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).

slide1
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).

slide1
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 Single Molecule FRET to Understand Substrate Binding Domains

By Bryony Ackroyd

Twitter: @BryonyAckroyd

ABC transporters can be either import or export systems for cells. They consist of two transmembrane domains (TMDs) and two cytoplasmic nucleotide binding domains (NBDs). ABC importers also use substrate binding domains (SBDs) or substrate binding proteins (SBPs). SBPs are separate proteins present in the periplasm, however SBDs are fused to the TMDs. Some ABC transporters even have two or three SBDs fused together in tandem. Although this is a known phenomenon, very little is understood about the system and how ABC transporters are able to interact with multiple and structurally distinct SBDs. In the work carried out in this particular paper the group focusses on GlnPQ from L. lactis, a Gram-positive bacterium, that imports asapargine, glutamine and glutamate via two different SBDs.

Although there are crystal structures and NMR data available for SBDs, not much is known about the mechanism of ligand binding e.g. induced fit or conformational selection. Bearing this is mind Poolman et al., used a unique combination of techniques to probe the conformational dynamics of the SBDs, single-molecule Forster resonance energy transfer (smFRET) coupled with isothermal titration calorimetry (ITC). Using this strategy the group was able to provide mechanistic insight into the transport mechanisms of ABC importers, showing that the SBDs of GlnPQ bind ligands via an induced-fit mechanism.

The SBDs can be in one of four states, closed-ligand bound (CL), open (O), partially closed (PC) or closed (C). The induced-fit mechanism of binding triggers the CL state from the O state, however in the conformation-selection model the SBD can be in the PC or C states without a ligand bound. Ligand binding stabilises the PC state and therefore pushes the SBD to the CL conformation. These differing conformational states were examined via smFRET and the changes between states was observed via FRET efficiency. The experiment was designed so that the O conformation of the SBD gave a low FRET efficiency and the closed conformations gave higher FRET efficiency. Fluorophores were designed on the SBDs to be between 3-6 nm apart in both the closed and open states.

The SBD1 of GlnPQ binds asparagine with high affinity and glutamine with low affinity whereas SBD2 solely binds glutamine with a high affinity.

slide1
Single molecule dynamics of SBDs probed with smFRET. (a) Schematic showing immobilisation of histidine tagged SBDs to a PEG-biotin coated surface in a flow cell. The surface scan on the right is shown in flase colour, orange indicates double-labelled SBDs, green is SBDs with only donor fluorophore and red is SBDs with only acceptor fluorophores. (b-d) Representative fluorescence time traces, blue is donor signal, red acceptor signal, grey FRET signal and orange is the fit. These graphs show that the FRET efficiency of SBD1 and SBD2 increased as the concentration of substrate increased. This indicates that closing of SBD1 and SBD2 increased as substrate concentration increased, in keeping with the induced-fit model.

By measuring the fluorescence emitted from an SBD immobilised on a surface when varying concentrations of substrate were added, it was possible to determine the conformational state of the SBD and therefore whether the induced-fit or conformational-selection model was being employed by the SBD. Poolman et al., showed that in the absence of ligand the SBDs of GlnPQ were continuously in the O conformation and not in the PC or C conformations, therefore demonstrating the induced-fit model is used by the SBDs of GlnPQ.

This clever and unique technique was able to beautifully show the different conformations of the SBDs and conformational changes that occur within SBDs during ligand binding. Hopefully this technique will be employed more widely in the future to elucidate ligands, binding mechanisms and conformations of other SBDs and SBPs.

 

Source: Poolman et al., (2015). Conformational dynamics in substrate-binding domains influences transport in the ABC importer GlnPQ. Nature Structural and Molecular Biology 22, 57–64.