Bacterial competition is a key ecological force shaping the composition, diversity, and evolution of microbial communities in natural environments. In Gram-negative bacteria, the Type VI secretion system (T6SS) mediates interbacterial antagonism by injecting toxic effectors, including rearrangement hotspot (Rhs) proteins, into neighbouring competitor cells. However, the structural organisation and delivery mechanisms of Rhs proteins, and their association with the T6SS, remain poorly characterised.
Here, we expressed and purified Rhs proteins from Escherichia coli K-12 (RhsA) and enterohemorrhagic E. coli (EHEC), and employed cryo-electron microscopy to determine their structures. Functional properties and delivery mechanisms were investigated using tethered Bilayer Lipid Membrane–Electrochemical Impedance Spectroscopy (tBLM–EIS) and interbacterial competition assays.
We show that E. coli K-12 RhsA adopts an anticlockwise β-barrel cage formed by YD-repeat domains, which encapsulates the C-terminal toxin and is sealed by structural “plug” elements at both ends. Rhs proteins harbour functionally distinct autocleavage sites at their N- and C-termini, with C-terminal autocleavage facilitating toxin encapsulation and N-terminal autocleavage implicated in toxin release. The C-terminal cleavage site is conserved across the Rhs family and involves two aspartate residues, whereas N-terminal autocleavage is more variable. Structural analysis reveals a distinct N-terminal cleavage site between His47 and Pro48, supported by a glutamate-dependent autoproteolytic mechanism involving Glu61, His349, His351, and a coordinated Zn²⁺ ion.
Structures of full-length RhsA (RhsA-FL) and a toxin-deleted variant (RhsAΔT) show that toxin removal does not alter the overall Rhs scaffold. Cryo-EM analysis reveals that, in both constructs, a subset of particles binds an endogenous E. coli chaperone, likely a peptidyl-prolyl cis–trans isomerase (PPIase), and N-terminal autocleavage occurs exclusively in the PPIase-bound population.
tBLM–EIS measurements indicate that the Rhs β-barrel cage does not form stable membrane pores but instead modulates membrane permeability, while interbacterial competition assays suggest that toxin release occurs through opening of the Rhs cage before delivery. In contrast, the EHEC Rhs protein (EDL933-0630) requires co-expression with its cognate EagR chaperone for stabilisation, highlighting distinct assembly requirements among Rhs effectors. Together, these findings provide structural and functional insights into Rhs-mediated interbacterial competition and toxin deployment in Gram-negative bacteria.