Elucidation of quantitative structural diversity of remarkable rearrangement regions, shufflons, in IncI2 plasmids

Abstract

A multiple DNA inversion system, the shufflon, exists in incompatibility (Inc) I1 and I2 plasmids. The shufflon generates variants of the PilV protein, a minor component of the thin pilus. The shufflon is one of the most difficult regions for de novo genome assembly because of its structural diversity even in an isolated bacterial clone. We determined complete genome sequences, including those of IncI2 plasmids carrying mcr-1, of three Escherichia coli strains using single-molecule, real-time (SMRT) sequencing and Illumina sequencing. The sequences assembled using only SMRT sequencing contained misassembled regions in the shufflon. A hybrid analysis using SMRT and Illumina sequencing resolved the misassembled region and revealed that the three IncI2 plasmids, excluding the shufflon region, were highly conserved. Moreover, the abundance ratio of whole-shufflon structures could be determined by quantitative structural variation analysis of the SMRT data, suggesting that a remarkable heterogeneity of whole-shufflon structural variations exists in IncI2 plasmids. These findings indicate that remarkable rearrangement regions should be validated using both long-read and short-read sequencing data and that the structural variation of PilV in the shufflon might be closely related to phenotypic heterogeneity of plasmid-mediated transconjugation involved in horizontal gene transfer even in bacterial clonal populations.

Expanding landscapes of the diversified mcr-1-bearing plasmid reservoirs

“…Although this type of plasmids lacks the insertion sequence IS Apl1 (Additional file 2: Figure S2), they consistently share a site-specific recombinase ( rci ) gene, two pilus-encoding genes ( pil and tra ), and four different pilli proteins (Additional file 2: Figure S2). In addition, we found that heterogeneity of whole-shufflon structural variations exists between the 3′ region of pilV gene and rci gene in mcr-1 -harboring IncI2 plasmids as recently reported [62]. The plasmid sequences yielded the following results: in pWH9-3 and pWH7-3, rci -A-A′-C-C′-B′-D′- pilV ; in pWH13-4, rci -A-A′-C-C′-D′-B′- pilV ; in LS142-1, rci -B′-D′-A-A′- pilV ; in 65-3, rci -A′-A-D′-B′-C-C′-B′-D′-A-A′- pilV ; in pGD23-3, rci -C-C′-D′-B′-A′-A-B′-D′- pilV ; in pGD53-3, rci -C-C′- B′-D′-D′-B′-A′-A- pilV ; and in hybrid plasmid pGD17-2, rci -C-C′-A-A′-D′-B′- pilV .…”

Pushing the limits of de novo genome assembly for complex prokaryotic genomes harboring very long, near identical repeats

“…Importantly, a highly conserved sequence motif of the inverted repeats could be identified for this shufflon system ( Figure 4C), where only the bases at position 14 and 16 of the 19 bp motif showed variation. The motif sequence was closely related to a repeat reported for an Inc2 plasmid shufflon (49) .…”

“…Illumina" track, Figure 4A) (49) . Importantly, rearrangements for most inverted repeat sites between the two recombination variants were observed (black arrows, Figure 4B), except for two repeat positions which did not show any rearrangement (white arrows).…”

Selective and non‐selective bottlenecks as drivers of the evolution of hypermutable bacterial loci

“…The ubiquity of mobile integrons, containing almost exclusively antimicrobial resistance genes, within healthcare settings demonstrates the importance of integrons for adaptive environmental responses. Shufflons are another site‐specific recombination system that can generate diversity in expression of specific genes (Sekizuka et al, ). The incompatibility (Inc) plasmids, I1 and I2, contain partial open reading frames for up to four different pilV alleles that can be rearranged by the recombinase Rci using a conserved region.…”

“…The incompatibility (Inc) plasmids, I1 and I2, contain partial open reading frames for up to four different pilV alleles that can be rearranged by the recombinase Rci using a conserved region. Variation in the expressed PilV protein contributes to recognition of recipient cells during plasmid transmission and hence facilitates plasmid exchange within and between a wide range of Enterobacteriaceae species (Sekizuka et al, ).…”