Identification of the periplasmic cobalamin-binding protein BtuF of Escherichia coli

Abstract

Cells of Escherichia coli take up vitamin B(12) (cyano-cobalamin [CN-Cbl]) and iron chelates by use of sequential active transport processes. Transport of CN-Cbl across the outer membrane and its accumulation in the periplasm is mediated by the TonB-dependent transporter BtuB. Transport across the cytoplasmic membrane (CM) requires the BtuC and BtuD proteins, which are most related in sequence to the transmembrane and ATP-binding cassette proteins of periplasmic permeases for iron-siderophore transport. Unlike the genetic organization of most periplasmic permeases, a candidate gene for a periplasmic Cbl-binding protein is not linked to the btuCED operon. The open reading frame termed yadT in the E. coli genomic sequence is related in sequence to the periplasmic binding proteins for iron-siderophore complexes and was previously implicated in CN-Cbl uptake in Salmonella. The E. coli yadT product, renamed BtuF, is shown here to participate in CN-Cbl uptake. BtuF protein, expressed with a C-terminal His(6) tag, was shown to be translocated to the periplasm concomitant with removal of a signal sequence. CN-Cbl-binding assays using radiolabeled substrate or isothermal titration calorimetry showed that purified BtuF binds CN-Cbl with a binding constant of around 15 nM. A null mutation in btuF, but not in the flanking genes pfs and yadS, strongly decreased CN-Cbl utilization and transport into the cytoplasm. The growth response to CN-Cbl of the btuF mutant was much stronger than the slight impairment previously described for btuC, btuD, or btuF mutants. Hence, null mutations in btuC and btuD were constructed and revealed that the btuC mutant had a strong impairment similar to that of the btuF mutant, whereas the btuD defect was less pronounced. All mutants with defective transport across the CM gave rise to frequent suppressor variants which were able to respond at lower levels of CN-Cbl but were still defective in transport across the CM. These results finally establish the identity of the periplasmic binding protein for Cbl uptake, which is one of few cases where the components of a periplasmic permease are genetically separated.

FIG. 1.

Representation of the pfs-btuF-yadS region of the E. coli chromosome map and description of deletion mutations. (A) The wild-type 2,723-bp region in plasmid pNC5. The expanded sequences show the overlap between the 3′ end of the pfs gene and the 5′ end of the btuF gene. Arrows indicate the direction of transcription. (B) The 3,318-bp insert in pΔpfs::Km, showing the 657-bp in-frame deletion of part of pfs gene and insertion of the Km cassette. (C and D) Structures of ΔbtuF::Km (C) and ΔyadS::Km (D).

FIG. 2.

Expression, purification, and cellular localization of BtuF-His protein. (A) SDS-PAGE analysis with Coomassie blue staining; (B) Western immunoblot with primary tetra-His monoclonal antibody (Qiagen). Whole-cell samples suspended and boiled in sample buffer were obtained from strain BL21(DE3) carrying no plasmid (lanes 1), the pBtuF-His plasmid without induction (lanes 2), or the pBtuF-His plasmid 3 h after induction with 0.25 mM IPTG (lanes 3). IPTG-induced cells were analyzed before osmotic shock treatment (lanes 4), and following osmotic shock, samples from the osmotic shock fluid (lanes 5) and pellet (lanes 6) were run. Induced cells were taken before (lanes 8) and after disruption in French pressure cell. The lysate was subjected to centrifugation, and the supernatant (lanes 9) and pellet (lanes 10) were resolved. Lanes 11 show the affinity-purified BtuF-His protein following elution from an Ni-nitrilotriacetic acid affinity matrix. On the left are shown the mobilities of molecular weight standards (in thousands), and on the right are indicated the positions of the precursor and mature forms of BtuF-His. The arrow points to oligomeric forms which are lost upon prolonged heating in sample buffer.

FIG. 3.

Binding of CN-[⁵⁷Co]Cbl to purified BtuF-His, measured in the charcoal filtration assay. Binding is plotted as the number of picomoles of CN-Cbl bound to BtuF as a function of the concentration of CN-Cbl added. The two symbols represent results from separate experiments. The curve was fit to a one-site hyperbolic model by the DeltaGraph curve-fitting feature.

FIG. 4.

Isothermal titration calorimetry of BtuF-His with CN-Cbl. (A) Specific heat versus time of titration of CN-Cbl into purified BtuF-His. The heat of dilution of CN-Cbl into the buffer has been subtracted. (B) Enthalpies per mole of CN-Cbl injected versus molar ratio (CN-Cbl/BtuF-His).

FIG. 5.

Uptake of CN-[⁵⁷Co]Cbl. Strains assayed were RK4379 (metE) (○), RK4936 (btuB) (•), RK5015 (tonB) (▴), RK6049 (btuC) (□), and NC17 (ΔbtuF:: Km) (▪). Results are expressed as picomoles of Cbl taken up per 10⁹ cells. At the time indicated by the arrow (30 min), a 100-fold molar excess of unlabeled CN-Cbl was added. Transport by the newly constructed btuC and btuD mutants was similar to that shown here for the btuC mutant.

FIG. 6.

Complementation of the defect in CN-Cbl transport in btuF and pfs mutants by deletion plasmids. Host strains were NC17 (metE ΔbtuF::Km) (A) and NC16 (metE Δpfs::Km) (B). Host strains carried the following plasmids: no plasmid (○), pYadT2 (pfs-btuF-yadS) (•), pΔpfs (▴), or pΔbtuF (▪). Experimental conditions were as described for Fig. 5.