Previous studies based on whole genome sequencing data using PAML have not identified aes to be under positive selection [17, 18]. Visual comparison of the phylogenetic history of aes with that of the six concatenated housekeeping genes, reflecting the species phylogeny, revealed a similar topology with four main phylogenetic groups (Fig. 2). Indeed, all strains belonging to the B2 Luminespib phylogenetic group were clustered in a monophyletic group (bootstrap 99%) with ECOR 66 at

its base, as observed in the MLST tree. Likewise, two sub-groups were observed for phylogenetic group D, one of which was associated with the phylogenetic group B2 (ECOR 35, 36, 38, 39, 40, 41) (bootstrap 85%), also observed in the MLST tree. Phylogenetic group A also constituted two sub-groups, although these were not sister groups. By contrast, the B1 phylogenetic group was monophyletic overall, with only two strains (ECOR 4 and ECOR 47) clearly misclassified (Fig. 2). Figure 2 Phylogenetic trees for the 72 ECOR strains and six E. coli reference strains. The trees were

constructed from (A) aes sequences and (B) multi-locus sequence typing of STI571 mw six housekeeping genes representing the species phylogeny (trpA, trpB, pabB, putP, icd and polB) [5], obtained using PHYML procedure [50]. E. fergusonii was used as an outgroup. Bootstraps are shown for values higher than 70%. Strains studied and belonging to phylogenetic groups A (blue boxed), B1 (green boxed), B2 (red boxed), D (yellow boxed) and UG (white boxed) are indicated. We used a recently developed technique (“”TreeOfTree”") allowing the level of congruence between phylogenetic trees to be tested [19]. We tested each individual housekeeping gene tree, the MLST tree, and Carbohydrate the aes tree. All the bootstraps are low enough (less than 67%) to suggest that all the gene trees can be view as not incongruent, the aes gene tree itself clustering with pabB and trpA

gene trees with very low bootstrap (44%) (Fig. 3). Thus, aes tree topology showed that aes is a powerful marker of the species phylogeny, as observed for each housekeeping gene used in the MLST scheme. Figure 3 Tree representing the distance matrix generated from comparisons between gene tree structures. Gene tree structure comparisons were between trees based on aes sequences, six individual housekeeping genes (trpA, trpB, pabB, putP, icd and polB) and multi-locus sequence typing (concatenation of the six housekeeping genes), with distances derived from path-length difference. Numbers are bootstraps. Aes B1 and B2 protein variants were then compared by protein modelling. We found that residues S 157, D 254 and H 284 had a geometry similar to that of the esterase catalytic site.