A soft Tus-Ter interaction is hiding a fail-safe lock in the replication fork trap of Dickeya paradisiaca.

A variety of replication fork traps have recently been characterised in Enterobacterales, unveiling two different types of architecture. Of these, the degenerate type II fork traps are commonly found in Enterobacteriaceae such as Escherichia coli. The newly characterised type I fork traps are found almost exclusively outside Enterobacteriaceae within Enterobacterales and include several archetypes of possible ancestral architectures. Dickeya paradisiaca harbours a somewhat degenerate type I fork trap with a unique Ter1 adjacent to tus gene on one side of the circular chromosome and three putative Ter2-4 sites on the other side of the fork trap. The two innermost Ter1 and Ter2 sites are only separated by 18 kb, which is the shortest distance between two innermost Ter sites of any chromosomal fork trap identified so far. Of note, the dif site is located between these two sites, coinciding with a sharp GC-skew flip. Here we examined and compared the binding modalities of E. coli and D. paradisiaca Tus proteins for these Ter sites. Surprisingly, while Ter1-3 were functional, no significant Tus binding was observed for Ter4 even in low salt conditions, which is in stark contrast with the significant non-specific protein-DNA interactions that occur with E. coli Tus. Even more surprising was the finding that D. paradisiaca Tus has a relatively moderate binding affinity to double-stranded Ter while retaining an extremely high affinity to Ter-lock sequences. Our data revealed major differences in the salt resistance and stability between the D. paradisiaca and E. coli Tus protein complexes, suggesting that while Tus protein evolution can be quite flexible regarding the initial Ter binding step, it requires a highly stringent purifying selection for its final locked complex formation.