PARTIAL SUPPRESSION OF AN ESCHERICHIA-COLI TONB TRANSMEMBRANE DOMAIN MUTATION (DELTA-V17) BY A MISSENSE MUTATION IN EXBB

Active transport of vitamin B-12 and Fe(lll)-siderophore complexes across the outer membrane of Escherichia coli appears to be dependent upon the ability of the Tone protein to couple cytoplasmic membrane-generated protonmotive force to outer membrane receptors. Tone is supported in this role by an auxiliary protein, ExbB, which, in addition to stabilizing Tone against the activities of endogenous envelope proteases, directly contributes to the energy transduction process. The topological partitioning of Tone and ExbB to either side of the cytoplasmic membrane restricts the sites of interaction between these proteins primarily to their transmembrane domains. In this study, deletion of valine 17 within the aminoterminal transmembrane anchor of Tone resulted in complete loss of Tone activity, as well as loss of detectable in vivo crosslinking into a 59 kDa complex believed to contain ExbB. The Delta V17 mutation had no effect on Tone export. The loss of crosslinking appeared to reflect conformational changes in the TonB/ExbB pair rather than loss of interaction since ExbB was still required for some stabilization of TonB Delta V17. Molecular modelling suggested that the Delta V17 mutation caused a significant change in the predicted conserved face of the Tone amino-terminal membrane anchor. TonB Delta V17 was unable to achieve the 23 kDa proteinase K-resistant form in lysed sphaeroplasts that is characteristic of active Tone. Wild-type Tone also failed to achieve the proteinase K-resistant configuration when ExbB was absent. Taken together these results suggested that the Delta V17 mutation interrupted productive Tone-ExbB interactions. The apparent ability to crosslink to ExbB as well as a limited ability to transduce energy were restored by a second mutation (A39E) in or near the first predicted transmembrane domain of the ExbB protein. Consistent with the weak suppression, a 23 kDa proteinase K-resistant form of TonB Delta V17 was not observed in the presence of ExbBA39E. Neither the ExbBA39E allele nor the absence of ExbB affected Tone or TonB Delta V17 export. Unlike the tonB Delta V17 mutation, the exbBA39E mutation did not greatly alter a modelled ExbB transmembrane domain structure. Furthermore, the suppressor ExbBA39E functioned normally with wild-type Tone, suggesting that the suppressor was not allele specific. Contrary to expectations, the TonB Delta V17, ExbBA39E pair resulted in a Tone with a greatly reduced half-life (approximate to 10 min). These results together with protease susceptibility studies suggest that ExbB functions by modulating the conformation of Tone.