Mutational sensitivity of CcdB mutants in their native operonic context
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 studies further support the molecular mechanisms inferred from deep sequencing data. Since autoregulation and rejuvenation are mechanistically intertwined in the CcdAB TA system, residues that are inferred as being important for antitoxin binding are also important for rejuvenating CcdB from the CcdB-Gyrase complex. In this study, we delineated protein-protein interaction interface residues for two different interacting partners, namely the antitoxin CcdA and cellular target Gyrase, and discriminated buried residues from residues involved in partner binding, with the help of differential mutational patterns observed for these different classes of residues. Chapter 3 describes studies which demonstrate that the majority of the mutants that were destabilised in the absence of the antitoxin, when expressed in the operonic context, displayed a phenotype like the WT in the RelE reporter stain, and exhibited toxicity less than WT in a strain sensitive to the toxicity of the CcdB toxin. In vivo studies showed that the folding defects of several buried site mutants were relieved in the presence of CcdA. In vitro characterisation of these destabilised mutants showed that they form a complex with CcdA similar to that of the WT. In vivo solubility assays confirmed that the folding defects inherently present in these buried site CcdB mutants were alleviated in the presence of its interacting partner, CcdA, likely via cotranslational folding. xiii In Chapter 4, we measured the effects of all possible single-site synonymous substitutions for each position of CcdB in their native operonic context. It is important to establish genotype-phenotype relationships under physiological conditions. The ability to perform genetic screens for both antitoxin binding and toxin binding to its cellular target helped to probe and understand effects of synonymous mutations on gene function. The data suggest that control of translational initiation is important for determining protein abundance inside the cell. There is an interplay of several factors, namely, codon usage, t-RNA abundance, generation of internal Shine-Dalgarno-like sequences, mRNA structure and evolutionary conservation, in dictating the translational efficiency of a gene. Amongst all the amino acids, synonymous mutations of Arginine displayed the maximum phenotypic and codon-specific effects on in vivo protein abundance in the ccdB gene, in its operonic context. In Chapter 5, we used the same methodology, to estimate the effects of single-site synonymous substitutions in combination with a mutation in the N-terminal region of CcdB, that likely alters the translation rate, thereby affecting the cotranslational folding process of the protein. We observed that introduction of potential pause sites in the middle of the gene because of the synonymous mutation, results in increased CcdB toxicity. Our data suggest that double-site synonymous mutations result in translational uncoupling, along with alteration in translational kinetics. This is possibly accompanied by improved in vivo protein stability or folding kinetics, in the case of CcdB protein. In summary, extensive analysis of the deep sequencing data obtained for both non-synonymous and synonymous CcdB substitutions helps understand the molecular basis of the observed mutant phenotypes. Such studies provide novel insights into gene regulation, protein folding, stability, and activity.