Dissecting the genetic components of adaptation of Escherichia coli to the mouse gut

Abstract

While pleiotropic adaptive mutations are thought to be central for evolution, little is known on the downstream molecular effects allowing adaptation to complex ecologically relevant environments. Here we show that Escherichia coli MG1655 adapts rapidly to the intestine of germ-free mice by single point mutations in EnvZ/OmpR two-component signal transduction system, which controls more than 100 genes. The selective advantage conferred by the mutations that modulate EnvZ/OmpR activities was the result of their independent and additive effects on flagellin expression and permeability. These results obtained in vivo thus suggest that global regulators may have evolved to coordinate activities that need to be fine-tuned simultaneously during adaptation to complex environments and that mutations in such regulators permit adjustment of the boundaries of physiological adaptation when switching between two very distinct environments.

Figure 1. A New Colony Morphotype Is Rapidly Selected during Colonisation

(A) Morphotypes observed on motility plates: large smooth (LS; similar to the ancestral MG1655 strain) and small granular (SG; selected mutant). (B) Evolution over time (in days) of SG colony forming units (mean (cfu) ± standard deviation) in the feces of 12 mice. (C) Evolution over time (in days) of the ratio of green to red cfu in the feces of mice inoculated with MG1655 ptet-GFP ompBSG1-cat (containing the SG1 envZ mutation) and MG1655 ptet-RFP ompB-cat (containing the WT envZ) mixed at initial ratios of 1:1, 1:100, and 1:1,000. The error bars represent the standard error of the mean of four mice.

Figure 2. Localization of the Selected Mutations

Mutations are depicted on a representation of EnvZ dimer, based on the transmembrane/periplasmic domain model [47] and the NMR structures of the homodimeric core [48] and the ATP-binding domain [49] with ATP bound molecules depicted in balls and sticks. Localizations of the amino acid where mutations had been identified are represented by red stars (linker and periplasmic domain) and with red side chain in the known EnvZ sub-structures. The side chain of active site His residues are in blue. Underlined amino acids are mutations previously described to increase the EnvZ kinase/phosphatase activity ratio in references [17] or [18].

Figure 3. SG Mutants Lack Flagellin and Inflammatory Properties and Exhibit an Altered Porin Profile

(A) IL-8 secretion measured by ELISA in supernatants of HT29-19A cells after a 16-h stimulation with WT, ΔfliC, SG (SG1 and 2), LS (LS1 and 2) strains, or medium alone (ctrl) (mean ± SEM for triplicates in one representative experiment out of five). (NF-κB DNA-binding activity and CCL-20 mRNA expression are presented in Figure S3). (B) Immunoblotting with an antibody against E. coli flagellin of proteins concentrated from culture supernatants (upper panel) or extracted from cell lysates (lower panel) of the same strains. (C) Immunoblotting with an antibody against E. coli OmpF and OmpC porins of outer membrane fraction of the same strains and control strains deleted of ompF or ompC.

Figure 4. Flagellin Expression Is Counterselected during Colonization

(A) fliC promoter activity monitored by flow cytometry: overlay fluorescence histograms in feces from germ-free mice (grey shading) and from two representative mice (see Figure S3 for the complete dataset) colonized with WT (solid lines) or ΔfliC (dotted lines) E. coli strains containing an YFP under the control of fliC promoter (pfliC-YFP), at different days post-inoculation. (B) Evolution over time (in days) of fluorescent cfu (circles) and LS cfu (filled triangle) in the feces of 12 mice inoculated with the WT pfliC-YFP strain (circles) and fluorescent cfu in the feces of 11 mice inoculated with the ΔfliC pfliC-YFP strain (square) (mean ± standard deviation).

Figure 5. In Vivo Selection of Mutations during Intestinal Colonization Results from Two Independent and Additive Effects on FliC and OmpF Expression

Evolution over time (in days) of non-fluorescent cfu in the feces of mice inoculated with the WT pfliC-YFP strain (filled triangles, n = 12), the ΔfliC pfliC-YFP strain (empty squares, n = 11), the ΔOmpF pfliC-YFP strain (empty diamonds, n = 5), or the double mutant ΔompF ΔfliC pfliC-YFP strain (crosses, n = 4) (mean ± standard deviation).

Figure 6. Deletions in the Region Downstream of flhDC Operon Are Selected for in Mice Colonized with the ΔOmpFpfliC-YFP Strain

Genetic map of the deletions detected in four non-motile clones isolated from four independent mice. All non-motile clones tested possess a deletion in the flhDC region.