Rene Baerentsen (Aarhus University)| Stine Nielsen (University of Copenhagen)| Taoran Li (Aarhus University)| Jeppe Lyngsø (Aarhus University)| Helena Rasmussen (Aarhus University)| Jan Pedersen (Aarhus University)| Michael Sørensen (University of Copenhagen)| Kenn Gerdes (University of Copenhagen)| Ditlev Brodersen (Aarhus University)
Prokaryotic type II toxin-antitoxin (TA) modules encode a toxin component that modulates bacterial growth and an antitoxin that neutralizes the toxin by direct protein-protein interaction. hipBA is one of the most well-studied TA modules in Escherichia coli, for which the activity of the toxin has been linked to increased formation of persister cells able to survive antibiotic treatment. The encoded toxin, HipA, regulates growth through phosphorylation and inhibition of glutamyl tRNA synthetase, and is regulated via auto-phosphorylation that inhibits the activity via a conformational shift of an internal loop.
A novel tripartite TA module homologous to hipBA, hipBST, was found in the enteropathogenic Escherichia coli strain O127:H6, which is unique since it encodes three protein components. Sequence alignment suggest that HipB has retained its DNA-binding motif, while HipS and HipT align to the N and C terminal parts of HipA, respectively, suggesting an evolutionary split of the toxin into two distinct proteins. Surprisingly, HipS functions as the antitoxin of the hipBST module.
We have determined crystal structures of the HipBST complex in both a phosphorylated and non-phosphorylated state of the HipT toxin. Despite a high level of structural similarity to the homologous HipBA complex, the phosphorylation does not lead to a conformational shift of the internal loop, which indicates that the system operates differently from its two-component counterpart. The inactive state is determined by HipS binding instead, leaving the role of the two phosphorylation sites uncertain. The current understanding of this tripartite TA system will be presented, and light will be shed on a hitherto undiscovered aspect of toxin-antitoxin systems.