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Journal of Bacteriology, March 2006, p. 2063-2072, Vol. 188, No. 6
0021-9193/06/$08.00+0     doi:10.1128/JB.188.6.2063-2072.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Putative Surface Proteins Encoded within a Novel Transferable Locus Confer a High-Biofilm Phenotype to Enterococcus faecalis{dagger}

Preeti M. Tendolkar,{ddagger} Arto S. Baghdayan,{ddagger} and Nathan Shankar*

Department of Pharmaceutical Sciences, University of Oklahoma Health Sciences Center, P.O. Box 26901, Oklahoma City, Oklahoma 73190

Received 26 July 2005/ Accepted 4 January 2006

Enterococci are opportunistic pathogens and among the leading causes of nosocomial infections. Enterococcus faecalis, the dominant species among infection-derived isolates, has recently been recognized as capable of forming biofilms on abiotic surfaces in vitro as well as on indwelling medical devices. A few bacterial factors known to contribute to biofilm formation in E. faecalis have been characterized. To identify additional factors which may be important to this process, we utilized a Tn917-based insertional mutagenesis strategy to generate a mutant bank in a high-biofilm-forming E. faecalis strain, E99. The resulting mutant bank was screened for mutants exhibiting a significantly reduced ability to form biofilms. One mutant, P101D12, which showed greater than 70% reduction in its ability to form biofilms compared to the wild-type parent, was further characterized. The single Tn917 insertion in P101D12 was mapped to a gene, bee-2, encoding a probable cell wall-anchored protein. Sequence information for the region flanking bee-2 revealed that this gene was a member of a locus (termed the bee locus for biofilm enhancer in enterococcus) comprised of five genes encoding three putative cell wall-anchored proteins and two probable sortases. Contour-clamped homogeneous electric field gel and Southern hybridization analyses suggested that the bee locus is likely harbored on a large conjugative plasmid. Filter mating assays using wild-type E99 or mutant P101D12 as a donor confirmed that the bee locus could transfer conjugally at high frequency to recipient E. faecalis strains. This represents the first instance of the identification of a mobile genetic element conferring biofilm-forming property in E. faecalis.

* Corresponding author. Mailing address: Department of Pharmaceutical Sciences, University of Oklahoma Health Sciences Center, P.O. Box 26901, Oklahoma City, OK 73190. Phone: (405) 271-6481. Fax: (405) 271-7505. E-mail: nathan-shankar{at}ouhsc.edu.

{ddagger} These authors contributed equally to this work.

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

Journal of Bacteriology, March 2006, p. 2063-2072, Vol. 188, No. 6
0021-9193/06/$08.00+0     doi:10.1128/JB.188.6.2063-2072.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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