title: From substrate specificity to promiscuity: hybrid ABC transporters for osmoprotectants.
abstract: The ABC-transporters OpuB and OpuC from Bacillus subtilis function as osmoprotectant import systems. Their structural genes have most likely evolved through a duplication event but the two transporters are remarkably different in their substrate profile. OpuB possesses narrow substrate specificity, while OpuC is promiscuous. We assessed the functionality of hybrids between these two ABC-transporters by reciprocally exchanging the coding regions for the OpuBC and OpuCC substrate-binding proteins between the corresponding opuB and opuC operons. Substantiating the critical role of the binding protein in setting the substrate specificity of ABC transporters, OpuB::OpuCC turned into a promiscuous system, while OpuC::OpuBC now exhibited narrow substrate specificity. Both hybrid transporters possessed a high affinity for their substrates but the transport capacity of the OpuB::OpuCC system was moderate due to the synthesis of only low amounts of the xenogenetic OpuCC protein. Suppressor mutations causing single amino acid substitutions in the GbsR repressor controlling the choline to glycine betaine biosynthesis pathway greatly improved OpuB::OpuCC-mediated compatible solute import through transcriptional up-regulation of the hybrid opuB::opuCC operon. Collectively, we demonstrate for the first time that one can synthetically switch the substrate specificity of a given ABC transporter by combining its core components with a xenogenetic ligand-binding protein.
authors.affiliation: Laboratory for Molecular Microbiology, Department of Biology, Philipps-University Marburg, Karl-von-Frisch Str. 8, Marburg, D-35043, Germany.
authors.forename: Laura
authors.initials: L
authors.lastname: Teichmann
authors.affiliation: Laboratory for Molecular Microbiology, Department of Biology, Philipps-University Marburg, Karl-von-Frisch Str. 8, Marburg, D-35043, Germany.|LOEWE-Center for Synthetic Microbiology, Philipps-University Marburg, Hans-Meerweinstr. 6, Marburg, D-35043, Germany.
authors.forename: Chiliang
authors.initials: C
authors.lastname: Chen
authors.affiliation: Laboratory for Molecular Microbiology, Department of Biology, Philipps-University Marburg, Karl-von-Frisch Str. 8, Marburg, D-35043, Germany.
authors.forename: Tamara
authors.initials: T
authors.lastname: Hoffmann
authors.affiliation: Institute of Biochemistry, Heinrich-Heine-University Düsseldorf, Universitätsstr. 1, Düsseldorf D-40225, Germany.
authors.forename: Sander H J
authors.initials: SHJ
authors.lastname: Smits
authors.affiliation: Institute of Biochemistry, Heinrich-Heine-University Düsseldorf, Universitätsstr. 1, Düsseldorf D-40225, Germany.
authors.forename: Lutz
authors.initials: L
authors.lastname: Schmitt
authors.affiliation: Laboratory for Molecular Microbiology, Department of Biology, Philipps-University Marburg, Karl-von-Frisch Str. 8, Marburg, D-35043, Germany.|LOEWE-Center for Synthetic Microbiology, Philipps-University Marburg, Hans-Meerweinstr. 6, Marburg, D-35043, Germany.
authors.forename: Erhard
authors.identifier: http://orcid.org/0000-0002-2225-7005
authors.initials: E
authors.lastname: Bremer
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000378:metabolism*; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism*; D001622:Betaine / Q000378:metabolism; D002794:Choline / Q000378:metabolism; D009876:Operon; D064587:Osmoregulation; D013379:Substrate Specificity; D014158:Transcription, Genetic; D015854:Up-Regulation
chemical_list: D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins; C119749:OpuB protein, Bacillus subtilis; C119750:OpuC protein, bacteria; D001622:Betaine; D002794:Choline