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Journal of Bacteriology, November 2004, p. 7280-7289, Vol. 186, No. 21
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.21.7280-7289.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Construction of an Enterococcus faecalis Tn917-Mediated-Gene-Disruption Library Offers Insight into Tn917 Insertion Patterns

Danielle A. Garsin,1* Jonathan Urbach,2 Jose C. Huguet-Tapia,3 Joseph E. Peters,3 and Frederick M. Ausubel2

Department of Microbiology and Molecular Genetics, University of Texas Health Science Center at Houston, Houston, Texas,1 Department of Molecular Biology, Massachusetts General Hospital, Boston, Massachusetts,2 Department of Microbiology, Cornell University, Ithaca, New York3

Received 16 June 2004/ Accepted 29 July 2004

Sequencing the insertion sites of 8,865 Tn917 insertions in Enterococcus faecalis strain OG1RF identified a hot spot in the replication terminus region corresponding to 6% of the genome where 65% of the transposons had inserted. In E. faecalis, Tn917 preferentially inserted at a 29-bp consensus sequence centered on TATAA, a 5-bp sequence that is duplicated during insertion. The regional insertion site preference at the chromosome terminus was not observed in another low-G+C gram-positive bacterium, Listeria monocytogenes, although the consensus insertion sequence was the same. The 8,865 Tn917 insertion sites sequenced in E. faecalis corresponded to only ~610 different open reading frames, far fewer than the predicted number of 2,400, assuming random insertion. There was no significant preference in orientation of the Tn917 insertions with either transcription or replication. Even though OG1RF has a smaller genome than strain V583 (2.8 Mb versus 3.2 Mb), the only E. faecalis strain whose sequence is in the public domain, over 10% of the Tn917 insertions appear to be in a OG1RF-specific sequence, suggesting that there are significant genomic differences among E. faecalis strains.

* Corresponding author. Mailing address: University of Texas Health Science Center at Houston, Department of Microbiology and Molecular Genetics, 6431 Fannin St., JFB 1.711, Houston, TX 77030. Phone: (713) 500-5454. Fax: (713) 500-5499. E-mail: Danielle.A.Garsin{at}uth.tmc.edu.

Journal of Bacteriology, November 2004, p. 7280-7289, Vol. 186, No. 21
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.21.7280-7289.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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