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. Author manuscript; available in PMC: 2022 Sep 8.
Published in final edited form as: Cell Host Microbe. 2021 Sep 1;29(9):1454–1468.e4. doi: 10.1016/j.chom.2021.08.003

Figure 3: Similar mutations are repeatedly selected for in the mouse gut, likely driven at least in part by the presence of raffinose, an abundant carbon source in the gut.

Figure 3:

A) All S1 strains except S1–36 were used to gavage 9 germ-free mice in three cages on day 0 and tracked via daily fecal sampling. Shown are relative abundances (top) and mutations identified from metagenomic sequencing (bottom) of the barcoded E. coli populations from a representative mouse in each cage over the first 20 days of colonization. Different barcodes emerged in each cage, with similar mutations in cage 1 and 2 but not cage 3.

B) By day 20, relative abundances of the S136− barcodes were similar within each cage, despite differences across cages.

C) Map of mutations identified via whole-genome sequencing of 189 S1–36 clones isolated from various mice organized by cage throughout the experiment. The most prevalent mutations, which include the [flhE-flhD] deletion (purple) and mutations mostly in metabolism-related genes (green), are labeled with the associated gene. Yellow regions highlight a lon promoter and ycjW alleles that co-occurred in multiple cages, indicating parallel evolution. The red region highlights a lacIG272V mutation that co-occurred with the lon promoter mutation in cage 2 mice and hence is more likely due to within-cage transfer than parallelism.

D) Growth curves of the wild-type parent, evolved S1–36, a lacI* mutant, and an [flhD-flhE] mutant in M9 supplemented with four carbon sources. In lactulose, lactitol, and raffinose, evolved S1–36 and the lacI* mutant grew much faster and with a shorter lag than the parent or the [flhD-flhE] mutant; in lactitol and raffinose, the parent and [flhD-flhE] mutant could not grow at all, suggesting that the ability to utilize raffinose is conferred by the lacI* mutation. In β-D allose, the parent grew better than the lacI or [flhD-flhE] mutants; evolved S1–36 exhibited no growth and hence is not shown.

E) Concentrations of various amino acids and carbon sources in the feces and ceca of germ-free mice measured using NMR. Feces and ceca exhibited high concentrations of raffinose and stachyose (boxes, n=3 mice).

F) The number of mutations detected in S1–36 clones increased over the duration of the experiment, suggesting continued selection. Data are mean values and error bars represent 1 standard deviation, with n=10–38 isolates on each day.