title: CydDC-mediated reductant export in Escherichia coli controls the transcriptional wiring of energy metabolism and combats nitrosative stress.
abstract: The glutathione/cysteine exporter CydDC maintains redox balance in Escherichia coli. A cydD mutant strain was used to probe the influence of CydDC upon reduced thiol export, gene expression, metabolic perturbations, intracellular pH homoeostasis and tolerance to nitric oxide (NO). Loss of CydDC was found to decrease extracytoplasmic thiol levels, whereas overexpression diminished the cytoplasmic thiol content. Transcriptomic analysis revealed a dramatic up-regulation of protein chaperones, protein degradation (via phenylpropionate/phenylacetate catabolism), β-oxidation of fatty acids and genes involved in nitrate/nitrite reduction. (1)H NMR metabolomics revealed elevated methionine and betaine and diminished acetate and NAD(+) in cydD cells, which was consistent with the transcriptomics-based metabolic model. The growth rate and ΔpH, however, were unaffected, although the cydD strain did exhibit sensitivity to the NO-releasing compound NOC-12. These observations are consistent with the hypothesis that the loss of CydDC-mediated reductant export promotes protein misfolding, adaptations to energy metabolism and sensitivity to NO. The addition of both glutathione and cysteine to the medium was found to complement the loss of bd-type cytochrome synthesis in a cydD strain (a key component of the pleiotropic cydDC phenotype), providing the first direct evidence that CydDC substrates are able to restore the correct assembly of this respiratory oxidase. These data provide an insight into the metabolic flexibility of E. coli, highlight the importance of bacterial redox homoeostasis during nitrosative stress, and report for the first time the ability of periplasmic low molecular weight thiols to restore haem incorporation into a cytochrome complex.
authors.affiliation: School of Biosciences, University of Kent, Canterbury CT2 7NJ, U.K.
authors.forename: Louise V
authors.initials: LV
authors.lastname: Holyoake
authors.affiliation: Department of Molecular Biology and Biotechnology, The University of Sheffield, Sheffield S10 2TN, U.K.
authors.forename: Stuart
authors.initials: S
authors.lastname: Hunt
authors.affiliation: School of Informatics, The University of Edinburgh, Informatics Forum, 10 Crichton Street, Edinburgh EH8 9AB, U.K.
authors.forename: Guido
authors.initials: G
authors.lastname: Sanguinetti
authors.affiliation: Department of Microbiology and Immunology, University of Otago, P.O. Box 56, 720 Cumberland Street, Dunedin 9054, New Zealand.
authors.forename: Gregory M
authors.initials: GM
authors.lastname: Cook
authors.affiliation: School of Biosciences, University of Kent, Canterbury CT2 7NJ, U.K.
authors.forename: Mark J
authors.initials: MJ
authors.lastname: Howard
authors.affiliation: School of Biosciences, University of Kent, Canterbury CT2 7NJ, U.K.
authors.forename: Michelle L
authors.initials: ML
authors.lastname: Rowe
authors.affiliation: Department of Molecular Biology and Biotechnology, The University of Sheffield, Sheffield S10 2TN, U.K.
authors.forename: Robert K
authors.initials: RK
authors.lastname: Poole
authors.affiliation: School of Biosciences, University of Kent, Canterbury CT2 7NJ, U.K. M.Shepherd@kent.ac.uk.
authors.forename: Mark
authors.initials: M
authors.lastname: Shepherd
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000235:genetics / Q000378:metabolism*; D001692:Biological Transport; D004734:Energy Metabolism / Q000502:physiology*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D017353:Gene Deletion; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology*; D008954:Models, Biological; D015538:Nitrosation; D010084:Oxidation-Reduction; D013312:Stress, Physiological; D014158:Transcription, Genetic
chemical_list: D018528:ATP-Binding Cassette Transporters; C089838:CydC protein, E coli; C089839:CydD protein, E coli; D029968:Escherichia coli Proteins
keywords: cysteine; cytochrome; glutathione; nitric oxide; nitrosative stress; redox regulation