Mutations that affect protein binding to a cognate partner primarily occur either at buried residues or at exposed residues directly involved in partner binding. Distinguishing between these two categories based solely on mutational phenotypes is challenging. The bacterial toxin CcdB kills cells by binding to DNA Gyrase. Cell death is prevented by binding to its cognate antitoxin CcdA, at an extended interface that partially overlaps with the GyrA binding site. Using the CcdAB toxin-antitoxin (TA) system as a model, a comprehensive site-saturation mutagenesis library of CcdB was generated in its native operonic context. The mutational sensitivity of each mutant was estimated by evaluating the relative abundance of each mutant in two strains, one resistant and the other sensitive to the toxic activity of the CcdB toxin, through deep sequencing. The ability to bind CcdA was inferred through a RelE reporter gene assay, since the CcdAB complex binds to its own promoter, repressing transcription. By analyzing mutant phenotypes in the CcdB-sensitive, CcdBresistant, and RelE reporter strains, it was possible to assign residues to buried, CcdA interacting or GyrA interacting sites. A few mutants were individually constructed, expressed, and biophysically characterized to validate molecular mechanisms responsible for the observed phenotypes. Residues inferred to be important for antitoxin binding, are also likely to be important for rejuvenating CcdB from the CcdB-Gyrase complex. Therefore, even in the absence of structural information, when coupled to appropriate genetic screens, such high-throughput strategies can be deployed for predicting structural and functional determinants of proteins.

Graphical Abstract 5In Brief: 6 The authors elucidate adaptation of SARS CoV-2 to different climates by studying 7 phylogenetics and the distribution of strains on Koppen's climate map. 8 Highlights: 9 • SARS CoV-2 follows inverse latitudinal gradient.

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Single-site synonymous mutations typically have only minor or no effects on gene function. Here, we estimate the effects on cell growth of ~200 single-site synonymous mutations in an operonic context by mutating each position of ccdB, the 101-residue long cytotoxin of the ccdAB Toxin-Antitoxin (TA) operon to all possible degenerate codons. Phenotypes were assayed by transforming the mutant library into CcdB sensitive and resistant E. coli strains, isolating plasmid pools, and subjecting them to deep sequencing. Since autoregulation is a hallmark of TA operons, phenotypes obtained for ccdB synonymous mutants after transformation in a RelE toxin reporter strain followed by deep sequencing provided information on the amount of CcdAB complex formed. Synonymous mutations in the N-terminal region involved in translation initiation showed the strongest non-neutral phenotypic effects. We observe an interplay of numerous factors, namely, location of the codon, codon usage, t-RNA abundance, formation of anti-Shine Dalgarno sequences, transcript secondary structure, and evolutionary conservation in determining phenotypic effects of ccdB synonymous mutations. Incorporation of an N-terminal, hyperactive synonymous mutation, in the background of the single-site synonymous mutant library sufficiently increased translation initiation, such that mutational effects on either folding or termination of translation became more apparent. Introduction of putative pause sites not only affects the translational rate but might also alter the folding kinetics of the protein in vivo. This information is useful in optimizing heterologous gene expression in E. coli and understanding the molecular bases for alteration in gene expression that arise due to synonymous mutations.

Mutations that affect protein binding to a cognate partner primarily occur either at buried residues or at exposed residues directly involved in partner binding. Distinguishing between these two categories based solely on mutational phenotypes is challenging. The bacterial toxin CcdB kills cells by binding to DNA Gyrase. Cell death is prevented by binding to its cognate antitoxin CcdA, at an extended interface that partially overlaps with the GyrA binding site. Using the CcdAB toxin-antitoxin (TA) system as a model, a comprehensive site-saturation mutagenesis library of CcdB was generated in its native operonic context. The mutational sensitivity of each mutant was estimated by evaluating the relative abundance of each mutant in two strains, one resistant and the other sensitive to the toxic activity of the CcdB toxin, through deep sequencing. The ability to bind CcdA was inferred through a RelE reporter gene assay, since the CcdAB complex binds to its own promoter, repressing transcription. By analysing mutant phenotypes in the CcdB sensitive, CcdB resistant and RelE reporter strains, it was possible to assign residues to buried, CcdA interacting or GyrA interacting sites. A few mutants were individually constructed, expressed, and biophysically characterised to validate molecular mechanisms responsible for the observed phenotypes. Residues inferred to be important for antitoxin binding, are also likely to be important for rejuvenating CcdB from the CcdB-Gyrase complex. Therefore, even in the absence of structural information, when coupled to appropriate genetic screens, such high-throughput strategies can be deployed for predicting structural and functional determinants of proteins.