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Updated: Aug 21, 2026
Generating Transposon Insertion Libraries in Gram-Negative Bacteria for High-Throughput Sequencing
Published on: July 7, 2020
Construction of an Enterococcus faecalis Tn917-mediated-gene-disruption library offers insight into Tn917 insertion
Danielle A Garsin1, Jonathan Urbach, Jose C Huguet-Tapia
1Department of Microbiology and Molecular Genetics, University of Texas Health Science Center at Houston, 6431 Fannin Street, JFB 1.711, Houston, TX 77030, USA. Danielle.A.Garsin@uth.tmc.edu
Abstract:
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 approximately 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.
