The obligate aerobe Streptomyces coelicolor A3(2) synthesizes three active respiratory nitrate reductases
The obligate aerobe Streptomyces coelicolor A3(2) synthesizes three active respiratory nitrate reductases
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DOI：
10.1099/mic.0.042572-0
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发表时间：
2010-10-01
期刊：
影响因子：
2.8
通讯作者：
Sawers, R. Gary
中科院分区：
文献类型：
--
作者：
Fischer, Marco;Alderson, Jesse;Sawers, R. Gary
天蓝色链霉菌A3（2）合成三种膜结合呼吸硝酸还原酶（Nars）。在液体培养基中的需氧生长期间，细菌能够化学计量地将50 mM硝酸盐还原为亚硝酸盐。构建并分析了缺失所有三个narGHJI操纵子的突变体，结果表明它不能还原硝酸盐。缺失编码MoaA的基因（MoaA催化钼辅因子生物合成的第一步）也阻止了硝酸盐还原，这与Nars是辅酶一致。与三重narGHJ/突变体相反，moaA突变体也不能利用硝酸盐作为唯一氮源，这表明S.腔棘鱼也依赖于海藻。分析S. coelicolor在固体培养基上的生长证明Nar活性存在于孢子和菌丝体（菌丝）中。用硝酸盐类似物氯酸盐进行的存活试验表明，野生型S。coelicolor孢子和菌丝体在厌氧培养后对氯酸盐敏感，与硝酸盐的存在无关，而moaA和三重nar突变体都是抗氯酸盐的。互补的三重nar突变体与个人narGHJI操纵子上提供的cosmetry显示，每个操纵子编码的酶，合成和活性在硝酸盐或氯酸盐还原。从这些研究中获得的数据允许Nan 1活性的孢子，Nar 2的孢子和菌丝体，和Nar 3专门菌丝体的暂定分配。
Streptomyces coelicolor A3(2) synthesizes three membrane-associated respiratory nitrate reductases (Nars). During aerobic growth in liquid medium the bacterium was able to reduce 50 mM nitrate stoichiometrically to nitrite. Construction and analysis of a mutant in which all three narGHJI operons were deleted showed that it failed to reduce nitrate. Deletion of the gene encoding MoaA, which catalyses the first step in molybdenum cofactor biosynthesis, also prevented nitrate reduction, consistent with the Nars being molybdoenzymes. In contrast to the triple narGHJ/mutant, the moaA mutant was also unable to use nitrate as sole nitrogen source, which indicates that the assimilatory nitrate reductases in S. coelicolor are also molybdenum-dependent. Analysis of S. coelicolor growth on solid medium demonstrated that Nar activity is present in both spores and mycelium (hypha). Development of a survival assay with the nitrate analogue chlorate revealed that wild-type S. coelicolor spores and mycelium were sensitive to chlorate after anaerobic incubation, independent of the presence of nitrate, while both the moaA and triple nar mutants were chlorate-resistant. Complementation of the triple nar mutant with the individual narGHJI operons delivered on cosmids revealed that each operon encoded an enzyme that was synthesized and active in nitrate or chlorate reduction. The data obtained from these studies allow a tentative assignment of Nan1 activity to spores, Nar2 to spores and mycelium, and Nar3 exclusively to mycelium.