A novel class of modular transporters for vitamins in prokaryotes

Abstract

The specific and tightly controlled transport of numerous nutrients and metabolites across cellular membranes is crucial to all forms of life. However, many of the transporter proteins involved have yet to be identified, including the vitamin transporters in various human pathogens, whose growth depends strictly on vitamin uptake. Comparative analysis of the ever-growing collection of microbial genomes coupled with experimental validation enables the discovery of such transporters. Here, we used this approach to discover an abundant class of vitamin transporters in prokaryotes with an unprecedented architecture. These transporters have energy-coupling modules comprised of a conserved transmembrane protein and two nucleotide binding proteins similar to those of ATP binding cassette (ABC) transporters, but unlike ABC transporters, they use small integral membrane proteins to capture specific substrates. We identified 21 families of these substrate capture proteins, each with a different specificity predicted by genome context analyses. Roughly half of the substrate capture proteins (335 cases) have a dedicated energizing module, but in 459 cases distributed among almost 100 gram-positive bacteria, including numerous human pathogens, different and unrelated substrate capture proteins share the same energy-coupling module. The shared use of energy-coupling modules was experimentally confirmed for folate, thiamine, and riboflavin transporters. We propose the name energy-coupling factor transporters for the new class of membrane transporters.

FIG. 1.

Distribution and comparative genomic analysis of the new class (ECF class) of prokaryotic transporters. (A) Classification and abundance of group I and group II ECF transporters. Group I transporters have a substrate-specific S component and a dedicated AT module encoded by linked genes. Group II transporters have individual S components and shared AT modules that are unlinked to S components. Composite bar colors indicate the contributions of transporters found in different taxa to the total transporter number. Note that the S components BioY, CbrT, HtsT, and QrtT (and, to a lesser extent, RibU, PanT, HmpT, ThiW, QueT, and CblT) occur in both groups. (B) Comparative genomic analysis of the identified transporter families including their domain compositions, names, predicted substrate specificities, and example gene identifications. Substrate-specific integral membrane components (S) are shown by black rectangles, conserved transmembrane components (T) are shown by blue rectangles, and ATPase domains (A) are shown by red circles. Examples of genome context evidence (e.g., gene coregulation or colocalization) supporting the predicted transporter function are shown on the right.

FIG. 2.

Genomic organization of the group I ECF transporters containing S components from the TrpP, RibU, PanT, HmpT, ThiW, QueT, and CblT families. Genes encoding substrate capture S components and A and T components of the dedicated energy-coupling modules are shown by black, red, and blue arrows, respectively.

FIG. 3.

Riboflavin uptake in Bacillus subtilis. Shown are data for the effect of disrupting ribU (triangles), ecfT (circles), or yceI (squares) on [³H]riboflavin uptake by B. subtilis. (The yceI gene served as a control; it encodes a protein unrelated to ECF transporters.) Cells were grown without riboflavin to an OD₆₀₀ value of 0.5, and [³H]riboflavin was added (17 nM final concentration). At the times indicated, cells were harvested by filtration and washed, and their ³H contents were determined. Values are means of duplicates; error bars indicate ranges.

FIG. 4.

Folate and thiamine uptake evidences. (A) Folate uptake by E. coli cells coexpressing L. mesenteroides folT and ecfAA′T. Recombinants containing empty vector or expression plasmids for the production of FolT, EcfAA′T, or FolT plus EcfAA′T were spotted (10 μl) after serial 10-fold dilutions onto nonsupplemented minimal medium and onto minimal medium containing 4-aminobenzoate (3.6 μM) or 5-formyltetrahydrofolate (11 μM). Plates were incubated for 48 h at 37°C. (B and C) Uptake of [³H]thiamine (B) and [³H]5-formyltetrahydrofolate (C) by L. lactis containing empty vectors (triangles) or carrying L. casei thiT or folT (circles), ecfAA′T (diamonds), or thiT or folT coexpressed with ecfAA′T (squares). Cells were energized with glucose (black symbols) or deenergized with 2-deoxyglucose (open symbols).

FIG. 5.

Physical interaction between S and ECFAA′T components from L. mesenteroides. Membranes of recombinant E. coli cells producing the proteins indicated in the upper six lines were solubilized with n-dodecyl-β-d-maltoside and subjected to Ni-chelate affinity chromatography prior to SDS-PAGE and Western blotting or were separated by SDS-PAGE without chromatography. The bottom shows strips of the blots probed with anti-penta-His or anti-FLAG antibodies.