AMB  >> Vol. 2 No. 4 (December 2013)

    Construction and Functional Analysis of osmC Gene Disruptant and ohr Gene Disruptant in Deinococcus radiodurans R1

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刘盈盈,陈 明,王 劲:中国农业科学院生物技术研究所,北京;

耐辐射异常球菌osmC与ohr基因突变株抗氧化D. radiodurans R1; osmC and ohr Gene; Mutant Stains; Antioxidant


渗透胁迫诱导蛋白OsmC与有机氢过氧化物抗性蛋白Ohr都属于OsmC超家族抗氧化物酶,在细菌抵御有机或无机过氧化反应的过程中起重要作用。耐辐射异常球菌(D. radiodurans R1)osmC基因(DR_1538)ohr基因(DR_1857)分别编码了OsmC超家族中的这两种抗氧化物酶。运用体外三段融合PCR方法和线性转化方法,首次构建了壮观霉素抗性完全缺失突变株ΔosmC和卡那霉素抗性完全缺失突变株ΔohrΔosmCΔohr突变株和野生型菌株分别都进行CHP(异丙苯基过氧化氢)H2O2 NaCl胁迫处理,结果显示,与野生型相比,ΔosmC突变株对H2O2异常敏感,而Δohr突变株CHP更敏感,两个突变株对NaCl胁迫都没有大的变化。QRT-PCR结果显示在H2O2NaCl胁迫下野生型菌株中osmC基因都上调,其中H2O2胁迫下基因上调水平显著;在CHPNaCl胁迫下野生型菌株中ohr基因基因上调大约2倍左右。根据结果推测同属于OsmC超家族的两个过氧化物酶选择底物的偏好性与作用方式可能并不一样,OsmC蛋白可能主要是以无机过氧化物作为底物,而Ohr蛋白可能主要是以有机过氧化物为底物。
The osmotic stress induced protein (OsmC) and organic hydroperoxide resistance protein (Ohr) were two antioxidant enzymes which play an important role in bacteria defensing organic or inorganic peroxidation. The osmC gene (DR_1538) and ohr gene (DR_1857) of Deinococcus radiodurans (D. radiodurans R1) encoded the two antioxidant enzymes in the OsmC superfamily. Fusion PCR of three sections in vitro and linear transformation method were used to firstly construct the spectinomycin-resistant mutant stain (ΔosmC) and the kanamycin-resistant mutant strain (Δohr). The ΔosmC, Δohr mutants strain and wild-type strain were treated with CHP (cumene hydroperoxide), H2O2 and NaCl stress treatments. The result showed that ΔosmC mutant is extremely sensitive to H2O2 compared with the wild type, while Δohr mutant is more sensitive to CHP and the two mutant strains had no changes after NaCl stress. QRT-PCR results displayed that the osmC gene of the wild-type strain were up-regulated in both H2O2 and NaCl stress treatments, and the osmC gene in H2O2 stress levels was significantly upregulated. The ohr genes in wild-type strain after CHP and NaCl stress treatments upregulated approximately 2-folds. From above, it was presumed that there may be different at the preference of two peroxidases in OsmC superfamily selecting substrates and the mode of those interacting with their substrate; OsmC proteins may mainly use inorganic peroxide as its substrate and Ohr protein may be primarily based on the organic peroxide as its substrate.

崔广艳, 鲜先毅, 刘盈盈, 代其林, 陈明, 王劲. 耐辐射异常球菌osmC与ohr基因突变株的构建及功能研究[J]. 微生物前沿, 2013, 2(4): 109-115.


[1] White, O., Eisen, J.A., Heidelberg, J.F., et al. (1999) Genome sequence of the radioresistant bacterium Deinococcus radiodu- rans R1. Science, 286, 1571-1577.
[2] Mattimore, V. and Battista, J.R. (1996) Radioresistance of Dei- nococcus radiodurans: Functions necessary to survive ionizing radiation are also necessary to survive prolonged desiccation. Journal of Bacteriology, 178, 633-637.
[3] Battista, J. (1997) Against all odds: The survival strategies of Deinococcus radiodurans. Annual Reviews in Microbiology, 1, 203-224.
[4] Heidelberg, J.F., Eisen, J.A., Nelson, W. C., et al. (2000) “DNA sequence of both chromosomes of the cholera pathogen Vibrio cholerae,” Nature, 406, 477-483.
[5] Daly, M.J., Gaidamakova, E.K., Matrosova, V.Y., et al. (2007) Protein oxidation implicated as the primary determinant of bac- terial radioresistance. PLoS Biology, 5, 769-779.
[6] Lesniak, J., Barton, W.A. and Nikolov, D.B. (2002) Structural and functional characterization of the Pseudomonas hydroper- oxide resistance protein Ohr. The EMBO Journal, 21, 6649- 6659.
[7] Dubbs, J.M. and Mongkolsuk, S. (2007) Peroxiredoxin systems. Springer, Berlin, 143-193.
[8] Gutierrez, C., Barondess, J., Manoil, C., et al. (1987) Analysis of osmotically regulated genes in Escherichia coli. Journal of Mo- lecular Biology, 195, 289-297.
[9] 李敏, 杨谦 (2007) 一种高效构建同源重组DNA片段的方法——融合PCR. 中国生物工程杂志, 27, 53-58.
[10] 常胜合, 舒海燕, 陆辉明 (2008) 耐辐射球菌基因DRB0099 缺失突变株的构建及逆境分析. 微生物学报, 48, 57-62.
[11] Mongkolsuk, S. and Helmann, J.D. (2002) Regulation of induc- ible peroxide stress responses. Molecular Microbiology, 45, 9- 15.
[12] Lim, Y.S., Cha, M.K., Kim, H.K., et al. (1994) The thiol-specific antioxidant protein from human brain: gene cloning and analysis of conserved cysteine regions. Gene, 140, 279-284.
[13] Chae, H.Z., Robison, K., Pooleo, L.B., et al. (1994) Cloning and sequencing of thiol-specific antioxidant from mammalian brain: Alkyl hydroperoxide reductase and thiol-specific antioxidant de- fine a large family of antioxidant enzymes. Proceedings of the National Academy of Sciences, 91, 7017-7021.
[14] Halliwell, B. and Gutteridge, J. (1984) Oxygen toxicity, oxygen radicals, transition metals and disease. Biochemical Journal, 219, 1-13.
[15] Atichartpongkul, S., Loprasert, S., Vattanaviboon, P., et al. (2001) Bacterial Ohr and OsmC paralogues define two protein families with distinct functions and patterns of expression. Microbiology, 147, 1775-1782.
[16] Lesniak, J., Barton, W.A. and Nikolov, D.B. (2003) Structural and functional features of the Escherichia coli hydroperoxide resistance protein OsmC. Protein Science, 12, 2838-2843.
[17] Rehse, P.H., Ohshima, N., Nodake, Y., et al. (2004) Crystallo- graphic structure and biochemical analysis of the Thermus ther- mophilus osmotically inducible protein C. Journal of Molecular Biology, 338, 959-968.
[18] Shin, D.H., Choi, I.G., Busso, D., et al. (2004) Structure of OsmC from Escherichia coli: A salt-shock-induced protein. Acta Crystallographica Section D: Biological Crystallography, 60, 903-911.