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

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

doi:10.1042/bj20150536

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.

Highlights

Phenotyping of Escherichia coli cydDC mutants reported that this locus was required for the correct assembly of cytochrome bd-type quinol oxidases [1,2]
The phenotype of the CydDC transporter (cydD) mutant strain is pleiotropic, exhibiting sensitivity to benzylpenicillin and dithiothreitol, loss of motility and the absence of holocytochrome c. These characteristics are reminiscent of E. coli dsb mutants [7], implicating defective disulfide bond formation/isomerization in the manifestation of these traits in cydDC mutant strains. These phenotypes, together with the loss of periplasmic cytochrome b562, can all be corrected by the addition of cysteine or reduced glutathione (GSH) to the growth medium [5,6,8], the addition of GSH or cysteine alone cannot restore the assembly of bd-type quinol oxidases [5,6]
Exponentially-growing wild-type, cydD and complemented cydD cells were grown in defined medium and the reduced thiol concentrations of the growth medium and spheroplast preparations were measured (Figure 1) to provide a measure of how CydDC affects redox balance across the cytoplasmic membrane

Summary

Introduction

Phenotyping of Escherichia coli cydDC mutants reported that this locus was required for the correct assembly of cytochrome bd-type quinol oxidases [1,2]. These characteristics are reminiscent of E. coli dsb mutants [7], implicating defective disulfide bond formation/isomerization in the manifestation of these traits in cydDC mutant strains These phenotypes, together with the loss of periplasmic cytochrome b562 (a typical cydDC phenotype), can all be corrected by the addition of cysteine or GSH to the growth medium [5,6,8], the addition of GSH or cysteine alone cannot restore the assembly of bd-type quinol oxidases [5,6]. Assigning a role for haem transport is tempting for CydDC, previous investigations indicate that this is not a physiological role for this transporter [12], indicating a likely role for the haem cofactor in redox sensing

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Conclusion