In vitro and in vivo stability of the ε2ζ2 protein complex of the broad host−range Streptococcus pyogenes pSM19035 addiction system

Streptococcus pyogenes pSM19035−encoded epsilon (10.7 kDa) and zeta (32.4 kDa) proteins are necessary to secure stable plasmid inheritance in bacteria, with zeta acting as toxin that kills plasmid−deprived cells and epsilon as an antitoxin that neutralises the activity of zeta. The epsilon and zeta proteins co−purify as a stable complex that, according to analytical ultracentrifugation and gel filtration, exists as epsilon2zeta2 heterotetramer in solution. Co−crystals of the epsilon2zeta2 complex contain epsilon and zeta in 1:1 molar ratio. Unfolding studies monitoring circular dichroic and fluorescence changes show that the zeta protein has a significantly lower thermodynamic stability than the epsilon protein both in free state and in the complex. Proteolytic studies indicate that zeta protein is more stable in the epsilon2zeta2 complex than in the free state. In vivo studies reveal a short half−life of the epsilon antitoxin (−18 min) and a long lifetime of the zeta toxin (>60 min). When transcription−translation of a plasmid containing the epsilon and zeta genes was inhibited, cell death was observed after a short lag phase that correlates with the disappearance of the epsilon protein from the backgroun