Functional mining of transporters using synthetic selections

Journal name:
Nature Chemical Biology
Volume:
12,
Pages:
1015–1022
Year published:
DOI:
doi:10.1038/nchembio.2189
Received
Accepted
Published online

Abstract

Only 25% of bacterial membrane transporters have functional annotation owing to the difficulty of experimental study and of accurate prediction of their function. Here we report a sequence-independent method for high-throughput mining of novel transporters. The method is based on ligand-responsive biosensor systems that enable selective growth of cells only if they encode a ligand-specific importer. We developed such a synthetic selection system for thiamine pyrophosphate and mined soil and gut metagenomes for thiamine-uptake functions. We identified several members of a novel class of thiamine transporters, PnuT, which is widely distributed across multiple bacterial phyla. We demonstrate that with modular replacement of the biosensor, we could expand our method to xanthine and identify xanthine permeases from gut and soil metagenomes. Our results demonstrate how synthetic-biology approaches can effectively be deployed to functionally mine metagenomes and elucidate sequence–function relationships of small-molecule transport systems in bacteria.

At a glance

Figures

  1. Synthetic selection system for thiamine uptake.
    Figure 1: Synthetic selection system for thiamine uptake.

    (a) The dual ribosome binding site (RBS) selection system controlling chloramphenicol-resistance and spectinomycin-resistance genes (cat and aadA). Translation of the resistance genes is enabled only after binding of TPP. The dual selection reduces the number of false positives, as false triggering (e.g., by mutation of one riboswitch) will not lead to cell growth. (b) Thiamine dose-response curves of the E. coli selection strain at three time points. (c) Intracellular concentrations of TMP and TPP in E. coli Dh10B grown in cultures fed with thiamine at different concentrations. A high extracellular thiamine level is reflected by a higher intracellular TMP/TPP level owing to diffusion and phosphorylation by kinases. Data in c and d represent averages of biological triplicate experiments, and error bars represent s.d. (n = 3). (d) Agar-plate-based growth assays of the E. coli selection strain under different selection conditions: chloramphenicol (CAM), spectinomycin (SPEC), chloramphenicol and spectinomycin (CAM + SPEC), and no antibiotics. When approximately 100 cells were plated, thiamine supplementation was required for growth under all selection conditions (top and middle rows). When many cells (>107) were plated in the absence of thiamine, the dual selection eradicated false positive growth completely (bottom row). The images shown are representative of biological replicates (n = 3).

  2. Functional metagenomic selection of thiamine transporters.
    Figure 2: Functional metagenomic selection of thiamine transporters.

    (a) Total DNA extracted from soil and gut fecal samples (metagenomic DNA) was fragmented into ~2-kb fragments, cloned into an expression vector and transformed into an E. coli host strain harboring the thiamine selection system. The cell library was plated on selective growth medium supplemented with low amounts of thiamine. Cells that expressed a thiamine-uptake transporter from the metagenomic DNA insert imported extracellular thiamine and had increased intracellular TPP concentrations, leading to induction of riboswitch-mediated antibiotic resistance. (b) Colony formation on selective agar plates after a 40-h incubation of ~25 million cells of the selection strain transformed with either a human fecal gut metagenomic library (right) or a control library (left). (c) Overview of functionally selected metagenomic fragments encoding thiamine transporters. Left, neighbor-joining tree based on multiple alignment of the pnuT nucleotide sequences. Right, phenotypic response (spot assays) of each selection strain when expressing the metagenomic insert in the presence of no or low amounts of thiamine. Images are representative of three replicates. (d) Thiamine dose-response curves of the wild-type strain (WT) and two strains expressing functionally selected thiamine transporters. (e,f) Intracellular concentrations of TPP (e) and TMP (f) in a functionally selected strain (CON31) and the wild-type strain at different concentrations of exogenously added thiamine. Data in df represent averages of biological triplicate experiments, and error bars represent s.d. (n = 3).

  3. Phylogenetic and functional relationship of Pnu transporters.
    Figure 3: Phylogenetic and functional relationship of Pnu transporters.

