections, in part due to its ability to form biofilms. A complete understanding of the genes required for E. faecalis biofilm formation as well as specific features of biofilm morphology related to nutrient availability and growth conditions is crucial for understanding how E. faecalis biofilm-associated infections develop and resist treatment in patients. We employed a comprehensive approach to analysis of biofilm determinants by combining TnSeq primary screens with secondary phenotypic validation using diverse biofilm assays. This enabled identification of numerous core (important under many conditions) and accessory (important under specific conditions) biofilm determinants in E. faecalis OG1RF. We found multiple genes whose disruption results in drastic changes to OG1RF biofilm morphology. These results expand our understanding of the genetic requirements for biofilm formation in E. faecalis that affect the time course of biofilm development as well as the response to specific nutritional conditions.
|Original language||English (US)|
|State||Published - 2021|
Bibliographical noteFunding Information:
We thank the University of Minnesota Genomics Center for assistance with TnSeq and the Minnesota Supercomputing Institute (MSI) at the University of Minnesota for providing computational resources. We thank Elizabeth Cameron for providing the pCIEtm::tig plasmid and Dawn Manias for assistance with constructing pP23::GFP and pP23::tdTomato. Parts of this work were carried out in the Characterization Facility, University of Minnesota, which receives partial support from the NSF through the MRSEC (award no. DMR-2011401) and the NNCI (award no. ECCS-2025124) programs. This work was supported by grant no. 1RO1AI122742 to G.M.D. from the NIH. J.L.E.W. was supported by American Heart Association grant no. 19POST34450124/Julia Willett/2018. M.L.K. was supported by grant no. TL1R002493 and UL1TR002494 from the NIH?s National Center for Advancing Translational Sciences. A.M.T.B. received support via NIH training grant no. AI055433 for portions of this work.
Parts of this work were carried out in the Characterization Facility, University of Minnesota, which receives partial support from the NSF through the MRSEC (award no. DMR-2011401) and the NNCI (award no. ECCS-2025124) programs. This work was supported by grant no. 1RO1AI122742 to G.M.D. from the NIH. J.L.E.W. was supported by American Heart Association grant no. 19POST34450124/Julia Willett/2018. M.L.K. was supported by grant no. TL1R002493 and UL1TR002494 from the NIH’s National Center for Advancing Translational Sciences. A.M.T.B. received support via NIH training grant no. AI055433 for portions of this work.
© 2021 Willett et al.
- Antibiotic resistance
- Biofilm infections
- Functional genomics
- Gene discovery
PubMed: MeSH publication types
- Comparative Study
- Journal Article
- Research Support, N.I.H., Extramural
- Research Support, Non-U.S. Gov't
- Research Support, U.S. Gov't, Non-P.H.S.
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- 1 Active
University of Minnesota Materials Research Science and Engineering Center (DMR-2011401)
9/1/20 → 8/31/26
Project: Research project