Characterization of the 13-kilobase ermF region of the Bacteroides conjugative transposon CTnDOT

Abstract

The conjugative transposon CTnDOT is virtually identical over most of its length to another conjugative transposon, CTnERL, except that CTnDOT carries an ermF gene that is not found on CTnERL. In this report, we show that the region containing ermF appears to consist of a 13-kb chimera composed of at least one class I composite transposon and a mobilizable transposon (MTn). Although the ermF region contains genes also carried on Bacteroides transposons Tn4351 and Tn4551, it does not contain the IS4351 element which is found on these transposons. In CTnDOT, insertion of the ermF region occurred near a stem-loop structure at the end of orf2, an open reading frame located immediately downstream of the integrase (int) gene of CTnDOT, and in a region known to be important for excision of CTnERL and CTnDOT. The chimera that comprises the ermF region can apparently no longer excise and circularize, but it contains a functional mobilization region related to that described for the Bacteroides MTn Tn4399. Analysis of 19 independent Bacteroides isolates showed that the ermF region is located in the same position in all of the strains analyzed and that the compositions of the ermF region are almost identical in these strains. Therefore, it appears that CTnDOT-like elements present in community and clinical isolates of Bacteroides were derived from a common ancestor and proliferated in the diverse Bacteroides population.

FIG. 1

Comparison of the Bacteroides CTns CTnERL and CTnDOT. CTnDOT is distinguished from CTnERL by a 13-kb insertion designated the ermF region which is present in CTnDOT (black box) but absent from CTnERL. Regions present in both CTnDOT and CTnERL are indicated by gray and white boxes. Open reading frames and their orientations are indicated. Cosmid clones p6E3 and p6E2, probes A and B used for localization of the right ermF region junction, and probe C used for localization of the left ermF region junction are also shown. Primers and their directions are represented by arrows; these primers were utilized for amplification of junction fragments (primers 1 and 2), for detection of a putative circular transposition intermediate (primers 3 to 5), and for amplification of the putative mobilization genes for mobilization experiments (primers 6 and 7). The putative mobilization genes were cloned onto Bacteroides mobilization-deficient vector pLYL7oriT_RK2, and the resulting construct was designated pGW39.1. Restriction sites for the following restriction enzymes are indicated: EcoRI (E), ClaI (C), and HindIII (H).

FIG. 2

ClustalW alignment of the CTnERL target site for integration of the ermF region and the CTnDOT junction sequences at the left (LJ) and right (RJ) junctions of the ermF region. An asterisk indicates 100% nucleotide identity. The TGA stop codon of orf2 is underlined, and this codon is the point of divergence between the CTnERL and CTnDOT sequences at the left end of the ermF region. Potential stem-loop structures downstream of orf2 in CTnDOT and CTnERL target sequences are indicated by boldface type, and the directions of the inverted repeats are indicated by arrows above the appropriate sequences. Sequences that are part of the ermF region and hence are not present in CTnERL are indicated by a gray background. The 89 nucleotides of CTnERL between the stop codon of orf2 and the left and right junctions of the ermF region are not present in CTnDOT.

FIG. 3

Comparison of the ermF region of CTnDOT with related mobilizable transposons (NBU1, NBU2, and Tn4399) and nonmobilizable transposons (Tn4351 and Tn4551) from Bacteroides. Regions of amino acid identity with proteins from MTns (cross-hatched boxes) and nonmobilizable transposons (dotted boxes) are indicated. Note that only parts of mobilizable transposons NBU1, NBU2, and Tn4399 are shown.

FIG. 4

Alignment of the putative nic sites of the mob region from the ermF region of CTnDOT, NBU1, and NBU2 and the determined nic sites of Tn4399, RP4, and R751. Nucleotide positions conserved in the family of oriT sequences are indicated by gray shading, and cleavage sites that have been determined are indicated by arrows. The consensus sequence described for the RP4 family of oriT nic sites is shown below the aligned sequences. GenBank accession numbers are as follows: NBU1, AF238307; NBU2, AF251288; RP4, L27758; and R751, X54458.