Biotin uptake in prokaryotes by solute transporters with an optional ATP-binding cassette-containing module

Abstract

BioMNY proteins are considered to constitute tripartite biotin transporters in prokaryotes. Recent comparative genomic and experimental analyses pointed to the similarity of BioMN to homologous modules of prokaryotic transporters mediating uptake of metals, amino acids, and vitamins. These systems resemble ATP-binding cassette-containing transporters and include typical ATPases (e.g., BioM). Absence of extracytoplasmic solute-binding proteins among the members of this group, however, is a distinctive feature. Genome context analyses uncovered that only one-third of the widespread bioY genes are linked to bioMN. Many bioY genes are located at loci encoding biotin biosynthesis, and others are unlinked to biotin metabolic or transport genes. Heterologous expression of the bioMNY operon and of the single bioY of the alpha-proteobacterium Rhodobacter capsulatus conferred biotin-transport activity on recombinant Escherichia coli cells. Kinetic analyses identified BioY as a high-capacity transporter that was converted into a high-affinity system in the presence of BioMN. BioMNY-mediated biotin uptake was severely impaired by replacement of the Walker A lysine residue in BioM, demonstrating dependency of high-affinity transport on a functional ATPase. Biochemical assays revealed that BioM, BioN, and BioY proteins form stable complexes in membranes of the heterologous host. Expression of truncated bio transport operons, each with one gene deleted, resulted in stable BioMN complexes but revealed only low amounts of BioMY and BioNY aggregates in the absence of the respective third partner. The results substantiate our earlier suggestion of a mechanistically novel group of membrane transporters.

Fig. 1.

Bioinformatics-based analysis of biotin-transport proteins. (A) Structure model for BioM (modeled by the 3D-JIGSAW server, based on similarity with other ABC proteins, and modified with CHIMERA) and topological models for BioN and BioY (based on PONGO and PREDICTPROTEIN predictions and multiple hydropathy profile alignments using PEPWINDOWALL). K42 and “B” indicate a Lys residue in the Walker A region and the Walker B region, respectively. (B) Genomic context of bioY genes. The most representative cases of positional clusters with genes encoding components of biotin transporters (bioM and bioN), biotin synthase (bioB), and other biotin biosynthesis genes (bioD and bioA), hypothetical lipoprotein signal peptidase (lpsA), and hypothetical fatty acid synthesis genes (yhfTS, fab) are shown.

Fig. 2.

Biotin uptake of recombinant E. coli. E. coli cells containing pRcBioMNY, pRcBioMY, pRcBioY, pRcBioNY, pRcBioMN, or an empty vector were incubated in glucose-containing phosphate buffer in the presence of 4 nM [³H]biotin. Cellular biotin contents were determined after 3.5 h. The values represent the means of double or triple independent assays.

Fig. 3.

Kinetics of biotin uptake. Initial velocities of biotin uptake into intact E. coli containing pRcBioY (A) or pRcBioMNY (B) were determined at various substrate concentrations. Apparent half saturation (k_M) values and maximal velocities (v_max) of transport were calculated with the help of GRAFIT.

Fig. 4.

Biotin-transport rates of recombinant E. coli. Initial velocities were calculated at biotin concentrations in the picomolar (Left) and nanomolar (Right) ranges. Transport rates of cells producing R. capsulatus BioMNY (black bars) were compared with those containing BioY (white bars in A), BioM_K42NNY (hatched bars in B), BioMY (horizontally hatched bars in C), and BioNY (gray bars in C). The values represent the means of at least two independent assays.

Fig. 5.

Complexes of biotin-transport proteins. Membranes of E. coli cells producing R. capsulatus BioM, BioN, and BioY proteins in combinations and with tags as indicated were solubilized by treatment with DDM. Proteins purified by anti-FLAG affinity chromatography (A) or metal-chelate affinity chromatography (A–D) were separated by SDS/PAGE and subsequently stained (Coomassie) or blotted onto nitrocellulose membranes that were treated with anti-penta-His (anti-His₅; Qiagen) or anti-FLAG (Sigma) antibodies. Numbers (in kDa) represent the positions of standard proteins. (E) Approximately 1 μg of purified protein was subjected to ATPase assays. ATPase activities represent the means of multiple assays with independently purified samples. BioMNY-containing samples yielded ≈400 nmol P_i × (mg protein)⁻¹ × min⁻¹. ATPase activity of all other samples was <50 nmol P_i × (mg protein)⁻¹ × min⁻¹.