title: Cysteine is exported from the Escherichia coli cytoplasm by CydDC, an ATP-binding cassette-type transporter required for cytochrome assembly.
abstract: Assembly of Escherichia coli cytochrome bd and periplasmic cytochromes requires the ATP-binding cassette transporter CydDC, whose substrate is unknown. Two-dimensional SDS-PAGE comparison of periplasm from wild-type and cydD mutant strains revealed that the latter was deficient in several periplasmic transport binding proteins, but no single major protein was missing in the cydD periplasm. Instead, CydDC exports from cytoplasm to periplasm the amino acid cysteine, demonstrated using everted membrane vesicles that transported radiolabeled cysteine inward in an ATP-dependent, uncoupler-independent manner. New pleiotropic cydD phenotypes are reported, including sensitivity to benzylpenicillin and dithiothreitol, and loss of motility, consistent with periplasmic defects in disulfide bond formation. Exogenous cysteine reversed these phenotypes and affected levels of periplasmic c-type cytochromes in cydD and wild-type strains but did not restore cytochrome d. Consistent with CydDC being a cysteine exporter, cydD mutant growth was hypersensitive to high cysteine concentrations and accumulated higher cytoplasmic cysteine levels, as did a mutant defective in orf299, encoding a transporter of the major facilitator superfamily. A cydD orf299 double mutant was extremely cysteine-sensitive and had higher cytoplasmic cysteine levels, whereas CydDC overexpression conferred resistance to high extracellular cysteine concentrations. We propose that CydDC exports cysteine, crucial for redox homeostasis in the periplasm.
authors.affiliation: Department of Molecular Biology and Biotechnology, Krebs Institute for Biomolecular Research, University of Sheffield, Firth Court, Western Bank, Sheffield S10 2TN, United Kingdom.
authors.forename: Marc S
authors.initials: MS
authors.lastname: Pittman
authors.forename: Hazel
authors.initials: H
authors.lastname: Corker
authors.forename: Guanghui
authors.initials: G
authors.lastname: Wu
authors.forename: Marie B
authors.initials: MB
authors.lastname: Binet
authors.forename: Arthur J G
authors.initials: AJ
authors.lastname: Moir
authors.forename: Robert K
authors.initials: RK
authors.lastname: Poole
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000737:chemistry / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D001692:Biological Transport; D002462:Cell Membrane / Q000378:metabolism; D002465:Cell Movement; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism*; D004220:Disulfides; D004229:Dithiothreitol / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins; D007785:Lactose / Q000378:metabolism; D009154:Mutation; D010084:Oxidation-Reduction; D010400:Penicillin G / Q000494:pharmacology; D019897:Periplasm / Q000378:metabolism; D010641:Phenotype; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D013347:Subcellular Fractions; D013997:Time Factors
chemical_list: D018528:ATP-Binding Cassette Transporters; C089838:CydC protein, E coli; C089839:CydD protein, E coli; D004220:Disulfides; D029968:Escherichia coli Proteins; D000255:Adenosine Triphosphate; D007785:Lactose; D003545:Cysteine; D010400:Penicillin G; D004229:Dithiothreitol