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 acr oss the outer membrane of Escherichia coil appears to be dependent upo n the ability of the Tone protein to couple cytoplasmic membrane-gener ated protonmotive force to outer membrane receptors. Tone is supported in this role by an auxiliary protein, ExbB, which, in addition to sta bilizing Tone against the activities of endogenous envelope proteases, directly contributes to the energy transduction process. The topologi cal partitioning of Tone and ExbB to either side of the cytoplasmic me mbrane restricts the sites of interaction between these proteins prima rily 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 a ppeared to reflect conformational changes in the TonB/ExbB pair rather than loss of interaction since ExbB was still required for some stabi lization of TonB Delta V17. Molecular modelling suggested that the Del ta V17 mutation caused a significant change in the predicted conserved face of the Tone amino-terminal membrane anchor. TonB Delta V17 was u nable to achieve the 23 kDa proteinase K-resistant form in lysed sphae roplasts that is characteristic of active Tone. Wild-type Tone also fa iled to achieve the proteinase K-resistant configuration when ExbB was absent. Taken together these results suggested that the Delta V17 mut ation interrupted productive Tone-ExbB interactions. The apparent abil ity to crosslink to ExbB as well as a limited ability to transduce ene rgy were restored by a second mutation (A39E) in or near the first pre dicted transmembrane domain of the ExbB protein. Consistent with the w eak suppression, a 23 kDa proteinase K-resistant form of TonB Delta V1 7 was not observed in the presence of ExbBA39E. Neither the ExbBA39E a llele nor the absence of ExbB affected Tone or TonB Delta V17 export. Unlike the tonB Delta V17 mutation, the exbBA39E mutation did not grea tly alter a modelled ExbB transmembrane domain structure. Furthermore, the suppressor ExbBA39E functioned normally with wild-type Tone, sugg esting that the suppressor was not allele specific. Contrary to expect ations, the TonB Delta V17, ExbBA39E pair resulted in a Tone with a gr eatly reduced half-life (approximate to 10 min). These results togethe r with protease susceptibility studies suggest that ExbB functions by modulating the conformation of Tone.