Mining the Sinorhizobium meliloti transportome to develop FRET biosensors for sugars, dicarboxylates and cyclic polyols

Abstract

Background: Förster resonance energy transfer (FRET) biosensors are powerful tools to detect biologically important ligands in real time. Currently FRET bisosensors are available for twenty-two compounds distributed in eight classes of chemicals (two pentoses, two hexoses, two disaccharides, four amino acids, one nucleobase, two nucleotides, six ions and three phytoestrogens). To expand the number of available FRET biosensors we used the induction profile of the Sinorhizobium meliloti transportome to systematically screen for new FRET biosensors.

Methodology/principal findings: Two new vectors were developed for cloning genes for solute-binding proteins (SBPs) between those encoding FRET partner fluorescent proteins. In addition to a vector with the widely used cyan and yellow fluorescent protein FRET partners, we developed a vector using orange (mOrange2) and red fluorescent protein (mKate2) FRET partners. From the sixty-nine SBPs tested, seven gave a detectable FRET signal change on binding substrate, resulting in biosensors for D-quinic acid, myo-inositol, L-rhamnose, L-fucose, β-diglucosides (cellobiose and gentiobiose), D-galactose and C4-dicarboxylates (malate, succinate, oxaloacetate and fumarate). To our knowledge, we describe the first two FRET biosensor constructs based on SBPs from Tripartite ATP-independent periplasmic (TRAP) transport systems.

Conclusions/significance: FRET based on orange (mOrange2) and red fluorescent protein (mKate2) partners allows the use of longer wavelength light, enabling deeper penetration of samples at lower energy and increased resolution with reduced back-ground auto-fluorescence. The FRET biosensors described in this paper for four new classes of compounds; (i) cyclic polyols, (ii) L-deoxy sugars, (iii) β-linked disaccharides and (iv) C4-dicarboxylates could be developed to study metabolism in vivo.

Figure 1. FRET vector and insertion sites for cloning SBPs.

(A) vector pCYS and cloning sites of (B) pCYS and (C) pROS. Direction of energy transfer between fluorescent proteins is shown. Fluorescent proteins are coloured coded; N-terminal His-tagged-eCFP (blue), Aphrodite (yellow), N-terminal His-tagged-mKate2 (red), and mOrange2 (orange). The position of core SBP insertion (green) is shown, together with the amino acid sequences of N-terminal (GTTS) and C-terminal (TSL/TSLE) linkers.

Figure 2. Normalised ligand binding isotherms for FRET biosensors.

(A) cyclic polyols; SMb20036-CY binding D-quinic acid and SMb20712-CY binding myo-inositol, (B) monosaccharides; SMc02774-CY binding L-fucose, RL2376-CY binding D-galactose, SMc02324-CY and SMc02324-RO binding L-rhamnose, (C) disaccharides; SMc04259-CY binding cellobiose and gentiobiose, (D) C4- dicarboxylates; rcc03024-CY binding L-malate, succinate, oxaloacetate and fumarate. The biosensor is designated by the gene encoding the SBP protein core, followed by the fluorescent protein pair (where C is CFP (eCFP), Y is YFP (Aphrodite), R is RFP (mKate2) and O is OFP (mOrange2)). All data points are an average of at least two independent protein purifications tested on technical quadruplicates.