Involvement of ExbB and TonB in transport across the outer membrane of Escherichia coli: phenotypic complementation of exb mutants by overexpressed tonB and physical stabilization of TonB by ExbB

Abstract

The exb locus in Escherichia coli consists of two genes, termed exbB and exbD. Exb functions are related to TonB function in that most TonB-dependent processes are enhanced by Exb Transport of iron(III) siderophores and vitamin B12 across the outer membrane of Escherichia coli (2, 4, 17, 21) stands in contrast to the passive diffusion of most substrates through porins (18). The activity of the receptor proteins for iron(III) siderophore and vitamin B12 transport depends on the TonB protein of the cytoplasmic membrane, since these substrates stay at the receptor proteins in tonB mutants and in unenergized tonB+ cells (3; H. Schoffler and V. Braun, Mol. Gen. Genet., in press). The group B colicins also remain bound to the outer membrane receptors unless tonB+ cells are energized (3,20). Mutations in a single codon of the tonB gene suppressed the btuB451 point mutation in the structural gene of the vitamin B12 receptor (14), and mutations in theJhuA gene (Schoffler and Braun, in press), which encodes the receptor for ferrichrome, the antibiotic albomycin, colicin M, and the bacteriophages T5, Ti, and +80. Suppression of mutations in receptor genes by tonB mutations suggests a direct interaction between the TonB protein and the receptor proteins. The TonB function seems to be involved only in transport processes across the outer membrane.Exb designates a function of Escherichia coli similar to TonB with the difference that TonB-dependent processes absolutely require TonB whereas Exb-dependent functions require Exb to a different extent. There are TonB-dependent functions which do not require Exb, but all Exb-dependent functions rely on TonB. For example, tonB mutants are insensitive to colicins B and M and to the antibiotic albomycin and are unable to take up ferrichrome. exb mutants have the same properties except that they exhibit reduced sensitivity to colicin B. The exb locus has been mapped at min 65 of the E. coli linkage map (20). Recently, we cloned (9) and sequenced (8) the exb region and identified two genes, termed exbB and exbD, which are required for complement-* Corresponding author.ing exb mutants. The exbB and exbD genes encode proteins with molecular masses of 26.1 and 15.5 kilodaltons (kDa), respectively, which were both preferentially localized in the cytoplasmic membrane (8). Since the ExbB and ExbD proteins affect the same cellular activities as the TonB protein, and the requirement for ExbB and ExbD differs in various TonB-dependent functions, we studied whether overproduction of TonB compensates for the lack of ExbB and ExbD and vice versa. Indeed, the results presented in this paper show that the low sensitivity of exb mutants to colicins B, D, and M could be greatly increased by overeXpression of tonB. In addition, suppression of the btuB451 mutation by a mutation in tonB was enhanced in an exb+ background. The ExbB protein prevented degradation of the TonB protein. Radioactive ferrichrome taken up by exb mutants was released by a surplus of unlabeled ferrichrome, indicating that ferrichrome...

In vivo evidence for FhuA outer membrane receptor interaction with the TonB inner membrane protein of Escherichia coli

“…Surprisingly, independently isolated tonB mutations which could suppress mutations in the TonB box of thefhuA gene (and of the btuB gene encoding the receptor for vitamin B12 transport) were all in the same triplet [2,5,6]. We describe here a new tonB mutation which is only two codons further upstream of the former.…”

“…The amount and the stability of the plasmidencoded TonB and FhuA proteins were studied in strain WM1576 which carried a temperature-inducible phage T7 RNA polymerase gene on plasmid pGP 1-2 [8]. Cells containing the fhuA/tonB gene combinations listed in Table I, were pulse-labeled with [3~S]methionine, and then chased with a 500-fold surplus of nonradioactive methionine for 0, 15, 30, and 60 min, as has been previously outlined in detail [2]. The proteins were separated by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE), and determined by fluorography.…”

“…Plasmid pGC8 carrying a tonB point mutation which results in a glutamine to leucine replacement at residue 160 of the TonB polypeptide (Q 160L), and plasmid pCG 15 (Q 160K) were previously described [2,6]. The newly isolated plasmid pCG128 (R158L) was obtained by selecting for tonB mutations which suppressed the fhuA mutation causing a FhuA 19P replacement [5].…”

“…After binding colicin M and ferrichrome have to be taken up into the periplasmic space and subsequently into (colicin M) or across the cytoplasmic membrane (ferrichrome). The vectorial release from the receptor across the outer membrane requires the TonB protein which is anchored in the cytoplasmic membrane but exposed to the periplasmic space [1,2]. Ferrichrome and colicin M bind to tonB mutants but remain on the cell surface receptors, suggesting a deficiency in the uptake across the outer membrane [2,3].…”

“…The vectorial release from the receptor across the outer membrane requires the TonB protein which is anchored in the cytoplasmic membrane but exposed to the periplasmic space [1,2]. Ferrichrome and colicin M bind to tonB mutants but remain on the cell surface receptors, suggesting a deficiency in the uptake across the outer membrane [2,3]. In addition, transport across the outer membrane requires energy although no energy-generating system nor energy-rich metabolites are known to reside outside the cytoplasmic membrane, leading to the concept that the TonB protein responds to the energized state of the cytoplasmic membrane, assuming an energized and an unenergized conformation, of which the energized state induces the release of the ligands at the FhuA receptor [4].…”