Possible origins of CTnBST, a conjugative transposon found recently in a human colonic Bacteroides strain

Abstract

A previous survey of Bacteroides isolates suggested that the ermB gene entered Bacteroides spp. recently. Previously, ermB had been found almost exclusively in gram-positive bacteria. In one Bacteroides strain, ermB was located on 100-kb conjugative transposon (CTn) CTnBST. To assess the possible origin of this CTn, we obtained the full DNA sequence of CTnBST and used this information to investigate its possible origins. Over one-half of CTnBST had high sequence identity to a putative CTn found in the genome of Bacteroides fragilis YCH46. This included the ends of the CTn and genes involved in integration, transfer, and excision. However, the region around the ermB gene contained genes that appeared to originate from gram-positive organisms. In particular, a 7-kb segment containing the ermB gene was 100% identical to an ermB region found in the genome of the gram-positive bacterium Arcanobacterium pyogenes. A screen of Bacteroides isolates whose DNA cross-hybridized with a CTnBST probe revealed that several isolates did not carry the 7-kb region, implying that the acquisition of this region may be more recent than the acquisition of the entire CTnBST element by Bacteroides spp. We have also identified other Bacteroides isolates that carry a slightly modified 7-kb region but have no other traces of CTnBST. Thus, it is possible that this 7-kb region could itself be part of a mobile element that has inserted in a Bacteroides CTn. Our results show that CTnBST is a hybrid element which has acquired a portion of its coding region from gram-positive bacteria but which may originally have come from Bacteroides spp. or some related species.

FIG. 1.

(A) The percent amino acid identity between the ORFs of CTnBST and CTn3-Bf defines two distinctly gram-positive organism-like regions within CTnBST. The ermB region, as depicted in the graph, is 100% identical on the nucleotide level to the same sequence in Arcanobacterium pyogenes OX-7. The region upstream of the ermB region, labeled as a mixed region, contains a mixture of Bacteroides-like and gram-positive organism-like ORFs. (B) The % G+C content of each ORF in CTnBST is shown. This graph helps to define both functional regions (transfer region) and putative insertions of gram-positive organism-like DNA (in both the mixed region and the ermB region). It should be noted that the average % G+C content of the Bacteroides chromosome is around 42%.

FIG. 2.

The ermB region of CTnBST and the corresponding region in Arcanobacterium pyogenes OX-7 share 100% identity on the nucleotide level. This identity continues past the published sequence for Arcanobacterium pyogenes OX-7 (shown here) to include the aadE homolog and the flanking partial ORFs (in gray).

FIG. 3.

The region that includes the genes necessary for excision of CTnBST. The numbers above the arrows indicate the base pair positions within CTnBST sequence. The “+” and “−” symbols correspond to whether or not complementation of the excisionless phenotype was achieved with the given construct. The results of this experiment lead us to conclude that the promoter for xisBST lies within and/or upstream of BST002.

FIG. 4.

The joined ends of the excised circular form of CTnBST were PCR amplified from each of the listed strains carrying each of the listed plasmids shown in Fig. 3 and described in Table 1. BT4020, a strain that carries a wild-type copy of CTnBST, was used as a positive control. BT4001 does not contain CTnBST and was used as a negative control in this experiment. As a positive control for the amplification reaction the gene that encodes sigma 70 in Bacteroides was successfully amplified from each strain but is not shown here.