Fig. 4
From: The genetic basis for the adaptation of E. coli to sugar synthesis from CO2
Leave-one-out suppressor analysis reveals the functionality of mutations in the minimal set. a The reconstructed strain has five essential mutations that enable sugar synthesis from CO2 using the non-native CBB cycle. We constructed "leave-one-out" strains, each missing one essential mutation from the minimal set of mutations that reproduce the hemiautotrophic phenotype, and grew them in permissive conditions to give a population in which many natural mutational variants exist due to DNA replication errors. The population was plated in selective conditions, where CBB activity is essential for growth (Methods), to reveal mutations that regenerate the hemiautotrophic phenotype. Colonies from the plate were sequenced to detect the compensating mutation(s). Several independent cultures (n > 4) were cultured and plated for each leave-one-out strain, and multiple colonies were sequenced from each plate. For the sake of graphical simplicity, a single plate is presented. b The compensating mutations to pgi occurred either in the pgi gene itself or in pgm, a downstream reaction to biomass (glycogen) production. Most mutations in the pgi gene are loss-of-function mutations. c Mutations in serA were located in the functional regions of the enzyme (e.g., active site and NAD-binding site). We also found that mutations in prs can serve as an alternative to the mutation in serA. d In the prs leave-one-out strain we found that compensating mutations in prs were located in the functional regions of the enzyme. Other compensating mutations are described in Supplementary Table 3. e For the crp WT strain, most compensating mutations occurred in cAMP biosynthesis and degradation, or in the crp gene itself. f ppsR WT managed to grow without further compensating mutations on solid plates, but mutations in ppsR took over the population very rapidly in liquid. The wild-type allele introduced in the leave-one-out assay are labeled red. Stars mark unique mutations. Full details for all mutations are listed in Supplementary Tables 1–5
