Fig 1.
The reactions catalyzed by ProA (γ-glutamyl phosphate reductase) and ArgC (N-acetyl glutamyl phosphate reductase).
Fig 2.
Restoration of growth rate of the ΔargC proA* strain to the level of the wild-type strain requires addition of both proline (0.4 mM) and arginine (5.2 mM).
Fig 3.
The effects of point mutations in the proBA* operon and near the head of the proA* mRNA discovered on the levels of proB (red) and proA* (gold) mRNA in strains carrying mutations discovered in the adaptive evolution experiment (A) and in strains with additional synonymous mutations created by genome editing (B). Error bars indicate 1 SD.
Fig 4.
Improvement of growth rate during evolution of four populations of the ΔargC proA* strain on M9/glucose.
Fig 5.
Mutations detected in the proBA* operon in four lineages of the ΔargC proA* strain after adaptation for approximately 250 generations in M9/glucose.
A) Locations of point mutations; B) presence of point mutations in the population at the indicated time points. Lower-case letters indicate that the mutation was present in a fraction of the population based on Sanger sequencing. Each of these mutations was subsequently detected in individual colonies from the population, as well. Upper-case letters denote mutations that had swept the population to the limit of detection by Sanger sequencing.
Table 1.
Mutations found in seven adapted clones.
Table 2.
Strains used in this work.
Fig 6.
Comparison of the growth rates of the ΔargC proA* strain in which the indicated mutations had been introduced by genome editing.
Error bars represent 1 SD.
Fig 7.
Growth rates and levels of ProA* (A) and ProB (B) in strains carrying the promoter mutation M1 and other mutations relative to those of the strain carrying just M1. Black, no other mutations in head region; blue, intergenic mutation M2; red, synonymous mutations in codon 2; white, synonymous mutation in codon 3; magenta, synonymous mutation in codon 4; cyan, synonymous mutations in codon 6; yellow, non-synonymous mutation M5. Data points representing strains with M1 and two additional mutations are shown with colored stripes corresponding to the colors used for individual mutations. Error bars represent 1 SD.
Fig 8.
Predicted lowest-free energy structure of the 53-nt fragment of the head of the wild-type proA* transcript generated by the Fold algorithm.
The Shine-Dalgarno sequence is indicated by the red box and the start codon by the blue box. The colors of the bases indicate the probability that each base is found in the depicted state.
Table 3.
Effects of mutations on growth rate relative to the parental strain containing the promoter mutation M1 and the minimal folding energy of 53-nucleotide fragments surrounding the proA* start codona.
Fig 9.
Predicted lowest-free energy structures of the region surrounding the AUG start codon (blue box) in wild-type and in mutant proA* transcripts in which synonymous mutations in codons 2 and 6 increase growth rate.
Numbers underneath the structures indicate the folding energy calculated by the Fold algorithm in kcal/mol.
Fig 10.
Predicted lowest-free energy structures of the region surrounding the AUG start codon (blue box) in wild-type and in mutant proA* transcripts in which synonymous mutations in codon 2 decrease growth rate.
Numbers underneath the structures indicate the folding energy calculated by the Fold algorithm in kcal/mol.
Fig 11.
Growth rate is proportional to the stability of the 53-nt head region of the proA* region in the ΔargC proA* strain carrying the promoter mutation M1 for strains containing synonymous mutations in codons 2 and 6.
Black dot indicates the strain that contains M1 only and has the wild-type sequence at the head of the proA* mRNA.
Fig 12.
M2 and synonymous mutations in codons 3 and 4 affect the probability that the region preceding the start codon is single-stranded.
The minimal folding energy of all four structures is -12.3 kcal/mol. RGR, relative growth rate.
Fig 13.
Fold-change in ProA* as a function of fold-change in the levels of proA* mRNA due to mutations in the head region of the proA* mRNA.