Fig. 1
Phenotypic effects of single synonymous codon mutants of CcdB in an operonic context. A Correlation between biological replicates. Following deep sequencing, the fraction of each mutant normalised to the WT value is estimated for all mutants in resistant (extreme left panel), sensitive (middle panel) and RelE reporter strains (extreme right panel). The (0,0) point represent the WT. B In vivo activity of CcdB mutants in both resistant (Top10Gyr) and sensitive (Top10) strains validate deep sequencing results in a low-throughput manner (left panel). Mutants are tenfold serially diluted and spotted on LB Agar plates. Corresponding ESCcdB scores of individual mutants obtained from deep sequencing are mentioned in grey in the bottom panel. The mutants are ordered according to the residue number from right to left. A correlation of r = 0.96 (P < .001) is observed between the ESCcdB scores and normalised CFU count of mutants relative to WT in the sensitive (Top10) strain obtained in the spotting assay. Error bars for duplicate experiments are also shown (right panel). C Effects of synonymous mutations on ESCcdB and ESRelE scores as a function of location within the gene. Top panel shows position averaged ESCcdB scores for synonymous mutants as a function of residue number. The bottom panel shows position averaged ESRelE scores for synonymous mutants as a function of residue number. The N-terminal region shows the largest variation. Black dashed line represents the WT score. The entire data is divided into N-terminal (1–13), middle (14–86) and C-terminal (87–101) region. The secondary structure of CcdB (from PDB id 3VUB) is shown at the bottom