Fig 1.
Genomic inversions are detected by clustering unusual read pairs.
(A) DNA inversion results in read pairs with same relative orientation and increased inner-mate distance. The invariable DNA segment is illustrated by a white box and the invertible segment by a black-and-white gradient box. Individual reads are illustrated for two distinct regions by colored arrows. (B) Top cartoon: reads that overlap the junction of invertible sites are trimmed either on the 5-prime end (dark green) or 3-prime end (light green) during the alignment step. The trimmed portion of the reads are shown as dashed. Only the left side of the inversion site is illustrated. Bottom cartoon: histogram illustration of the frequency of starting position of all trimmed reads. Read trimmed at 5-prime end all start at the same genomic position and present high enrichment. Starting position of 3-prime end trimmed read is variable and present low enrichment.
Fig 2.
C. difficile R20291 genome harbors seven inversion sites.
(A) Seven distinct clusters (black circles) composed of unidirectional read pairs are detected in C. difficile R20291 genome. Each cluster is numbered following a previously described convention. (B-H) Illustration of unidirectional reads enrichment relative to the genomic on-scale context. The y axis represents read number and the x axis represent genomic position. Associated open reading frames are shown with arrowed boxes and labeled by the numeric value from their respective locus_tag number. The position of the invertible segment is illustrated by the dark green box relative to the gene content. The enrichment of 5-prime end clipped reads is shown in the separate plot at the top right corner. The two major dark green bands correspond to left and right boundary of the invertible site.
Fig 3.
Terminal inverted DNA repeats border inversion sites in C. difficile.
LIR stands for “left inverted repeat” and RIR stands for “right inverted repeat”. Gray shading illustrates sequence homology. The asterisk (*) points to the nucleotide where recombination is taking place as identified by enrichment of 5-prime end clipped reads.
Fig 4.
Detection and quantification of inversion events in C. difficile.
(A) Schematic representation of orientation-specific PCR and qPCR strategies. Invariable DNA region is illustrated by white and invertible region by black-and-white gradient box. Primers used for PCR amplification are shown as arrows. (B) The agarose gel showing PCR products for either published (Pub) or inverted (Inv) orientation. For Cdi4 and Cdi1, permissive (ON) and restrictive (OFF) orientations for downstream gene expression have been determined elsewhere. DNA from 3 biological replicates was tested and one representative example is shown. (C) Quantification of inverted orientation relative to the published orientation by qPCR. Presented are means from 3 biological replicates. Error bars correspond to 2 standard deviations.
Fig 5.
Expression of CDR20291_3128 is consistent with phase variation.
Liquid culture samples from C. difficile R20291 and ∆3128::SNAP-tag were analyzed by light and fluorescence microscopy following staining with SNAP-Cell TMR-Star substrate. The left column shows the view of total cells in bright field light (BF), the middle column shows the SNAP-tag expressing cells pseudocolored in red, and the right column shows the merged pictures of BF and fluorescence.
Fig 6.
Impact of recV loss on orientation of invertible sites in C. difficile.
Agarose gels showing PCR products from published (Pub) and inverted (Inv) orientations in the wild type (WT), the recV null mutant (RecV-) and following complementation from a plasmid (RecV+).
Fig 7.
Impact of recV overexpression on the orientation frequency in C. difficile.
recV was overexpressed from a plasmid in the wild type background. The y axis represents the percentage of inverted orientation relative to published orientation as determined in the wild type context carrying the empty pRPF185 plasmid (black filled circles) and during recV overexpression (orange filled squares) by qPCR. Reported are medians, upper and lower quartiles and extremes.