CydDC-mediated reductant export in Escherichia coli controls the transcriptional wiring of energy metabolism and combats nitrosative stress

Louise V Holyoake, Stuart Hunt, Guido Sanguinetti, Gregory M Cook, Mark J Howard, Michelle L Rowe, Robert K Poole, Mark Shepherd

Research output: Contribution to journalArticlepeer-review

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 homeostasis, 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. 1H 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 homeostasis 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.
Original languageEnglish
Pages (from-to)693-701
Number of pages9
JournalBiochemical Journal
Volume473
Issue number6
DOIs
Publication statusPublished - 15 Mar 2016

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