Kim Minwook, Jeong Sunwook, Lim Sangyong, Sim Jeonggu, Rhie Ho-Gun, Lee Sung-Jae
Department of Biology, Kyung Hee University, Seoul, 02447, Republic of Korea.
Division of Biotechnology, Korea Atomic Energy Research Institute, Jeongeup, 56212, Republic of Korea.
J Microbiol. 2017 Feb;55(2):137-146. doi: 10.1007/s12275-017-6382-y. Epub 2017 Jan 26.
A cystine-dependent anti-oxidative stress response is characterized in Deinococcus geothermalis for the first time. Nevertheless, the same transcriptional directed Δdgeo_1985F mutant strain was revealed to have an identical phenotype to the wild-type strain, while the reverse transcriptional directed Δdgeo_1985R mutant strain was more resistant to oxidative stress at a certain concentration of HO than the wild-type strain. The wild-type and mutant strains expressed equal levels of superoxide dismutase and catalase under HO-induced stress. Although the expression levels of the general DNA-damage response-related genes recA, pprA, ddrA, and ddrB were up-regulated by more than five-fold in the wild-type strain relative to the Δdgeo_1985R mutant strain, the mutant strain had a higher survival rate than the wild-type under HO stress. The Δdgeo_1985R mutant strain highly expressed a cystine-transporter gene (dgeo_1986), at levels 150-fold higher than the wild-type strain, leading to the conclusion that this cystine transporter might be involved in the defensive response to HO stress. In this study, the cystine transporter was identified and characterized through membrane protein expression analysis, a cystine-binding assay, and assays of intracellular HO, cysteine, and thiol levels. The genedisrupted mutant strain of the cystine importer revealed high sensitivity to HO and less absorbed cystine, resulting in low concentrations of total thiol. Thus, the absorbed cystine via this cystine-specific importer may be converted into cysteine, which acts as a primitive defense substrate that non-enzymatically scavenges oxidative stress agents in D. geothermalis.
首次在地热栖热放线菌中发现了一种依赖胱氨酸的抗氧化应激反应。然而,研究发现,转录方向正向的Δdgeo_1985F突变株与野生型菌株具有相同的表型,而转录方向反向的Δdgeo_1985R突变株在一定浓度的HO下比野生型菌株对氧化应激更具抗性。在HO诱导的应激条件下,野生型和突变株中超氧化物歧化酶和过氧化氢酶的表达水平相当。尽管相对于Δdgeo_1985R突变株,野生型菌株中一般DNA损伤反应相关基因recA、pprA、ddrA和ddrB的表达水平上调了五倍以上,但在HO应激下,突变株的存活率高于野生型。Δdgeo_1985R突变株中一个胱氨酸转运基因(dgeo_1986)的表达量极高,比野生型菌株高150倍,由此得出结论,这种胱氨酸转运蛋白可能参与了对HO应激的防御反应。在本研究中,通过膜蛋白表达分析、胱氨酸结合试验以及细胞内HO、半胱氨酸和硫醇水平的测定,对胱氨酸转运蛋白进行了鉴定和特性分析。胱氨酸导入蛋白的基因敲除突变株对HO高度敏感,吸收的胱氨酸较少,导致总硫醇浓度较低。因此,通过这种特异性胱氨酸导入蛋白吸收的胱氨酸可能会转化为半胱氨酸,半胱氨酸作为一种原始的防御底物,可在非酶促条件下清除地热栖热放线菌中的氧化应激剂。