Fig 1.
Region of increased coverage observed in WT BtE264 re-sequencing analyses.
(A) Graphical representation of genomic coverage after mapping NGS reads to chromosomes I and II of the BtE264 reference genome. Left end of the region with increased coverage in chromosome I is marked as α, and the right end as β. Location of all six IS2-like elements (light blue), csu operon (purple), and bcpAIOB operon (red) are indicated with arrowheads. (B) Diagram depicting a closer view of the beginning and end of the sequence with increased coverage. Elongated pentagons represent genes outside (light grey) or inside (dark blue) of the high-coverage region. IS2-like elements are composed of orfAB (light blue) flanked by two inverted repeats (yellow). The single nucleotide variation between IS2α and IS2β is shown as a red asterisk. (C) Alignment using ClustalW of the IS2 right and left imperfect repeats. Shaded nucleotides are conserved in both repeats.
Fig 2.
The bcpAIOB Locus is part of a composite transposon that forms an extrachromosomal megacircle.
(A) Schematic representation of binding sites for primers Circ1 and Circ2 in the chromosome, as well as the IS2-like megacircle. (B) Agarose gel electrophoresis analyses show detection of the megacircle junction in WT BtE264 by PCR. (C) Diagram of the IS2-like megacircle junction based on sequencing data. (D) Diagram of the putative IS2-like composite transposon. The predicted IS2 translocatable unit (TU) is shown below.
Fig 3.
Megacircle production is disrupted in the absence of orfABβ.
(A) PCR analyses to detect the megacircle junction using primers Circ1 and Circ2 (top), or primers that bind within the putative composite transposon (bottom), in WT and strains with defective IS2α or IS2β elements. During construction of IS2α::nptII and IS2β::nptII, several kanamycin-resistant colonies were analyzed; a representative strain is shown. (B) Graphical representation of detection (or lack of) of a PCR product with primers Circ1 and Circ2 from an extrachromosomal megacircle (top) compared to detection of a product from the interphase of multiple copies of the element present in tandem repeats.
Fig 4.
PCR analyses to detect the megacircle junction in BtE264 mutant strains.
Detection of the megacircle junction using primers Circ1 and Circ2 (top) or primers that bind within the putative composite transposon (bottom) in WT and mutant strains lacking the bcpAIOB locus (Δbcp) or producing catalytically inactive (BcpAEKAA) or chimeric (Bt-bp) BcpA.
Fig 5.
Megacircle production and community-associated behaviors, but not CDI, are disrupted in the absence of orfABβ.
(A) Models illustrating the possible correlations between CDS and megacircle formation. Delivered C-term of the BcpA toxin, dBcpA-CT. (B) Contact-dependent growth inhibition (CDI) is not affected by disruption of IS2α or IS2β. Inhibitors (WT, IS2α::nptII or IS2β::nptII) were co-cultured with ΔbcpAIOB (a strain susceptible to killing via CDI) for 24 hours then plated on selective media to calculate the competitive index (C.I.). When only WT bacteria were recovered, the data point is displayed in red; in this case the actual C.I. is greater than or equal to the represented value. Differences in C.I. are not statistically significant. (C) Ability of WT, IS2α::nptII or IS2β::nptII to display phenotypes linked to community-associated behaviors. These phenotypes include: production of pigments by colony biofilms (top), autoaggregation in M63 minimal medium (middle), or the proportion of Congo red (CR) binding colonies. P values were obtained using Mann-Whitney U test comparing mutant strains to WT. Results are shown as mean +/- SEM of three independent experiments (n = 6). **P < 0.01.
Fig 6.
Evidence for intracellular movement of the IS2-like megacircle.
(A) Diagram depicting the strategy used to replace the first ~48 kb (Region 1) of the bcpAIOB-containing putative composite transposon (dark blue) with a cassette containing the gene that confers kanamycin resistance, nptII, and FRT-binding sites (pink). (B) PCR analyses to confirm removal of Region 1 in strain Reg1::nptII using primers P1 and P2 (shown as green arrows in panel A) which are close enough to generate a product only after deletion of the ~48 kb region. (C) PCR analyses of WT BtE264 and strain Reg1::nptII using primers P7 and P8, which would amplify two genes from within the Region 1 sequence. (D) Integration of a suicide plasmid (red arrowhead) within the Region 1 sequence of the mobilized element, followed by plasmid rescue studies, suggests that the mobilized megacircle inserted adjacent to the truncated composite transposon carrying Reg1::nptII mutation. (E) Comparison of the transformation efficiencies upon deletion of Region 1 in strains that are positive (WT) or deficient (ΔIS2β and Bt-Bp chimera) in the production of the megacircle. No DNA, black bars; DNA to introduce the Reg1::nptII mutation, red bars; DNA to introduce an nptII cassette outside of the putative composite transposon, blue bars; DNA to introduce an nptII cassette inside the putative composite transposon, green bars. Horizontal dashed line represents the average lowest limit of detection. P values were obtained using Mann-Whitney U test comparing mutant strains to WT when the same DNA (or no DNA) was added. Results are shown as mean +/- SEM of three independent experiments with three technical replicates each (n = 9). *P < 0.05; **P < 0.01.
Fig 7.
The IS2-like Translocatable unit mobilized to chromosome II in the 131–10 strain.
(A) Graphical representation of the region in chromosome 1 where the bcpAIOB-containing composite transposon is located (top). Graphical representation of strain 131–10 in which the entire composite transposon has been replaced by an nptII cassette (bottom). (B) PCR analyses to confirm removal of the MGE in strain 131–10 using primers P5 and P6 (shown as green arrows in panel A). PCR analyses detect DNA corresponding to genes within the composite transposon in strain 131–10. (C) IS2β5 in chromosome II in a WT background (top), or upon insertion of the IS2-like translocatable unit (bottom). PCR analyses of WT and strain 131–10 to confirm the insertion of the IS2-like translocatable unit into chromosome II. The distance between the binding site of primers β5-F and β5-R (grey arrows) is approximately 1.8 kb in WT DNA. Primers Circ1 and Circ2 (red arrows) bind specifically to the ends of the mobilized bcpAIOB-containing element. The insertion site of pABT73-TMP is denoted with a black arrowhead.