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 pGAT400DeltaBglII 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.

FIG. 1.

Isolation of large DNA fragment insertions in pGAT400ΔBglII from B. fragilis LV23. Restriction enzyme analysis of transconjugant plasmid DNA from matings was performed. Large fragments and those corresponding to Tn5520 are indicated (arrows), along with the time point during growth at which they were isolated. All transconjugant DNA was digested with EcoRI. DNA molecular size markers (MW) are indicated.

FIG. 2.

Restriction map of BTF-37. Insertion into pGAT400ΔBglII occurred at pRK231 oriT in the 3.5-kb PstI-EcoRI fragment. E, EcoRI; H, HindIII; P, PstI; S, Sau3AI.

FIG. 3.

Tn5520 is linked to BTF-37. Matings were performed using B. fragilis LV23, B. thetaiotaomicron, or E. coli HB101, all harboring BTF-37 as the donor, and B. fragilis TM4000, E. coli HB101, or E. coli DW1030 as the recipient. (A) A 3-kb Tn5520-specific DdeI fragment that included the bipH (transposase) and bmpH (mobilization) genes was used as a probe for colony hybridizations of total transconjugant DNA. (B) Ten transconjugants from each mating (a to j) were tested, except for the E. coli-to-E. coli matings, where 3 transconjugants were tested. An example of a negative control (TM4000) is shown. BT4001 and the E. coli strains alone showed no hybridization signal with the probe (not shown).

FIG. 4.

Detection of cell surface structures using scanning EM. All strains were subjected to tetracycline induction and 6 h of incubation on mating filters (see Materials and Methods). (A) Clinical isolate LV23, wild-type control TM4000, and TM4000 harboring BTF-37. Arrows, pilus-like structures. (B) Other Tet element-containing strains. Magnification, ×37,000 to 43,000. All samples were viewed at 15 kV. Panels are representative of visualization of at least 1,000 cells for each sample and of 8 to 10 fields photographed. Approximately 10 to 15% of cells express the surface structure. Bars, 200 nm.

FIG. 5.

Transmission electron micrograph of TM4000(BTF-37). A possible central channel in the pilus-like structure can be seen. The panel is representative of at least 2,000 cells visualized for the sample and of six fields photographed. Approximately 1% of cells visualized express the cell surface structure. Magnification (after size correction of the image), ×54,000.

FIG. 6.

(A) Cell surface structures are expressed upon tetracycline induction of LV23 and TM4000(BTF-37). (B and C) Lengths of cell-surface structures vary depending on total time on mating filters. Similar results were obtained for LV23 and TM4000(BTF-37). Magnification, ×37,000 to 43,000. All samples were viewed at 15 kV. Panels are representative of visualization of at least 1,000 cells for each sample and of 8 to 10 fields photographed. Bars, 200 nm. Arrows, pilus-like structures.

FIG. 7.

E. coli HB101 harboring BTF-37 expresses pilus-like cell surface structures. (A) HB101, no plasmid. (B) HB101(BTF-37) and HB101(R751) (self-transferable R plasmid) as a control. Arrows, pilus-like structures. Magnification, ×37,000 to 43,000. All samples were viewed at 15 kV. Panels are representative of visualization of at least 1,000 cells for each sample and of 8 to 10 fields photographed. Bar, 200 nm.