Genetic and structural analysis of the Bacteroides conjugative transposon CTn341

Abstract

The genetic structure and functional organization of a Bacteroides conjugative transposon (CTn), CTn341, were determined. CTn341 was originally isolated from a tetracycline-resistant clinical isolate of Bacteroides vulgatus. The element was 51,993 bp long, which included a 5-bp coupling sequence that linked the transposon ends in the circular form. There were 46 genes, and the corresponding gene products fell into three major functional groups: DNA metabolism, regulation and antibiotic resistance, and conjugation. The G + C content and codon usage observed in the functional groups suggested that the groups belong to different genetic lineages, indicating that CTn341 is a composite, modular element. Mutational analysis of genes representing the different functional groups provided evidence for the gene assignments and showed that the basic conjugation and excision genes are conserved among Bacteroides spp. A group IIA1 intron, designated B.f.I1, was found to be inserted into the bmhA methylase gene. Reverse transcriptase PCR analysis of CTn341 RNA showed that B.fr.I1 was functional and was spliced out of the bmhA gene. Six related CTn-like elements were found in the genome sequences of Bacteroides fragilis NCTC9343 and Bacteroides thetaiotaomicron VPI5482. The putative elements were similar to CTn341 primarily in the tra and mob regions and in the exc gene, and several appeared to contain intron elements. Our data provide the first reported sequence for a complete Bacteroides CTn, and they should be of considerable benefit to further functional and genetic analyses of antibiotic resistance elements and genome evolution in Bacteroides.

FIG. 1.

DNA sequence alignment of the CTn341, XBU4422, and CTnDOT ends, as found in the closed circular intermediate. The 5-bp coupling sequence also is shown where it is known. The conserved 10-bp region of homology between the transposon right end and the insertion target site is enclosed in a box, and the imperfect indirect repeats at the ends are indicated by the dashed arrows above the sequence. xxxxx represents the bases of an unknown coupling sequence. The sequence of XBU4422 was obtained from GenBank accession numbers S75303 and S75304, and the CTnDOT sequence was obtained from reference .

FIG. 2.

Genetic organization and ORF map of CTn341. The ORFs are indicated by arrows, which show the orientation of transcription. Arrows with light diagonal lines, transfer region; arrows with dark diagonal lines, regulation and resistance; arrows with vertical lines, DNA metabolism; arrows with stippling, UF-A region with unknown function; dark grey arrows, UF-B region with unknown function. The group II intron B.f.I.1 is indicated by a light stippled box, and the associated intron-encoded protein (maturase) is indicated by the labeled grey arrow.

FIG. 3.

Plot of G+C content of CTn341 along the length of the molecule. The G+C content was determined by the Window program in the GCG analysis package. attL and attR are represented at the ends of the map by the stickball symbols. ORFs are represented by arrows except for four small ORFs which are indicated by thick vertical lines. The functional regions of the conjugative transposon are indicated by the bars above the ORF map.

FIG. 4.

Analysis of CTn341 insertion sites by Southern hybridization. Chromosomal DNA from independent B. fragilis (A) and B. thetaiotaomicron (B) transconjugants was digested with AvaII, subjected to Southern blot analysis, and probed with a biotinylated probe containing the CTn341 ends. Lane C contained the parent strain without CTn341; lane λ contained the molecular size standard phage λ DNA digested with HindIII. Lanes 1 through 10 contained independent transconjugants.

FIG. 5.

Schematic diagram of intron area and agarose gel of PCR products from amplified cDNA. The small arrow indicates the priming site used for bmhA cDNA synthesis. The large arrows flanking the intron region indicate the primer binding sites used to detect the presence of the intron in bmhA mRNA. Lane 1, amplification product produced from chromosomal DNA template; lane 2, control without reverse transcriptase; lane 3, 1-kb ladder molecular size standard; lanes 4 and 5, amplification products from cDNA template.

FIG. 6.

Summary of percent identity plots showing regions of nucleotide similarity for CTn341 and several B. fragilis and B. thetaiotaomicron chromosomal segments with homology to the tra genes. The plots measured similarity between ungapped, aligned regions consisting of at least 100 bp (green, 55 to 70% identity; red, >70% identity). The contiguous sequence compared is indicated by a genetic map at the top of each panel, and other sequences are represented by open boxes.