A novel conjugative transposon carrying an autonomously amplified plasmid

Abstract

Tetracyclines serve as broad-spectrum antibiotics to treat bacterial infections. The discovery of new tetracycline resistance genes has led to new questions about the underlying mechanisms of resistance, gene transfer, and their relevance to human health. We tracked changes in the abundance of a 55-kbp conjugative transposon (CTn214) carrying tetQ, a tetracycline resistance gene, within a Bacteroides fragilis metagenome-assembled genome derived from shotgun sequencing of microbial DNA extracted from the ileal pouch of a patient with ulcerative colitis. The mapping of metagenomic reads to CTn214 revealed the multi-copy nature of a 17,044-nt region containing tetQ in samples collected during inflammation and uninflamed visits. B. fragilis cultivars isolated from the same patient during periods of inflammation harbored CTn214 integrated into the chromosome or both a circular, multi-copy, extrachromosomal region of the CTn214 containing tetQ and the corresponding integrated form. The tetracycline-dependent mechanism for the transmission of CTn214 is nearly identical to a common conjugative transposon found in the genome of B. fragilis (CTnDOT), but the autonomously amplified nature of a circular 17,044-nt region of CTn214 that codes for tetQ and the integration of the same sequence in the linear chromosome within the same cell is a novel observation. Genome and transcriptome sequencing of B. fragilis cultivars grown under different concentrations of tetracycline and ciprofloxacin indicates that tetQ in strains containing the circular form remains actively expressed regardless of treatment, while the expression of tetQ in strains containing the linear form increases only in the presence of tetracycline.IMPORTANCEThe exchange of antibiotic production and resistance genes between microorganisms can lead to the emergence of new pathogens. In this study, short-read mapping of metagenomic samples taken over time from the illeal pouch of a patient with ulcerative colitis to a Bacteroides fragilis metagenome-assembled genome revealed two distinct genomic arrangements of a novel conjugative transposon, CTn214, that encodes tetracycline resistance. The autonomous amplification of a plasmid-like circular form from CTn214 that includes tetQ potentially provides consistent ribosome protection against tetracycline. This mode of antibiotic resistance offers a novel mechanism for understanding the emergence of pathobionts like B. fragilis and their persistence for extended periods of time in patients with inflammatory bowel disease.

Keywords: Bacteroides fragilis; CTnDOT; antibiotic resistance; conjugal transposon; host–microbe interactions; microbial evolution.

Fig 1

Bacteroides fragilis RAST annotation of CTn214 linear form and coverage profiles of short read mapping to CTn214. CTn214 begins with the purple integrase protein, followed by several green hypothetical proteins; the blue annotations are the putative excision operon, followed by the turquoise genes involved in excitation, orange genes for mobilization, purple conjugation genes (TraA-TraG), yellow BtgA and BtgB plasmid mobilization genes, and purple conjugation genes (TraG-TraQ). The black histogram of coverage for each base is displayed for each sample from patient 214. The earliest visit is shown at the top “5 luminal,” and subsequent visits of the longitudinal sampling are plotted below. The date since pouch activation corresponding to the longitudinal sample number is found in Table S2. The average coverage of each nucleotide position of the 55-kbp conjugative transposon is displayed on the left.

Fig 2

MAUVE alignment and gene annotation of CTn214, B. fragilis conjugative transposons derived from cultivars, and Alloprevotella tannerae cultivar. Genes are shown as different color blocks, and the color of the block corresponds to the categories of genes outlined in Fig. 1.

Fig 3

The average read coverage of single copy, 16S and 23S, and the tetQ genes are compared using box whisker plots for longitudinal samples collected from patient 214. The timepoint and source (luminal or mucosal) are displayed above each plot.

Fig 4

Three regions of the CTn (A, B, C) that are targeted for DNA sequence confirmation. (A) The primers are designed to amplify a short fragment of DNA that spans the gap between the TetQ and integrase genes that exists only if the region is circularized. (B) Primer design to confirm the left flanking region of the clindamycin/tyrosine site-specific recombinase gene insert into the CTn machinery. (C) Primer design to confirm the right flanking region of the clindamycin/tyrosine site-specific recombinase gene insert into the CTn machinery.

Fig 5

Genomic and transcriptomic sequencing results for two separate cultivars isolated from patient 214, grown in the presence of tetracycline. The annotation of CTn214 is provided at the top of the figure. The panels below the annotation describe the genomic (DNA) or transcriptomic (RNA) average coverage of each nucleotide position of the CTn214 recovered from patient 214 visit 8. The coverage of tetQ is shown between the two dotted vertical lines. The coverage displayed was calculated by mapping reads to the visit 8 CTn214 (genome—p214_V8GG_col2_contigs) for all tetracycline experiments, regardless of concentration.