Requirement of purine and pyrimidine synthesis for colonization of the mouse intestine by Escherichia coli

Abstract

To study the adaptation of an intestinal bacterium to its natural environment, germfree mice were associated with commensal Escherichia coli MG1655. Two-dimensional gel electrophoresis was used to identify proteins differentially expressed in E. coli MG1655 collected from either cecal contents or anaerobic in vitro cultures. Fourteen differentially expressed proteins (>3-fold; P < 0.05) were identified, nine of which were upregulated in cecal versus in vitro-grown E. coli. Four of these proteins were investigated further for their role in gut colonization. After deletion of the corresponding genes, the resulting E. coli mutants were tested for their ability to colonize the intestines of gnotobiotic mice in competition with the wild-type strain. A mutant devoid of ydjG, which encodes a putative NADH-dependent methylglyoxal reductase, reached a 1.2-log-lower cecal concentration than the wild type. Deletion of the nanA gene encoding N-acetylneuraminate lyase affected the colonization and persistence of E. coli in the intestines of the gnotobiotic mice only slightly. A mutant devoid of 5'-phosphoribosyl 4-(N-succinocarboxamide)-5-aminoimidazole synthase, a key enzyme of purine synthesis, displayed intestinal cell counts >4 logs lower than those of the wild type. Deletion of the gene encoding aspartate carbamoyltransferase, a key enzyme of pyrimidine synthesis, even resulted in the washout of the corresponding mutant from the mouse intestinal tract. These findings indicate that E. coli needs to synthesize purines and pyrimidines to successfully colonize the mouse intestine.

FIG. 1.

Time course of the competition experiment between nanA mutant and wild-type E. coli. Individually housed germfree mice were associated with equal numbers of WT (white diamonds) and mutant (black squares) bacteria. At the indicated time points, fecal samples were collected and analyzed by plate counting for wild-type and mutant cell numbers. Data are expressed as means ± standard deviations for an experiment with 4 mice. The asterisks indicate significant differences, with P values of ≤0.05. dw, dry weight.

FIG. 2.

Analysis of the intestinal cell numbers at the end of the competition experiment between the nanA mutant and wild-type E. coli. White bars, WT; gray bars, nanA mutant. Data are expressed as means ± standard deviations for an experiment with 4 mice. The asterisks indicate significant differences, with P values of ≤0.05.

FIG. 3.

Time course of the competition experiment between the ydj operon deletion mutant and wild-type E. coli. Individually housed germfree mice were associated with equal numbers of WT (white diamonds) and mutant (black squares) bacteria. At the indicated time points, fecal samples were collected and analyzed by plate counting for wild-type and mutant cell numbers in order to follow the colonization process through time. Data are expressed as means ± standard deviations for an experiment with 4 mice. The asterisks indicate significant differences, with P values of ≤0.05.

FIG. 4.

Analysis of the intestinal cell numbers at the end of the competition experiment between the ydj operon mutant and wild-type E. coli. White bars, WT; gray bars, ydj operon mutant. Data are expressed as means ± standard deviations for an experiment with 4 mice. The asterisk indicates a significant difference, with a P value of ≤0.05.

FIG. 5.

Time course of the competition experiment between the purC mutant and wild-type E. coli. Individually housed germfree mice were associated with equal numbers of WT (white diamonds) and mutant (black squares) bacteria. At the indicated time points, fecal samples were collected and analyzed by plate counting for wild-type and mutant cell numbers in order to follow the colonization process through time. Data are expressed as means ± standard deviations for an experiment with 4 mice. The asterisks indicate significant differences with P ≤ 0.05.

FIG. 6.

Analysis of the intestinal cell numbers at the end of the competition experiment between the purC mutant and wild-type E. coli. White bars, WT; gray bars, purC mutant. Data are expressed as means ± standard deviations for an experiment with 4 mice. The asterisks indicate significant differences, with P values of ≤0.05.

FIG. 7.

Time course of the competition experiment between the pyrBI mutant and wild-type E. coli. Individually housed germfree mice were associated with equal numbers of WT (white diamonds) and mutant (black squares) bacteria. At the indicated time points, fecal samples were collected and analyzed by plate counting for wild-type and mutant cell numbers in order to follow the colonization process through time. Black squares with down arrows indicate sampling points with mutant cell numbers below the detection limit of 10³ CFU per g dry feces. Data are expressed as means ± standard deviations for an experiment with 4 mice. The asterisks indicate significant differences, with P values of ≤0.05.

FIG. 8.

Analysis of the intestinal cell numbers at the end of the competition experiment between the pyrBI mutant and wild-type E. coli. White bars, WT. Black squares with down arrows indicate mutant cell numbers below the detection limit of 10³ CFU per g dry feces. Data are expressed as means ± standard deviations for an experiment with 4 mice. The asterisks indicate significant differences, with P values of ≤0.05.

FIG. 9.

Concentrations of uracil (diamonds), uridine (squares), thymidine (circles), and guanosine (triangles) in the small intestines of monoassociated mice.

FIG. 10.

Development of cell titers in a competition experiment between E. coli MG1655 (diamonds) and MG1655ΔpyrBI (triangles). Minimal media supplemented with 25 μM (open symbols) and 50 μM uracil (closed symbols) were inoculated with equal cell numbers of the two strains. Every 12 h, the cell numbers were determined and 0.5% of the cell culture was transferred to fresh medium.

FIG. 11.

Optical densities (OD₄₂₀) of pure cultures as determined after 24 h of growth in minimal media supplemented with the indicated concentrations of uracil. Diamonds, MG1655; triangles, MG1655ΔpyrBI.