Mutational sensitivity of CcdB mutants in their native operonic context

Abstract

Determining the precise effects of thousands of point mutants on protein structure, stability, and activity is a challenging task. Chapter 1 introduces how next-generation sequencing technologies exploit phenotypic screens of mutant libraries to simultaneously measure the effects of thousands of mutants in a single experiment. In the recent past, several approaches have been developed to predict fitness effects of individual members of mutational libraries constructed on a target gene by treating them under different conditions, optionally indexing them based on their barcodes and finally, generating a fitness landscape to ascertain the effect of each mutant. The model system used in our work is the ccdAB operon which is tightly autoregulated by the CcdAB complex. CcdAB is a TypeII toxin-antitoxin (TA) system composed of a labile CcdA antitoxin and a stable CcdB toxin. At low toxin: antitoxin ratio, the operator/promoter region is repressed by a multimeric chain of alternating CcdA2-CcdB2 modules spiralling around the promoter DNA. Degradation of the labile antitoxin causes a decrease in the toxin: antitoxin ratio, which results in formation of a V-shaped derepressing CcdB2-CcdA2-CcdB2 heterohexamer. xii In Chapter 2, we developed a facile method to deduce the structural and functional determinants of the globular, cytotoxic protein, CcdB from E.coli, solely from mutational data. We generated a comprehensive site-saturation mutagenesis library of CcdB in its native operon. We examined the effects of mutations on Gyrase binding activity of the CcdB toxin in an E.coli strain which was sensitive to the toxic activity of the toxin. In conjunction with this screen, we also developed a RelE reporter assay in which the ccd promoter precedes the RelE gene to examine the effects of mutations in CcdB on CcdA binding activity, in a strain resistant to the toxic action of CcdB but sensitive to RelE toxicity. In vivo studies with individual point mutants of CcdB were used to validate our deep sequencing results. In vitro ...