Identification and characterization of conjugative transposons CTn86 and CTn9343 in Bacteroides fragilis strains

Abstract

The related genetic elements flanking the Bacteroides fragilis pathogenicity island (PAI) in enterotoxigenic B. fragilis (ETBF) 86-5443-2-2 and also present in pattern III nontoxigenic B. fragilis (NTBF) NCTC 9343 were defined as putative conjugative transposons (CTns), designated CTn86 and CTn9343, respectively (A. A. Franco, J. Bacteriol. 181:6623-6633, 2004). CTn86 and CTn9343 have the same basic structures except that their encoded transposases have low similarity and CTn9343 lacks the B. fragilis PAI and contains an extra 7-kb region not present in CTn86. In this study, using DNA hybridization and PCR analysis, we characterized the genetic element flanking the PAI in a collection of ETBF strains and the related genetic elements in a collection of NTBF pattern III strains. We found that in all 123 ETBF strains, the PAI is contained in a genetic element similar to CTn86. Of 73 pattern III strains, 26 (36%) present a genetic element similar to CTn9343, 38 (52%) present a genetic element similar to CTn9343 but lack the 7-kb region that is also absent in CTn86 (CTn9343-like element), and 9 (12%) present a genetic element similar to CTn86 but lacking the PAI (CTn86-like element). In addition to containing CTn86, ETBF strains can also contain CTn9343, CTn9343-like, or CTn86-like elements. CTn86, CTn9343, CTn86-like, and CTn9343-like elements were found exclusively in B. fragilis strains and predominantly in division I, cepA-positive strains.

FIG. 1.

Characterization of the genetic element flanking the B. fragilis PAI in ETBF strains and the related genetic elements in NTBF pattern III strains. (A) Schematic map of CTn9343 showing the relative locations of the PCR products used as probes. The hybridization results for CTn86 are indicated beneath each probe (P, positive; N, negative). The double-lined bar indicates the 12-kb region previously demonstrated to be present in 100% of pattern I and III strains and absent from pattern II strains (7). The B. fragilis PAI is located between oriT-bfmA-bfmB (oriT) and bfmC in ETBF strains. (B) Hybridization results for ETBF strains. (C) Hybridization results for NTBF pattern III strains.

FIG. 2.

Schematic maps of the CTn9343 and CTn86 left ends showing the relative locations of the primers used to identify the CTn86, CTn86-like, CTn9343, and CTn9343-like elements. Primers 86CTn1R and Tn9B yield a ∼1.8-kb PCR product in genetic elements that have the CTn86 left end and lack the 7-kb region, primers Tn25B and Tn9B yield a ∼1.8-kb PCR product in genetic elements that have the CTn9343 left end and lack the 7-kb region, primers Tn25B and Tn25C yield a ∼1-kb PCR product in genetic elements that have the CTn9343 left end and contain the 7-kb region, and primers P1T3 and P1T7 yield a ∼1.6-kb PCR product in genetic elements that lack the B. fragilis PAI (BfPAI) (pattern III strains). Primer pair P1T3 and P1T3-1 and primer pair P1T7 and P1T7-1 yield 1.1-kb and 1.3-kb PCR products, respectively, in strains that have the B. fragilis PAI integrated between bfmB (ORF 19) and bfmC (ORF 20). The thick bar shows the 7-kb region deleted in the CTn86, CTn86-like, and CTn9343-like elements.

FIG. 3.

Southern blot hybridization of the HindIII-digested chromosome of pattern I strain 86-5443-2-2 (lane 1), pattern I.3 strain DS-93 (lane 2), pattern III.1 strain I-1345 (lane 3), and pattern I.2 strain K641 (lane 4). The digested chromosomes were hybridized with probes derived from the CTn86 left end (A) and the CTn9343 left end (B). ETBF 86-5443-2-2 carries two copies of CTn86 (9).

FIG. 4.

Identification of CTn86 and CTn9343 circular intermediates by inverse PCR using primers derived from the CTn9343 ends (lanes 1 to 5) and CTn86 ends (lane 6 to 10). Lanes 1 and 6, ETBF 86-5443-2-2; lanes 2 and 7, NCTC 9343; lanes 3 and 8, K641 (pattern I.2); lanes 4 and 9, DS-93 (pattern I.3); lanes 5 and 10, I-1345 (pattern III.1). M, 1-kb DNA ladder.

FIG. 5.

Insertion sites of CTn9343 (A) and the copies of CTn86 (B) in NCTC 9343 and ETBF 86-5443-2-2, respectively. The relative positions of primers Tn24/Tn25 and Tn22/Tn23, as well as of Tn22/86CTn27 and Tn22/86CTn9, to identify the integration sites of CTn9343 and CTn86 in pattern III (NTBF) and I (ETBF) strains, respectively, are shown.