Ra. Larsen et al., PARTIAL SUPPRESSION OF AN ESCHERICHIA-COLI TONB TRANSMEMBRANE DOMAIN MUTATION (DELTA-V17) BY A MISSENSE MUTATION IN EXBB, Molecular microbiology, 13(4), 1994, pp. 627-640
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.