In vitro and in vivo stability of the ε2ξ2 protein complex of the broad host-range Streptococcus pyogenes pSM19035 addiction system

Streptococcus pyogenes pSM19035-encoded ε (10.7 kDa) and ζ (32.4 kDa) proteins are necessary to secure stable plasmid inheritance in bacteria, with ζ acting as toxin that kills plasmid-deprived cells and ε as an antitoxin that neutralises the activity of ζ. The ε and ζ proteins co-purify as a stable complex that, according to analytical ultracentrifugation and gel filtration, exists as ε2ζ2 heterotetramer in solution. Cocrystals of the ε2ζ2 complex contain ε and ζ in 1:1 molar ratio. Unfolding studies monitoring circular dichroic and fluorescence changes show that the ζ protein has a significantly lower thermodynamic stability than the ε protein both in free state and in the complex. Proteolytic studies indicate that ε protein is more stable in the ε2ζ2 complex than in the free state. In vivo studies reveal a short half-life of the e antitoxin (∼18 min) and a long lifetime of the ζ toxin (>60 min). When transcription-translation of a plasmid containing the ε and ζ genes was inhibited, cell death was observed after a short lag phase that correlates with the disappearance of the ε protein from the background.