Extended Data Fig. 9: DoTA-seq elucidates diverse genetic subpopulations in B. fragilis generated via promoter inversion.

(a) Schematic of the potential diversity generated by B. fragilis CPS operons. There are a total of 8 CPS operons referred to as A-H, 7 of which contain promoters flanked by inverted repeats (triangles). These promoters switch ON and OFF through recombination at the inverted repeats driven by an endogenous recombinase (mpi). (b) Schematic of DoTA-seq reaction with primers designed to flank all 7 invertible promoters. Primers are represented by half-headed arrows. Gray vertical lines represent region of complementarity between amplicons and barcodes. P5 and P7 represent the Illumina sequencing adaptor sequences. (c) Bar plot of the relative frequencies of unique CPS promoter states in a single B. fragilis colony. Promoter states are represented by a 7-letter code, where the letters (A-H) denote that a given promoter is turned ON, and ‘-‘ denote the given promoter is turned OFF. Data points represent technical replicates. Error bars represent 1 s.d. from the mean of technical replicates (n = 3). Bar height represents the mean. Since a subset of combinatorial promoter states were rare in the population and not observed in all technical replicates, we computed the stochastic limit of detection (Supplementary Note 4). (d) An undirected graph network representation of the CPS promoter state subpopulations in (c). Nodes represent CPS promoter combinatorial states where diameter is proportional to relative frequency, and edges connect nodes that are one promoter flip away from each other. (e) Network representation of the measured combinatorial promoter states where the recombinase (mpi) responsible for promoter inversions was deleted. In this strain, the entire population is locked in a single state (A–E-H). The diameter of the node and edges are the same as (d).