Sequence-independent assay for importers results in validation of novel thiamine uptake system

By Ivan Gyulev

Twitter: @IvanGyulev

A study published in October 2016 in Nature Chemical Biology by Prof Morten Sommer and colleagues reported the use of a sequence-independent screen for the identification of novel bacterial small molecule transporters. The assay is based on a synthetic selection system that relies on riboswitch biosensors. A riboswitch (small molecule-binding RNAs) is located in the 5’UTR of an antibiotic resistance gene and inhibits its translation by sequestering its ribosome binding sites. However, when the riboswitch’s ligand is present in sufficient concentration intracellularly, the translational repression is alleviated and the gene is expressed, thereby conferring resistance against its respective antibiotic. By using two antibiotics and two resistance genes, the researchers dramatically reduced the rate of false positive mutants. Using this assay, one can screen a library of metagenomic fragments for ligand importers. To screen for importers of a new ligand researchers only need to change the riboswitch. Genee et al. demonstrated the modularity of their design by implementing it in the discovery of thiamine and xanthine importers.

The outline of the synthetic selection system in the case of selection for thiamine importers (using the ThiM19 riboswitch) is shown below (taken from Figure 1a from the paper).

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Figure 1. Synthetic selection system for thiamine uptake. (a) The dual ribosome binding site (RB S) 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.

After validating the synthetic selection system the authors then screened metagenomic DNA libraries from soil and gut fecal samples for thiamine importers and discovered a novel class of thiamine importer – PnuT (screen strategy outlined in Figure 2a from the paper).

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Figure 2. Functional metagenomic selection of thiamine transporter. (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.

PnuT has homology to the nicotinamide riboside and nicotinamide mononucleotide transporter PnuC and had been previously predicted to be involved in thiamine uptake. PnuT’s function as a thiamine transporter was validated by selective growth and intracellular thiamine quantification by HPLC. Further bioinformatics analysis, revealed that PnuT is very common in the Bacteroidetes phylum. The authors then looked at phylogeny and the pattern of Pnu transporters’ co-localization with genes from thiamine salvage or biosynthesis pathway across genomes.

Finally, a previously published synthetic riboswitch (derived from aptamer identified by SELEX) was utilized to select for xanthine importers. The screen resulted in the isolation of two unique ORFs with more than 99% sequence identity at the amino acid level with known xanthine permeases from the NAT/NCS2 nucleobase-ascorbate transporter family. In both screens, fragments containing multi-drug resistance proteins were isolated

The authors highlight several limitations of the current screen technique –firstly, the discovery of transporters relying on multiple protein complexes (such as the thiamine importer from Bacillus/Clostridium ECF-ThiT or the E.coli ThiBPQ) would require larger metagenomic (or genomic) fragments (in the present study the range was between 1kb and 3kb but it is possible to use larger fragments). Secondly, these proteins are not necessarily encoded in the same chromosomal region. Thirdly, naturally-occurring riboswitches and allosteric transcription factors are the go-to choice for small-molecule biosensors but synthetic riboswitches are more difficult to develop synthetically. Reportedly, one way to go around this is to construct a metabolic pathway bridging an undetectable compound to a detectable one.

Altogether, the novel synthetic selection strategy is a powerful tool for the isolation and validation of novel importers from metagenomic libraries or putative transporters from genomic sequences. It is also impressive that in its first implementation the assay led to the experimental validation of a novel import system.

 

Source: Genee, H.J. et al., (2016).Functional mining of transporters using synthetic selections. Nat. Chem. Biol.  12, 1015-1022.

Using a periplasmic binding protein as a biosensor for thiamine

By Sophie Rugg

Twitter: @sophiejrugg

Thiamine, also known as vitamin B1, is an essential micronutrient with an important role in metabolism for all life forms. Thiamine can’t be synthesised by animals, and so has to be obtained from their diet. Until recently, detecting thiamine was limited to either the use of expensive high-performance liquid chromatography (HPLC), or a slow assay involving microbial growth. This is because there is no antibody available that is specific for thiamine, making commonly used high throughput detection techniques such as enzyme-linked immunosorbent assay (ELISA) impossible.

Periplasmic binding proteins are components of the ABC transporters of Gram-negative bacteria, and bind their substrates with high affinity and specificity to enable them to be transported into the bacterial cell. These properties of high affinity binding and specificity make periplasmic binding proteins ideal for use as the recognition element in a biosensor. As bacterial ABC transporters are used for the import of nutrients, including a transporter for thiamine, many of these binding proteins have evolved to recognise small molecules which it may be difficult to raise an antibody against.

Edwards et al., (2016) developed a biosensor for thiamine based on the periplasmic binding protein for thiamine from Escherichia coli. This binding protein was incorporated into dye-encapsulating liposomes in order to amplify the signal. Immobilised to the surface of a streptavidin coated plate is biotin conjugated thiamine analogue. The thiamine analogue is connected to the biotin via a long polyethylene glycol (PEG)linker, so that the thiamine analogue doesn’t get in the way of the biotin binding the streptavidin coating. The immobilised thiamine analogue binds to the periplasmic binding protein with lower affinity than thiamine. After the thiamine containing sample has been added, any material not bound to the surface is removed. Any liposomes still stuck to the surface of the plate are lysed and the resulting dye concentration is inversely proportional to the thiamine concentration.

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Overview of assay for thiamine detection taken from Edwards et al., (2016). Competitive assay with biotin conjugated thiamine analogue immobilized via streptavidin in microtiter plates and detected via periplasmic binding protein for thiamine conjugated to the lipid bilayer of dye encapsulating liposomes (left). After competition with sample thiamine, unbound materials are removed (middle) and liposomes remaining bound are lysed to release dye yielding a signal inversely proportional to thiamine concentration (right).

This work shows that periplasmic binding proteins can be used effectively in biosensors , particularly where there is no antibody available. With the wide range of periplasmic binding proteins evolved by bacteria to be able to transport nutrients into their cells, this technique is open to use across a wide range of applications provided that a suitable analogue of the substrate can be immobilised to the surface of a plate.

Source: Edwards et al., 2016. High-Throughput Detection of Thiamine Using Periplasmic Binding Protein-Based Biorecognition. Analytical  Chemisty88 (16), pp 8248–8256. DOI: 10.1021/acs.analchem.6b02092