Identification of the Periplasmic Cobalamin-Binding Protein BtuF ofEscherichia 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.The outer membrane (OM) of gram-negative bacteria forms a permeability barrier which restricts passage of both nutrients and toxic environmental agents (19,25). Most nutrients cross the OM into the periplasmic space by diffusion through general or specific porins, such as OmpF or LamB. Nutrients which are too large or scarce to enter efficiently through the porins are taken into the periplasm via specific, high-affinity active transport systems. The transport systems for passage across the OM of ferric iron complexed with siderophores, heme, or host iron-binding proteins, and of cobalamins (Cbls) such as vitamin B 12 (CN-Cbl), consist of a substrate-specific TonB-dependent OM transporter, the transperiplasmic energy-coupling protein TonB, and its ancillary proteins ExbB and ExbD in the cytoplasmic membrane (CM).Most nutrients are transported across the CM by active transport systems coupled to a ...

Structure and Inhibition of a Quorum Sensing Target from Streptococcus pneumoniae

“…Similar values were observed in comparing p-F-PhT-ImmA [9] and p-F-PhT-DADMe-ImmA [30] where the DADMe-Immucillin binds 102 fold more tightly. Likewise, PhT-DADMeImmA [27] binds 167 fold more tightly than PhT-ImmA [8]. Finally, the BnT-group achieved an extra 100 fold affinity from being in the DADMe context.…”

“…Thus, EtT-ImmA ) indicates that aromatic residues including Phe207, Tyr107 and Phe105 surround the methylthio group of MT-ImmA [11]. The PhT-ImmA [8] contains a planar hydrophobic 5′-group and binds with a K i of 335 nM, a three fold improvement in the binding compared to MT-ImmA. Increasing the size of the aromatic group from phenyl to napthyl marginally improved binding affinity to give a K i *of 220 nM for NapT-ImmA [7], and this was the only inhibitor of the Immucillin series to show slow-onset inhibition.…”

“…In bacteria, accumulation of these metabolites is avoided through the function of the pfs gene. Deletion of pfs in E. coli causes a severe growth defect (8). Quorum sensing is important in pathogenicity since deletion of the LuxS quorum sensing gene in S. pneumoniae results in attenuation of pneumococcal infections (9).…”

A single intact ATPase site of the ABC transporter BtuCD drives 5% transport activity yet supports full in vivo vitamin B 12 utilization

“…Two E. coli methionine synthases are able to catalyze the last step in methionine biosynthesis: MetE is vitamin B 12 independent, and MetH fully depends on vitamin B 12 as a cofactor (34). Thus, in a strain deleted of metE, under conditions of methionine depletion, high-affinity acquisition of extracellular vitamin B 12 is essential for growth, and BtuC 2 D 2 -F becomes indispensable (35). We deleted btuD from a metE − strain creating a metE − /btuD − strain.…”

Comparative Genomics of the Vitamin B12 Metabolism and Regulation in Prokaryotes

“…Further, the ATP-dependent transport system CbiMNQO, encoded by the CBL biosynthetic operon in S. typhimurium, probably mediates high affinity transport of cobalt ions for the B 12 synthesis (14). Vitamin B 12 , cobinamide, and other corrinoids are actively transported in enteric bacteria using the TonB-dependent outer membrane receptor BtuB in the complex with the ABC transport system BtuFCD (15).…”