The high mutational sensitivity of ccdA antitoxin is linked to codon optimality

Abstract

Deep mutational scanning studies suggest that single synonymous mutations are typically silent and that most exposed, non active-site residues are tolerant to mutations. Here we show that the ccdA antitoxin component of the E.coli ccdAB toxin-antitoxin operonic system is unusually sensitive to mutations when studied in the operonic context. A large fraction (~80%) of single codon mutations, including many synonymous mutations in the ccdA gene show inactive phenotypes that are correlated with the E.coli codon usage frequency but retain native-like binding affinity towards cognate toxin, CcdB. Therefore, the observed phenotypic effects are largely not due to alterations in protein structure or stability, consistent with the fact that a large region of CcdA is intrinsically disordered. In select cases, proteomics studies reveal altered ratios of CcdA:CcdB protein levels in vivo, suggesting that these mutations likely alter relative translation efficiencies of the two genes in the operon. We extend these results by predicting and validating single synonymous mutations that lead to loss of function phenotypes in the relBE operon upon introduction of rarer codons. Thus, in their native context, genes are likely to be more sensitive to both synonymous and non-synonymous point mutations than inferred from previous saturation mutagenesis studies.

Structural and functional determinants inferred from deep mutational scans

“…A previously generated CcdB SSM library 26 was cloned in pUC57 vector via Gibson Assembly according to the manufacturer's protocol 37 . The Gibson product was transformed into high efficiency (10 9 CFU/μg of pUC57 plasmid DNA) electrocompetent E. coli Top10Gyr cells 38 . The cells were plated on LB agar plates containing 100 μg/mL ampicillin, for selection of transformants and incubated for 12 hr at 37°C.…”

“…37 The Gibson product was transformed into high efficiency (10 9 CFU/μg of pUC57 plasmid DNA) electrocompetent E. coli Top10Gyr cells. 38 The cells were plated on LB agar plates containing 100 μg/mL ampicillin, for selection of transformants and incubated for 12 hr at 37 C. Pooled plasmid was purified using a Qiagen plasmid maxiprep kit as per the manufacturer's instructions.…”

Structural and functional determinants inferred from deep mutational scans

“…A previously generated CcdB SSM library 26 was cloned in pUC57 vector via Gibson Assembly according to the manufacturer’s protocol 37 . The Gibson product was transformed into high efficiency (10 9 CFU/μg of pUC57 plasmid DNA) electro-competent E. coli Top10Gyr cells 38 . The cells were plated on LB agar plates containing 100μg/mL ampicillin, for selection of transformants and incubated for 12 hours at 37°C.…”

“…A previously generated CcdB SSM library 26 was cloned in pUC57 vector via Gibson Assembly according to the manufacturer's protocol 37 . The Gibson product was transformed into high efficiency (10 9 CFU/μg of pUC57 plasmid DNA) electro-competent E. coli Top10Gyr cells 38 .…”

“…The RelE gene downstream of the ccd promoter was synthesised in pBT vector at Genscript. Two E. coli host strains, i.e., Top10Gyr, strain containing R462C mutation in the GyrA subunit which prevents CcdB from binding to Gyrase 2 and Top10, strain sensitive to the action of CcdB were used for phenotypic screening of CcdB mutants. The WT and mutant ccdB genes were cloned under the control of the P BAD promoter in pBAD24 vector 3 and expressed and purified from the Top10Gyr strain.…”