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Applied and Environmental Microbiology, June 2008, p. 3377-3386, Vol. 74, No. 11
0099-2240/08/$08.00+0     doi:10.1128/AEM.02665-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Development and Use of an Efficient System for Random mariner Transposon Mutagenesis To Identify Novel Genetic Determinants of Biofilm Formation in the Core Enterococcus faecalis Genome{triangledown} ,{dagger}

Christopher J. Kristich,1,2 Vy T. Nguyen,1 Thinh Le,1 Aaron M. T. Barnes,1 Suzanne Grindle,1 and Gary M. Dunny1*

Department of Microbiology, University of Minnesota Medical School, Minneapolis, Minnesota 55455,1 Department of Microbiology and Molecular Genetics, Medical College of Wisconsin, Milwaukee, Wisconsin 532262

Received 14 November 2007/ Accepted 28 March 2008

Enterococcus faecalis is a gram-positive commensal bacterium of the gastrointestinal tract and an important opportunistic pathogen. Despite the increasing clinical significance of the enterococci, most of the genetic analysis of these organisms has focused on mobile genetic elements, and existing tools for manipulation and analysis of the core E. faecalis chromosome are limited. We are interested in a comprehensive analysis of the genetic determinants for biofilm formation encoded within the core E. faecalis genome. To identify such determinants, we developed a substantially improved system for transposon mutagenesis in E. faecalis based on a mini-mariner transposable element. Mutagenesis of wild-type E. faecalis with this element yielded predominantly mutants carrying a single copy of the transposable element, and insertions were distributed around the entire chromosome in an apparently random fashion. We constructed a library of E. faecalis transposon insertion mutants and screened this library to identify mutants exhibiting a defect in biofilm formation. Biofilm-defective mutants were found to carry transposon insertions both in genes that were previously known to play a role in biofilm formation and in new genes lacking any known function; for several genes identified in the screen, complementation analysis confirmed a direct role in biofilm formation. These results provide significant new information about the genetics of enterococcal biofilm formation and demonstrate the general utility of our transposon system for functional genomic analysis of E. faecalis.

* Corresponding author. Mailing address: 420 Delaware St. SE, MMC196, Minneapolis, MN 55455. Phone: (612) 625-9930. Fax: (612) 626-0623. E-mail: dunny001{at}umn.edu

{triangledown} Published ahead of print on 11 April 2008.

{dagger} Supplemental material for this article may be found at http://aem.asm.org/.

Applied and Environmental Microbiology, June 2008, p. 3377-3386, Vol. 74, No. 11
0099-2240/08/$08.00+0     doi:10.1128/AEM.02665-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



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