Fig. 2. Identification of the toxin-encoding gene on a mobile plasmid.

a Agar overlay assays of toxin-producing strain PvCL04 and two transposon mutants that abrogate its ability to inhibit the growth of PvCL10 and PvCL09. b Genetic context of the gene into which the two transposons inserted. c Agar overlay assays showing that deletion of the gene identified by transposon mutagenesis from strains PvCL04 and PdCL02 abrogates toxin activity, which is restored when the gene is added back in trans to these strains. d Agar spot overlay assay showing that placement of CS034_04058 in trans in B. thetaiotaomicron VPI 5482 confers toxin activity to the strain. e Orf map of the contig of the sequenced genome of PvCL04 containing CS034_04058. Primers used to determine if the contig is a circular plasmid and to identify the toxin-producing gene in other strains are shown. Sites of the transposon insertions of mutants Tn10 and Tn12 are shown. An insertion sequence present in a similar contig of strain PvCL10 is shown under the map with its insertion site mapped relative to the contig of PvCL04. f EtBr-stained gel showing the results of the PCR using the primers shown in panel E with each of the 18 P. vulgatus and P. dorei strains listed in Fig. 1a. PCR analyses were performed twice. g The circular 9117 pBCPT plasmid of strain PvCL04 showing the portion of the contig that is similar to the Bacteroidales mobile plasmid pBUN24 (pink outer line). The gene shown in green encodes a plasmid replication protein, the genes in blue encode mobilization proteins, and the genes in orange encode a putative toxin-antitoxin system. h Overlay assays showing that B. fragilis ΔT6SSermG and B. ovatus D2 that conjugally received pBCPT-tetQ-ttr from PvCL04 pBCPT-tetQ-ttr acquire the ability to inhibit the growth of PvCL10. All overlay assays were performed at least twice. Source data are provided as a Source Data file.