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 pH, 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.

Restoration of biofuel production levels and increased tolerance under ionic liquid stress is enabled by a mutation in the essential Escherichia coli gene cydC

“…2 b). As CydC is functional only when in a heterodimeric complex with its partner protein, CydD [ 26 ], our result also suggests that previous studies examining ∆ cydC or ∆ cydD mutants need to be interpreted with caution, and that mutant alleles thought to be total loss-of-function alleles may be partially active [ 23 , 32 ]. We conclude from this result that cydC - D86G could not be encoding a nonfunctional protein in the strain isolated in our study, and that cydC - D86G necessarily retains the activity required for cell viability equivalent to the wild-type gene.…”

NMR metabolomics reveals effects of Cryptosporidium infections on host cell metabolome

“…The following protocol was adapted from published and well-established metabolic extraction methods used for NMR-based untargeted analysis of cell extracts [62][63][64][65]. Samples collected from the mouse experiments were retrieved from the contents of the ileum and surrounding intestinal structure.…”

The cytochrome bd-I respiratory oxidase augments survival of multidrug-resistant Escherichia coli during infection

“…In addition, E. coli also utilises the diiron protein YtfE to repair iron-sulphur clusters damaged by nitrosative stress 18 , and possesses the NO-inducible cytochrome bd -I respiratory oxidase that confers resistance to NO 19 20 . Finally, efflux of glutathione and cysteine by the ABC transporter CydDC has also been shown to provide tolerance to nitric oxide 21 (presumably via reaction of NO with these thiols), although the majority of the NO-tolerance effects result from the requirement of CydDC for the assembly of the NO-tolerant cytochrome bd -I terminal oxidase. The cytochrome bd -I complex, encoded by the cydABX operon, is expressed maximally in microaerobic environments under the dual control of the transcription factors ArcA and FNR 22 23 , and is up-regulated in response to NO 20 .…”