Escherichia coli isolate for studying colonization of the mouse intestine and its application to two-component signaling knockouts

Abstract

The biology of Escherichia coli in its primary niche, the animal intestinal tract, is remarkably unexplored. Studies with the streptomycin-treated mouse model have produced important insights into the metabolic requirements for Escherichia coli to colonize mice. However, we still know relatively little about the physiology of this bacterium growing in the complex environment of an intestine that is permissive for the growth of competing flora. We have developed a system for studying colonization using an E. coli strain, MP1, isolated from a mouse. MP1 is genetically tractable and does not require continuous antibiotic treatment for stable colonization. As an application of this system, we separately knocked out each two-component system response regulator in MP1 and performed competitions against the wild-type strain. We found that only three response regulators, ArcA, CpxR, and RcsB, produce strong colonization defects, suggesting that in addition to anaerobiosis, adaptation to cell envelope stress is a critical requirement for E. coli colonization of the mouse intestine. We also show that the response regulator OmpR, which had previously been hypothesized to be important for adaptation between in vivo and ex vivo environments, is not required for MP1 colonization due to the presence of a third major porin.

FIG 1

Colonization of mice with E. coli strain MP1. (A) Schematic of the colonization protocol. Mice were fed streptomycin (Strep.) in their drinking water for 72 h. The water with streptomycin was then removed and replaced with antibiotic-free water, and there was no further antibiotic treatment. After an additional 24 h, mice were orally inoculated with approximately 10⁹ CFU of E. coli cells by gavage. (B) Circles show data for 16 mice colonized with MP7, an mcherry-marked MP1 derivative (MP1 att_λ::pML8). CFU were measured on the indicated days following inoculation. Triangles show measurements of total E. coli (Lac⁺ colonies on MacConkey plates) for three mice that were not treated with streptomycin and were not exposed to MP1. (C) Colonies of MP7 and MP13, a gfp-marked MP1 derivative (MP1 att_λ::pAS07). The image is an overlay of red and green fluorescence images of colonies growing on LB agar containing 15 μg/ml tetracycline.

FIG 2

Mouse colonization competitions between MP1 and HS or Nissle. Each symbol represents CFU measurements for a single mouse, taken at the indicated days postinoculation. The dashed lines indicate the detection limit. The strains used for colonization were MP7 (MP1 att_λ::pML8), HS att_λ::pAS07, and Nissle att_λ::pAS07.

FIG 3

MLST-based tree of 67 E. coli strains. The gray boxes denote each phylogenetic group, and the ST of each strain is indicated.

FIG 4

Mouse colonization competitions between response regulator deletion strains and wild type. The competitive index (CI) is shown for competitions with the indicated response regulator deletion. Response regulator deletions were in the strain MP13 (MP1 att_λ::pAS07) and competed against the marked wild-type strain MP7 (MP1 att_λ::pML8). Each symbol represents measurements for a single mouse, taken at least 20 days postinoculation, and the horizontal bars indicate the geometric means. The lower dashed line is the detection limit (10⁻⁴).

FIG 5

Complementation of arcA, rcsB, and arcA deletions in mouse colonization competition assays. The complemented genes were inserted at the phage HK022 attachment site, as described in Materials and Methods. Each symbol represents an individual mouse, and the bars indicate the geometric means. The competitive indices were determined from the numbers of CFU in feces at 21 days (cpxR and arcA) or 14 days (rcsB) postinoculation.

FIG 6

Outer membrane profiles for wild-type (WT) strains and the indicated mutants. Outer membranes were resolved by urea–SDS-PAGE followed by staining with Coomassie brilliant blue.

FIG 7

Mouse colonization competitions between WT MP1 and the ΔnmpD and ΔnmpD ΔompR strains. Each symbol denotes an individual mouse, and the bars indicate the geometric means. The competitive indices were determined from numbers of CFU in feces at 21 days postinoculation.