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Mol Microbiol. 1994 Aug;13(4):627-40.

Partial suppression of an Escherichia coli TonB transmembrane domain mutation (delta V17) by a missense mutation in ExbB.

Author information

1
Department of Microbiology, Washington State University, Pullman 99164.

Abstract

Active transport of vitamin B12 and Fe(III)-siderophore complexes across the outer membrane of Escherichia coli appears to be dependent upon the ability of the TonB protein to couple cytoplasmic membrane-generated protonmotive force to outer membrane receptors. TonB is supported in this role by an auxiliary protein, ExbB, which, in addition to stabilizing TonB against the activities of endogenous envelope proteases, directly contributes to the energy transduction process. The topological partitioning of TonB 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 TonB resulted in complete loss of TonB 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 TonB 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 modeling suggested that the delta V17 mutation caused a significant change in the predicted conserved face of the TonB 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 TonB. Wild-type TonB 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 TonB-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 TonB 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 TonB, suggesting that the suppressor was not allele specific. Contrary to expectations, the TonB delta V17, ExbBA39E pair resulted in a TonB with a greatly reduced half-life (approximately 10 min). These results together with protease susceptibility studies suggest that ExbB functions by modulating the conformation of TonB.

[Indexed for MEDLINE]

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