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Bacteroides fragilis mobilizable transposon Tn 5520 requires a 71 base pair origin of transfer sequence and a single mobilization protein for relaxosome formation during conjugation
Author(s) -
Vedantam Gayatri,
Knopf Sarah,
Hecht David W.
Publication year - 2006
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1111/j.1365-2958.2005.04934.x
Subject(s) - biology , transposable element , plasmid , inverted repeat , genetics , gene , mutant , consensus sequence , insertion sequence , microbiology and biotechnology , peptide sequence , genome
Summary Tn 5520 is the smallest known bacterial mobilizable transposon and was isolated from an antibiotic resistant Bacteroides fragilis clinical isolate. When a conjugation apparatus is provided in trans , Tn 5520 is mobilized (transferred) efficiently within, and from, both Bacteroides spp. and Escherichia coli. Only two genes are present on Tn 5520 ; one encodes an integrase, and the other a multifunctional mobilization (Mob) protein BmpH. BmpH is essential for Tn 5520 mobility. The focus of this study was to identify the Tn 5520 origin of conjugative transfer ( oriT ) and to study BmpH‐ oriT binding. We delimited the functional Tn 5520 oriT to a 71 bp sequence upstream of the bmpH gene. A plasmid vector harbouring this minimal 71 bp oriT was mobilized at the same frequency as that of intact Tn 5520 . The minimal oriT contains one 17 bp inverted repeat (IR) sequence. We constructed and tested multiple IR mutants and showed that the IR was essential in its entirety for mobilization. A nick site sequence (5′‐GCTAC‐3′) was also identified within the minimal oriT ; this sequence resembled nick sites found in plasmids of Gram positive origin. We further showed that mutation of a highly conserved GC dinucleotide in the nick site sequence completely abolished mobilization. We also purified BmpH and showed that it specifically bound a Tn 5520 oriT fragment in electrophoretic mobility shift assays. We also identified non‐nick site sequences within the minimal oriT that were essential for mobilization. We hypothesize that transposon‐based single Mob protein systems may contribute to efficient gene dissemination from Bacteroides spp., because fewer DNA processing proteins are required for relaxosome formation.

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