CydDC-mediated reductant export in Escherichia coli controls the transcriptional wiring of energy metabolism and combats nitrosative stress.
CydDC-mediated reductant export in Escherichia coli controls the transcriptional wiring of energy metabolism and combats nitrosative stress.
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DOI：
10.1042/bj20150536
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发表时间：
2016-03-15
期刊：
影响因子：
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通讯作者：
Shepherd M
中科院分区：
文献类型：
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作者：
Holyoake LV;Hunt S;Sanguinetti G;Cook GM;Howard MJ;Rowe ML;Poole RK;Shepherd M
E.大肠杆菌输出谷胱甘肽和半胱氨酸。cydDC的丧失会导致能量代谢的适应，并诱导对NO的敏感性。因此，CydDC在感染期间的生长和存活中可能起作用。谷胱甘肽/半胱氨酸输出者CydDC维持大肠杆菌中的氧化还原平衡。一个cydD突变株被用来探测CydDC后减少硫醇输出，基因表达，代谢扰动，细胞内pH稳态和耐受一氧化氮（NO）的影响。发现CydDC的丢失降低了细胞质外巯基水平，而过表达降低了细胞质巯基含量。转录组学分析揭示了蛋白伴侣、蛋白降解（通过苯丙酸/苯乙酸催化剂）、脂肪酸的β-氧化和涉及硝酸盐/亚硝酸盐还原的基因的显著上调。1H NMR代谢组学显示cydD细胞中甲硫氨酸和甜菜碱升高，乙酸盐和NAD+减少，这与基于转录组学的代谢模型一致。然而，生长速率和ΔpH不受影响，尽管cydD菌株确实表现出对NO释放化合物NOC-12的敏感性。这些观察结果与以下假设一致，即CydD介导的还原剂输出的损失促进蛋白质错误折叠，适应能量代谢和对NO的敏感性。发现向培养基中添加谷胱甘肽和半胱氨酸可以补充cydD菌株中bd型细胞色素合成的损失（多效性cydDC表型的关键组分），提供了CydDC底物能够恢复该呼吸氧化酶的正确组装的第一个直接证据。这些数据提供了对E.大肠杆菌，突出的重要性，在亚硝化应激细菌氧化还原稳态，并首次报告的能力，周质低分子量硫醇恢复血红素掺入到细胞色素复合物。
The CydDC ABC transporter of E. coli exports glutathione and cysteine. Loss of cydDC elicits adaptations in energy metabolism and induces sensitivity to NO. CydDC therefore has a likely role in growth and survival during infection. 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. 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 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.