is a natural inhabitant of aquatic environments and converts to a pathogen upon infection by a filamentous phage, CTXΦ, that transmits the cholera toxin-encoding genes. This toxigenic conversion of has evident implication in both genome plasticity and epidemic risk, but the early stages of the infection have not been thoroughly studied. CTXΦ transit across the bacterial periplasm requires binding between the minor coat protein named pIII and a bacterial inner-membrane receptor, TolA, which is part of the conserved Tol-Pal molecular motor. To gain insight into the TolA-pIII complex, we developed a bacterial two-hybrid approach, named Oxi-BTH, suited for studying the interactions between disulfide bond-folded proteins in the bacterial cytoplasm of an reporter strain. We found that two of the four disulfide bonds of pIII are required for its interaction with TolA. By combining Oxi-BTH assays, NMR, and genetic studies, we also demonstrate that two intermolecular salt bridges between TolA and pIII provide the driving forces of the complex interaction. Moreover, we show that TolA residue Arg-325 involved in one of the two salt bridges is critical for proper functioning of the Tol-Pal system. Our results imply that to prevent host evasion, CTXΦ uses an infection strategy that targets a highly conserved protein of Gram-negative bacteria essential for the fitness of in its natural environment.

No abstract

There was an error in the genome description of the strain used to construct the OxiBTH strain. Shuffle T7 express was used to make the OxiBTH strain and not Shuffle T7 as originally reported. In Table S1

Vibrio cholerae is the bacterial causative agent of the human disease cholera. Non-pathogenic bacterium can be converted to pathogenic following infection by a filamentous phage, CTXΦ, that carries the cholera toxin encoding genes. A crucial step during phage infection requires a direct interaction between the CTXΦ minor coat protein (pIII(CTX)) and the C-terminal domain of V. cholerae TolA protein (TolAIIIvc). In order to get a better understanding of TolA function during the infection process, we have initiated a study of the V. cholerae TolAIII domain by 2D and 3D heteronuclear NMR. With the exception of the His-tag (H123-H128), 97 % of backbone (1)H, (15)N and (13)C resonances were assigned and the side chain assignments for 92 % of the protein were obtained (BMRB deposit with accession number 25689).