Isolation and characterization of cLV25, a Bacteroides fragilis chromosomal transfer factor resembling multiple Bacteroides sp. mobilizable transposons

Abstract

Horizontal DNA transfer contributes significantly to the dissemination of antibiotic resistance genes in Bacteroides fragilis. To further our understanding of DNA transfer in B. fragilis, we isolated and characterized a new transfer factor, cLV25. cLV25 was isolated from B. fragilis LV25 by its capture on the nonmobilizable Escherichia coli-Bacteroides shuttle vector pGAT400DeltaBglII. Similar to other Bacteroides sp. transfer factors, cLV25 was mobilized in E. coli by the conjugative plasmid R751. Using Tn1000 mutagenesis and deletion analysis of cLV25, two mobilization genes, bmgA and bmgB, were identified, whose predicted proteins have similarity to DNA relaxases and mobilization proteins, respectively. In particular, BmgA and BmgB were homologous to MocA and MocB, respectively, the two mobilization proteins of the B. fragilis mobilizable transposon Tn4399. A cis-acting origin of transfer (oriT) was localized to a 353-bp region that included nearly all of the intergenic region between bmgB and orf22 and overlapped with the 3' end of orf22. This oriT contained a putative nic site sequence but showed no significant similarity to the oriT regions of other transfer factors, including Tn4399. Despite the lack of sequence similarity between the oriTs of cLV25 and Tn4399, a mutation in the cLV25 putative DNA relaxase, bmgA, was partially complemented by Tn4399. In addition to the functional cross-reaction with Tn4399, a second distinguishing feature of cLV25 is that predicted proteins have similarity to proteins encoded not only by Tn4399 but by several Bacteroides sp. transfer factors, including NBU1, NBU2, CTnDOT, Tn4555, and Tn5520.

FIG. 1.

New DNA in pGAT400ΔBglII acquired from B. fragilis LV25. (A) Restriction map of pGAT400ΔBglII. bla, ampicillin resistance; ermF, clindamycin resistance; tetX, aerobic tetracycline resistance; A, AvaI; C, ClaI; E, EcoRI; H, HindIII; RV, EcoRV. The solid triangles indicate the locations of cLV25 in pGAT400ΔBglII. (B) Agarose gel of HindIII restriction enzyme digestions of pGAT400ΔBglII and transconjugant plasmid DNA from a B. fragilis LV25-to-E. coli HB101 mating. Lanes: 1, pGAT400ΔBglII; 2, p25Δ.1; 3, p25Δ.2; 4, p25Δ.3; 5, p25Δ.4; 6, p25Δ.5; 7, p25Δ.6; 8, high-molecular-weight DNA markers; 9, 1-kb DNA ladder. Molecular size markers in kilobases are indicated on the right. The solid lines on the left indicate pGAT400ΔBglII HindIII fragments in kilobases. The arrowhead indicates the presence of 15 kb of new DNA in pGAT400ΔBglII. (C) Sequence analysis of the cLV25 termini. Shown is an alignment of the 20-bp imperfect inverted repeat (underlined) and the sequence of the additional 8 bp detected at the left end of cLV25 after insertion in pGAT400ΔBglII. (D) DNA sequence analysis of the left and right junctions of transconjugant plasmid DNA p25Δ.2. The pGAT400ΔBglII sequence is in uppercase letters, and the cLV25 sequence is in lowercase letters. The 8-bp target site repeat is underlined.

FIG. 2.

Southern hybridization analysis demonstrating that cLV25 originated from the chromosome of B. fragilis LV25. LV25 chromosomal and plasmid DNAs were digested with EcoRI and hybridized with a 3.6-kb internal cLV25 EcoRI fragment from p25Δ.2. Lanes: 1, biotinylated lambda DNA HindIII markers; 2, pGAT400ΔBglII; 3, p25Δ.2; 4, LV25 chromosomal DNA; 5, LV25 plasmid DNA. The solid lines indicate the positions of molecular size markers in kilobases. The arrowhead indicates the 3.6-kb cLV25 EcoRI fragment.

FIG. 3.

Partial restriction map of cLV25 and locations of Tn1000 insertions (solid circles, Mob⁻; open circles, Mob⁺; hatched circles, reduced frequency of mobilization). The vertical arrow indicates the position of the putative nic site. Fifteen potential open reading frames (horizontal arrows), the location of the oriT, and the regions of cLV25 tested for mobilization are shown below the map. B, BstXI; C, ClaI; E, EcoRI; RV, EcoRV.

FIG. 4.

Nucleotide sequence of the 353-bp cLV25 oriT. The vertical arrow indicates the position of the putative nic site. The putative nic site sequence, the proposed start codon for bmgB (orf23), and the proposed stop codon for orf22 are underlined. bmgB and orf22 are coded by the cDNA strand. The solid arrows above the sequence indicate two sets of inverted repeats (IR). The positions of the oriTL1 and MOB-R primers used to amplify the oriT are indicated above the sequence by the dashed arrows. The location of the Tn1000 insertion in pAC148 is indicated.