title: Functional testing of putative oligopeptide permease (Opp) proteins of Borrelia burgdorferi: a complementation model in opp(-) Escherichia coli.
abstract: Studies of the protein function of Borrelia burgdorferi have been limited by a lack of tools for manipulating borrelial DNA. We devised a system to study the function of a B. burgdorferi oligopeptide permease (Opp) orthologue by complementation with Escherichia coli Opp proteins. The Opp system of E. coli has been extensively studied and has well defined substrate specificities. The system is of interest in B. burgdorferi because analysis of its genome has revealed little identifiable machinery for synthesis or transport of amino acids and only a single intact peptide transporter operon. As such, peptide uptake may play a major role in nutrition for the organism. Substrate specificity for ABC peptide transporters in other organisms is determined by their substrate binding protein. The B. burgdorferi Opp operon differs from the E. coli Opp operon in that it has three separate substrate binding proteins, OppA-1, -2 and -3. In addition, B. burgdorferi has two OppA orthologues, OppA-4 and -5, encoded on separate plasmids. The substrate binding proteins interact with integral membrane proteins, OppB and OppC, to transport peptides into the cell. The process is driven by two ATP binding proteins, OppD and OppF. Using opp-deleted E. coli mutants, we transformed cells with B. burgdorferi oppA-1, -2, -4 or -5 and E. coli oppBCDF. All of the B. burgdorferi OppA proteins are able to complement E. coli OppBCDF to form a functional Opp transport system capable of transporting peptides for nutritional use. Although there is overlap in substrate specificities, the substrate specificities for B. burgdorferi OppAs are not identical to that of E. coli OppA. Transport of toxic peptides by B. burgdorferi grown in nutrient-rich medium parallels borrelial OppA substrate specificity in the complementation system. Use of this complementation system will pave the way for more detailed studies of B. burgdorferi peptide transport than currently available tools for manipulating borrelial DNA will allow.
authors.affiliation: Tupper Research Institute, New England Medical Center, Tufts University School of Medicine, Boston MA 02111, USA.
authors.forename: B
authors.initials: B
authors.lastname: Lin
authors.forename: S A
authors.initials: SA
authors.lastname: Short
authors.forename: M
authors.initials: M
authors.lastname: Eskildsen
authors.forename: M S
authors.initials: MS
authors.lastname: Klempner
authors.forename: L T
authors.initials: LT
authors.lastname: Hu
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D001693:Biological Transport, Active; D015748:Borrelia burgdorferi Group / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics* / Q000254:growth & development; D017353:Gene Deletion; D005798:Genes, Bacterial; D005816:Genetic Complementation Test; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D009842:Oligopeptides / Q000737:chemistry / Q000378:metabolism; D009876:Operon; D010957:Plasmids / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D026901:Membrane Transport Proteins; D009842:Oligopeptides; C037230:oligopeptide permease, Bacteria