Isolation and Characterization of BTF-37: Chromosomal DNA Captured from Bacteroides fragilis That Confers Self-Transferability and Expresses a Pilus-Like Structure in Bacteroides spp. and Escherichia coli

Abstract

We report the isolation and preliminary characterization of BTF-37, a new 52-kb transfer factor isolated from Bacteroides fragilis clinical isolate LV23. BTF-37 was obtained by the capture of new DNA in the nonmobilizable Bacteroides-Escherichia coli shuttle vector pGAT400⌬BglII using a functional assay. BTF-37 is self-transferable within and from Bacteroides and also self-transfers in E. coli. Partial DNA sequencing, colony hybridization, and PCR revealed the presence of Tet element-specific sequences in BTF-37. In addition, Tn5520, a small mobilizable transposon that we described previously (G. Vedantam, T. J. Novicki, and D. W. Hecht, J. Bacteriol. 181:2564-2571, 1999), was also coisolated within BTF-37. Scanning and transmission electron microscopy of Tet element-containing Bacteroides spp. and BTF-37-harboring Bacteroides and E. coli strains revealed the presence of pilus-like cell surface structures. These structures were visualized in Bacteroides spp. only when BTF-37 and Tet element strains were induced with subinhibitory concentrations of tetracycline and resembled those encoded by E. coli broad-host-range plasmids. We conclude that we have captured a new, self-transferable transfer factor from B. fragilis LV23 and that this new factor encodes a tetracycline-inducible Bacteroides sp. conjugation apparatus.Horizontal DNA transfer by conjugation is widespread in the bacterial world and has been responsible in part for the dissemination of antibiotic resistance genes (22, 59). Conjugation between bacterial genera and species and also interkingdom conjugation (bacteria to yeast and bacteria to plants [6)]) have been shown to occur. While mobile genetic elements such as plasmids and transposons are most frequently transferred by conjugation, segments of chromosomal DNA can also be transferred by this process (17,31).Members of the genus Bacteroides are obligate, gram-negative, colonic anaerobes. Bacteroides spp. possess a plethora of mobile transfer factors, many of which harbor antibiotic resistance genes. These factors have been shown to transfer within and from Bacteroides, thus implicating these organisms as reservoirs of antibiotic resistance (A. A. Salyers, Letter, ASM News 65:459-460, 1999).DNA transfer by conjugation involves two major sets of processes: initiation and mating apparatus formation. Initiation results in the formation of a relaxosome, an ordered assembly of proteins that nick the DNA to be transferred in a site-and strand-specific manner (32,44,45,65). This nicked DNA is unwound and transmitted with 5Ј-3Ј polarity from the donor to the recipient. In Escherichia coli, the passage from donor to recipient is thought to occur through a specialized membrane-traversing channel. The channel and all accessory proteins required for mating pair stabilization, cell-cell contact, surface and entry exclusion, and DNA transfer are collectively referred to as the mating apparatus. For the E. coli F and RP4 plasmids and the Agrobacterium tumefaciens Ti plasmid, it is thought that at least 21, 12, and 1...

Interaction of Bacteroides fragilis pLV22a relaxase and transfer DNA with Escherichia coli RP4‐TraG coupling protein

“…, 1994; Lanka and Wilkins, 1995; Dang and Christie, 1997; Middleton et al ., 2005). However, OrfG does appear to be highly conserved among other Bacteroides transfer factors including TraG (CTn341) and BctA (pBF4 and BTF‐37) (Morgan and Macrina, 1997; Vedantam and Hecht, 2002; Bacic et al ., 2005; Hecht et al ., 2007) and also exhibits low similarity to VirB4 from the Ti plasmid of Agrobacterium (Dang and Christie, 1997; Middleton et al ., 2005). Whether OrfG is a Bacteroides CP‐like protein remains to be determined.…”

Bacteroides fragilis mobilizable transposon Tn5520 requires a 71 base pair origin of transfer sequence and a single mobilization protein for relaxosome formation during conjugation

“…There are three examples of this latter property – Tn 5520 can integrate into, and confer mobility on the clindamycin resistance plasmid pGAT400ΔBglII (Vedantam et al ., 1999). We have also found that Tn 5520 is associated with a large conjugative transposon (cTn) in B. fragilis (Vedantam and Hecht, 2002, and unpublished observations). Further, the Salyers’ group has found a Tn 5520 Mob gene homologue associated with a cTn in B. thetaiotaomicron (Bonheyo et al .…”

Epidemiological- and antibiotic susceptibility investigation of Hungarian Bacteroides fragilis group clinical isolates

“…The conjugative transposons, sized 52-150 kb are highly distributed among Bacteroides isolates, more than 80% of them harbour at least one CTn[62]. During their transfer they produce a covalently closed circular intermediate containing oriT sequence to allow conjugation[63]. CTn-s are able to transfer other genetic elements, such as plasmids and transposons (Tn4399 or cefoxitin resistance gene cfxA gene carrier Tn4555)[58]; they can mediate the excision and mobilization of Nonreplicating Bacteroides Units (NBUs) as well.…”