Abstract
BackgroundFörster 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 FindingsTwo 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/SignificanceFRET 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.
Highlights
Bacteria have evolved a wide variety of metabolic strategies to cope with varied environments
For S. meliloti data came from a comprehensive study using reporter fusions [9], while for R. leguminosarum, data was available from microarray experiments
The gateway-compatible Forster resonance energy transfer (FRET) vector pGWF1 is available for the cloning of genes encoding SBPs between two fluorescent protein partners (N-terminal cyan fluorescent protein (CFP) and Cterminal yellow fluorescent protein (YFP)) but this creates long linkers of 20 and 21 amino acids between the fluorescent proteins and SBP [21,22,23]
Summary
Bacteria have evolved a wide variety of metabolic strategies to cope with varied environments. The number of SBP-dependent transporters in rhizobia is much higher than in the majority of other bacteria so far sequenced. This may be due to the need for the acquisition of a broad range of growth-limiting nutrients from soil, rhizosphere and during symbiosis, facilitated by the high affinity of SBPs for specific solutes [1,2,3,4]. The known SBPdependent secondary transporters are currently placed in two families, the TRAP transporters and a smaller family of tripartite tricarboxylate transporters (TTT) Both families have a similar tripartite structure comprising an SBP and two unequally sized integral membrane components, but have unrelated amino acid sequences [7,8]. 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
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