Import of colicins across the outer membrane of Escherichia coli involves multiple protein interactions in the periplasm

Several proteins of the Tol/Pal system are required for group A colicin imp ort into Escherichia coli. Colicin A interacts with TolA and TolB via disti nct regions of its N-terminal domain. Both interactions are required for co licin translocation. Using in vivo and in vitro approaches, we show in this study that colicin A also interacts with a third component of the Tol/Pal system required for colicin import, TolR. This interaction is specific to c olicins dependent on TolR for their translocation, strongly suggesting a di rect involvement of the interaction in the colicin translocation step. TolR is anchored to the inner membrane by a single transmembrane segment and pr otrudes into the periplasm. The interaction involves part of the periplasmi c domain of TolR and a small region of the colicin A N-terminal domain. Thi s region and the other regions responsible for the interaction with TolA an d TolB have been mapped precisely within the colicin A N-terminal domain an d appear to be arranged linearly in the colicin sequence. Multiple contacts with periplasmic-exposed Tol proteins are therefore a general principle re quired for group A colicin translocation.