Extended Data Fig. 6: Evolutionary repurposing of TFs.

All-vs-all comparison of gene sets targeted by a given TF in each species reveals distinct clusters of conservation in different bacterial clades, suggesting TF functional rewiring. An ancestral form of the E. coli TF AscG appears to have diverged to regulate new metabolic functions in the Pseudomonas clade, while the DNA binding specificity has been maintained. P-values were determined by comparing to a mock set of target genes randomly selected from each genome for 10,000 iterations (see Methods for details). (a) The E. coli TF AscG provides an example of two distinct clusters of target genes: one cluster mainly limited to the Enterobacteria, and another extending across the genus Pseudomonas and into the class β-Proteobacteria. (b) A closer inspection of the AscG target operons in the model organisms E. coli MG1655 and P. aeruginosa PAO1, along with predicted orthologs of these genes in other species, suggests that the TF’s function has diverged between the two clusters. Genes are colored according to their orthogroup: E. coli genes and orthologs in solid colors, and those of P. aeruginosa with stripes. Functional predictions or gene names from RefSeq annotations are shown in legend. (c) Comparison of the Pseudomonas aeruginosa PAO1 PtxS DNA binding sequence motif (top) to that of the Escherichia coli MG1655 TF AscG (bottom) shows high similarity (p-value = 2e-7 as calculated by Tomtom).(d) Despite the nearly identical binding motifs, alignment of the AscG and PtxS amino acid sequences reveals they only share an average 24% amino acid identity across the entire protein sequence (95% coverage). The helix-turn-helix DNA binding domain is conserved at a higher 43% identity, while the C-terminal ligand binding domain shows only 21% amino acid identity. While AscG is known to be induced by the ligand salicin, PtxS is induced by 2-ketogluconate.