Fig. 3: Genetic and functional analysis of AA usage ex vivo.

a Initial hits from the MGD screen (see Supplementary Fig. 3a) were tested in the same conditions, and competitive index (C.I.) was calculated by dividing mutant/wt CFUs in each well. n = 5 mice, mean ± SD. Only mutants with a p < 0.05 by one-sample two-tailed t-test versus C.I. of 0 are shown. Arrows indicate the suspected or confirmed (bold) single-gene cause of the defect. Mutants with growth defects in minimal medium (see Supplementary Fig. 4) are indicated with filled circles. b Cecal homogenates were prepared from n = 4 mice and left whole or filtered. Filtrates with or without S. Tm and whole homogenates were incubated anaerobically at 37 °C. At 12 h, all samples were centrifuged, supernatants filtered, and AAs quantitated by targeted capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS). Levels in the sterile control filtrate incubation were subtracted from each sample and mean ± SD are shown. P values are the result of one-sample two-tailed t-test versus 0. c Cecal homogenates were left whole, diluted 32x in their own filtrate, filtered, or had 9 AAs or Asn/Lys/Ser added (as in Fig. 2f). Then the indicated mutants were grown in competition with the wt parent strain and CFUs at 16 h counted to calculate the C.I. Cecal contents from n = 9 mice, combined from multiple experiments. Bars are mean ± SD. P values are the result of one-sample two-tailed t-test versus 0. See also Supplementary Fig. 3d. Source data are provided as a Source Data file.