Genetic suppression demonstrates interaction of TonB protein with outer membrane transport proteins in Escherichia coli

Abstract

Energy-coupled reactions of the Escherichia coli outer membrane transport proteins BtuB and Cir require the tonB product. Some point mutations in a region of btuB and cir that is highly conserved in TonB-dependent transport proteins led to loss of TonB-coupled uptake of vitamin B12 and colicin Ia, whereas binding was unaffected. Most other point mutations in this region had no detectable effect on transport activity. Mutations in tonB that suppressed the transport defect phenotype of these btuB mutations were isolated. All carried changes of glutamine 165 to leucine, lysine, or proline. The various tonB mutations differed markedly in their suppression activities on different btuB or cir mutations. This allele specificity of suppression indicates that TonB interacts directly with the outer membrane transport proteins in a manner that recognizes the local conformation but not specific side chains within this conserved region. An effect of the context of the remainder of the protein was seen, since the same substitution (valine 1O-*glycine) in btuB and cir responded differently to the suppressors. This finding supports the proposal that TonB interacts with more of the transport proteins than the first conserved domain alone.The outer membrane of Escherichia coli contains several high-affinity, active-transport systems for substrates, such as ferric-siderophore complexes and cobalamins, which are too large to diffuse effectively through the nonspecific porin channels. Transport is mediated by minor outer membrane proteins that bind these substrates with high affinity and specificity and that can serve as receptors for the adsorption and entry of specific bacteriophages and bacteriocins. The BtuB polypeptide is responsible for the uptake of vitamin B12 and other cobalamins, bacteriophage BF23, and colicins A and E. The FepA protein transports ferric enterochelin and colicins B and D. The iron-repressible Cir protein was initially identified as the receptor for colicin I, has been implicated in the uptake of catechol-substituted cephalosporins, and may mediate uptake of ferric complexes of other 2,3-dihydroxybenzoate derivatives (4).Active transport of most of the substrates listed above also requires functioning of the tonB product and is stimulated by the products of exbB and exbD (1, 7-9, 12). In the absence of tonB function, the outer membrane transport proteins are produced in normal or larger amounts and still bind their substrates effectively, but they are unable to carry out the energy-dependent steps of substrate accumulation (7). Analysis of cobalamin uptake in btuC mutants blocked in the cytoplasmic membrane transport system suggested that tonB-dependent active transport across the outer membrane is driven by the proton motive force (23). It was suggested that TonB couples energy from the proton motive force across the cytoplasmic membrane to the outer membrane transporters (7,12,23).The deduced sequences of tonB-dependent transport proteins possess short, highly conserved regions separated by long variable...

Differential substrate-induced signaling through the TonB-dependent transporter BtuB

“…TonB action involves its recognition of the Ton box on the transporters, probably in response to a conformational transition that follows substrate binding. TonB may also interact with other parts of the transporters, as implied from genetic suppression patterns (15). Claims that FepA and FhuA variants lacking the hatch domain and Ton box (32,33) still exhibit TonB-dependent activity have been challenged by the recognition that the empty barrels can be complemented by the hatch domains encoded by the mutated chromosomal alleles (34).…”

“…This region is the site of mutations, such as the substitution of Pro for Leu-8 or Val-10 in BtuB, which result in loss of Cbl transport but do not affect Cbl binding or entry of the TonB-independent phage BF23 and E colicins (13,14). The transport defect in Ton box mutants is partially reversed by suppressor mutations affecting nearby residues in the Ton box or residue Gln-160 of TonB (15,16).…”

TonB protein appears to transduce energy by shuttling between the cytoplasmic membrane and the outer membrane in Escherichia coli

“…Although TonB has been localized to the cytoplasmic membrane by several groups (Hannavy et al, 1990;Plastow and Holland, 1979;Roof et al, 1991), its association with the outer membrane has never been investigated even though there have been numerous suggestions that such an interaction takes place (Bell et al, 1990;Braun et al, 1991;Gü nter and Braun, 1990;Reynolds et al, 1980;Schö ffler and Braun, 1989;Tuckman and Osburne, 1992), including the detection, by in vivo cross-linking, of direct physical interaction with the outer membrane receptor FepA (Skare et al, 1993).…”

Studies on colicin B translocation: FepA is gated by TonB

“…Transport involves direct contact between TonB protein and a semiconserved region at the extreme aminoterminus of the transporter, called the TonB box (Heller et al, 1988;Bell et al, 1990;Anton and Heller, 1993;Bruske and Heller, 1993;Endriss et al, 2003;Ogierman and Braun, 2003;Sean Peacock et al, 2005;Vakharia et al, 2007). In the absence of TonB, the CM pmf, or the TonB box of the transporter, ligands continue to bind the transporters with high affinity, but are not transported across the OM.…”