Exploring the parameters of post-segregational killing using heterologous expression of secreted toxin barnase and antitoxin barstar in an Escherichia coli case study

Abstract

Post-segregational killing (PSK) is a phenotype determined by plasmids using a toxin and an antitoxin gene pair. Loss of the genes depletes the cell's reserve of antitoxin and allows the toxin to act upon the cell. PSK benefits mobile elements when it increases reproductive success relative to other mobile competitors. A side effect of PSK is that plasmids become refractory to displacement from the cell during growth as a monoculture. Most PSK systems use a cytoplasmic toxin, but the external toxins of bacteriocins also have a PSK-like effect. It may be that any toxin and antitoxin gene pair can demonstrate PSK when it is on a plasmid. The secreted ribonuclease barnase and its protein inhibitor barstar have features in common with PSK modules, though their native context is chromosomal. We hypothesized that their recruitment to a plasmid could produce an emergent PSK phenotype. Others had shown that secreted barnase could exert a lethal effect on susceptible bacteria similarly to bacteriocins. However, barnase toxicity did not occur under the conditions tested, suggesting that barnase is toxic to neighbouring cells only under very specific conditions. Bacteriocins are only produced under some conditions, and some conditionality on toxin function or release may be advantageous in general to PSKs with external toxins because it would prevent killing of potential plasmid-naive hosts. Too much conditionality, however, would limit how advantageous the gene pair was to mobile elements, making the genes unlikely to be recruited as a PSK system.

Auxotrophy to Xeno-DNA: an exploration of combinatorial mechanisms for a high-fidelity biosafety system for synthetic biology applications

“…Barnase is encoded on a plasmid, preferably under control of an inducible or repressible promoter. Furthermore, the fusion of the barnase to secretion signals like that of PhoA allows the secretion into the periplasm [ 115 – 121 ]. Secreted barnase can be toxic to bacteria in the proximity of the producing cells if they do not express the inhibitor barstar, but the exact mechanism remains unknown [ 120 , 121 ].…”

“…Furthermore, the fusion of the barnase to secretion signals like that of PhoA allows the secretion into the periplasm [ 115 – 121 ]. Secreted barnase can be toxic to bacteria in the proximity of the producing cells if they do not express the inhibitor barstar, but the exact mechanism remains unknown [ 120 , 121 ]. In combination with another biosafety systems which represses the expression of barnase through arabinose-inducible promoter like P BAD , the expression of barnase can be induced if that biosafety mechanism fails, hence killing the host cell [ 122 , 123 ].…”