title: 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.
  
  
  RESULTS
  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
  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.
authors.affiliation: Department of Molecular Microbiology, John Innes Centre, Norwich, United Kingdom.
authors.forename: Alexandre
authors.initials: A
authors.lastname: Bourdès
authors.forename: Steven
authors.initials: S
authors.lastname: Rudder
authors.forename: Alison K
authors.initials: AK
authors.lastname: East
authors.forename: Philip S
authors.initials: PS
authors.lastname: Poole
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D001692:Biological Transport; D015374:Biosensing Techniques; D050260:Carbohydrate Metabolism; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D003998:Dicarboxylic Acids / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D023061:Gene Order; D005822:Genetic Vectors / Q000235:genetics; D008024:Ligands; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D011108:Polymers / Q000378:metabolism*; D011485:Protein Binding; D016962:Sinorhizobium meliloti / Q000235:genetics / Q000378:metabolism*; D013816:Thermodynamics
chemical_list: D002352:Carrier Proteins; D003998:Dicarboxylic Acids; D008024:Ligands; D026901:Membrane Transport Proteins; D011108:Polymers; C024617:polyol