Exogeneous protein over-expression in E. coli generates an analogous response to the development of antimicrobial resistance

The E. coli pET system is the most widely utilised bacterial protein over-expression system worldwide. Using this system, it is believed that all cells produce the exogeneous protein and that integral membrane protein (IMPs) over-expression is generally more toxic than their soluble counterparts. Colony forming units (CFU) were counted in the presence of varying concentrations of IPTG. Over-expression of either soluble or IMPs using >0.1 mM IPTG resulted in the death of >99.9 % of all viable cells. The survivors display three phenotypes: large green, large white and small colony variants. Genome sequencing of various phenotypes, selected from 0.01 to 1.0 mM IPTG exposure for both soluble protein and IMP proteins in BL21-Gold(DE3)pLysS revealed that the large white and large green colonies were formed from either the loss of T7 RNAP activity or its downregulation. Replica plating demonstrated that recombinant protein over-expression is bactericidal. Thus, target protein over-expression generates a selective pressure so that the <0.03 % of surviving cells, that ultimately produce the exogeneous protein, are genetically modified organisms or phenotypic variants, adapted to stop or reduce T7 RNAP activity. This applies to both soluble and membrane proteins. Hence, it is the act of over-expression of exogeneous proteins in E. coli using the pET system that is toxic whether it is an IMP or a soluble protein, resulting in an adaptative survival response analogous to the development of antibiotic resistance.