<scp>CcdA</scp> chaperones <scp>CcdB</scp> against irreversible misfolding and aggregation via a cotranslational folding mechanism

Cotranslational subunit assembly is thought to be a prominent feature throughout the proteome, but, in bacteria, there are only a limited number of experimentally confirmed examples and most involve the addition of extraneous tag sequences for experimental convenience. Toxin CcdA and Antitoxin CcdB are components of the ccdAB operon. They assemble in a hetero-multimeric complex in vivo. Building on a previously characterised saturation mutagenesis dataset of CcdB, we investigated how operonic gene organisation influences the cotranslational folding and assembly of the toxin-antitoxin complex. We compared the phenotypic effects of CcdB mutations when expressed alone versus in the native operonic context downstream of CcdA. Although several charged and polar mutations in the CcdB core result in loss of function in the absence of CcdA, many of these are functionally rescued in the operonic context. Furthermore, we show that the efficiency of rescue is substantially reduced when ccdA and ccdB are expressed from separate mRNAs rather than from a single polycistronic transcript. Our results highlight a direct role for cotranslational interactions in enabling correct folding of CcdB and suggest that bacterial operon structure may have evolved, in part, to facilitate such chaperone-like rescue of unstable protein variants. Gene organisation in operons in bacteria may thus reflect a fundamental cotranslational mechanism that is important for the effective assembly of protein complexes and can potentially buffer substantial genetic variation.