    (a) Phylogenetic relationships of Pnu transporters derived from soil and gut microbiomes (labeled CON1CON26) and other selected Pnu transporters available from GenBank, displayed in an unrooted neighbor-joining tree. Colors in the inner circle denote genetic colocalization of Pnu transporters with genes involved in salvage or biosynthesis pathways of specified molecules. Dashed lines indicate the presence of homologs of previously characterized thiamine transporters in the genome; solid lines indicate that no known thiamine transporter is present in genome. Open circles at the outer edge of the tree denote an incomplete thiamine pathway, leading to dependency on exogenous thiamine uptake. Red dots indicate thiamine transporters that were functionally validated in this study. (b) Characterization of heterologous Pnu transporters from indicated species expressed in the E. coli selection strain. Each strain was serially diluted tenfold and spotted on the specified selective medium. Growth at low thiamine concentrations in selective medium indicated an active thiamine transporter. Shown are representative images of biological replicates (n = 3).

  4. Riboswitch-based xanthine alkaloid selection system.
    Figure 4: Riboswitch-based xanthine alkaloid selection system.

    (a) The theophylline ribosome binding site (RBS) coupled to genes for chloramphenicol resistance (cat) and spectinomycin resistance (aadA). (b) Addition of theophylline induces resistance to chloramphenicol (CAM) and spectinomycin (SPEC) individually or in combination. (c) Mean diameter of colonies of the selection strain at various theophylline concentrations, with or without selection (30 μg/ml chloramphenicol and 80 μg/ml spectinomycin), after 22 h of growth at 37 °C. Error bars indicate ±s.d. of biological replicates (n = 3). (d) Plating of ~108 cells with single or dual selection in the absence of inducer ligand. Dual selection eliminated false positive growth at high cell densities. Images in b and d are representative of biological replicates (n = 3).

  5. Functional metagenomic selections identify xanthine uptake transporters.
    Figure 5: Functional metagenomic selections identify xanthine uptake transporters.

    (a) Total DNA extracted from soil and gut fecal samples (metagenomic DNA) was fragmented into 1- to 3-kb fragments, cloned into an expression vector and transformed into an E. coli host strain harboring the xanthine alkaloid selection system. The cell library was plated on LB agar plates under selective conditions. (b) Cells expressing functional xanthine uptake transporters from the metagenomic DNA inserts import extracellular xanthine and have increased intracellular xanthine concentrations, leading to induction of ribosome binding site (RBS)-mediated antibiotic resistance. (c) Xanthine dose-response curves for strains expressing functionally selected xanthine transporters (insert 3 and insert 4) and the wild-type strain (WT; harboring the selection plasmid but with no xanthine permease overexpressed), grown under selective conditions. (d) Intracellular concentrations of xanthine measured by LC-MS at 0 μM and 400 μM exogenously added xanthine. Error bars in c and d represent ±s.d. of biological replicates (n = 3).

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Author information

Affiliations

  1. The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kongens Lyngby, Denmark.

    • Hans J Genee,
    • Anne P Bali,
    • Søren D Petersen,
    • Solvej Siedler,
    • Mads T Bonde,
    • Mette Kristensen,
    • Scott J Harrison &
    • Morten O A Sommer
  2. Biosyntia ApS, Copenhagen, Denmark.

    • Hans J Genee,
    • Anne P Bali &
    • Luisa S Gronenberg

Contributions

H.J.G. and M.O.A.S. conceived the study. H.J.G. and S.D.P. developed the thiamine functional selection system, and H.J.G. and A.P.B. performed functional metagenomic selections for thiamine uptake. H.J.G., M.K. and L.S.G. developed the HPLC assay for measurement of thiamines. H.J.G. and A.P.B. cloned heterologous genes. H.J.G. and M.T.B. developed the xanthine alkaloid selection system, and H.J.G. performed functional metagenomic selections and analysis. H.J.G. and S.S. performed dose-response characterizations of selected xanthine importers. S.J.H. developed the LC-MS method for xanthine alkaloids and performed measurements of samples prepared by S.S. and H.J.G. H.J.G. wrote the manuscript with contributions from all other authors.

Competing financial interests

H.J.G. and M.O.A.S. are named on a pending patent application relating to dual genetic selection systems (WO 2014/187829 A1).

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Supplementary information

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  1. Supplementary Text and Figures (1,972 KB)

    Supplementary Results, Supplementary Figures 1–4 and Supplementary Tables 1–10.

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    Comparative genomics of thiamine biosynthesis, salvage and transport of 1752 complete bacterial genomes.

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