Genes required for rapid expression of nitrogenase activity in Azotobacter vinelandii
Genes required for rapid expression of nitrogenase activity in Azotobacter vinelandii
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DOI：
10.1073/pnas.0501216102
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发表时间：
2005-05-03
影响因子：
11.1
通讯作者：
Rubio, LM
中科院分区：
文献类型：
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作者：
Curatti, L;Brown, CS;Rubio, LM
Rnf蛋白被认为可以形成膜蛋白复合物，参与靶蛋白的还原，如转录调节因子SoxR或氮酶的二氮酶还原酶组分。在这项工作中，我们研究了rnf基因在固氮细菌固氮菌vinelandii中的作用。我们发现A. vinelandii有两个rnf样基因簇：rnf1，其表达受nif调控，rnf2，其表达不受培养基中氮源的影响。这些基因簇中的每一个的缺失都会产生固氮能力的时间延迟，从而导致重氮营养生长的延迟。Delta rnf突变对氮酶系统有两种明显的影响：(1)nifHDK基因表达减慢；(h)氮酶功能受损。在这些突变体中，二氮化酶还原酶活性降低，而二氮化酶活性基本保持不变。进一步分析表明，δ rnf突变体积累了一种无活性的缺铁形式的NifH，因为它们的[4Fe-4S]与NifH的结合率较低。rnf突变也会导致乌头酶活性显著降低；然而，它们不会产生一般的氧化应激或铁代谢的改变。我们的研究结果表明，葡萄球菌中存在一种氧化还原调节机制，通过Rnf蛋白复合物的活性来控制氮酶的表达率和成熟率。Rnf1在该方案中起着主要且更具体的作用，但Rnf1和rnf2突变的加性效应表明两个同源系统之间存在功能互补。
Rnf proteins are proposed to form membrane-protein complexes involved in the reduction of target proteins such as the transcriptional regulator SoxR or the dinitrogenase reductase component of nitrogenase. In this work, we investigate the role of rnf genes in the nitrogen-fixing bacterium Azotobacter vinelandii. We show that A. vinelandii has two clusters of rnf-like genes: rnf1, whose expression is nif-regulated, and rnf2, which is expressed independently of the nitrogen source in the medium. Deletion of each of these gene clusters produces a time delay in nitrogen-fixing capacity and, consequently, in diazotrophic growth. Delta rnf mutations cause two distinguishable effects on the nitrogenase system: (1), slower nifHDK gene expression and (h), impairment of nitrogenase function. In these mutants, dinitrogenase reductase activity is lowered, whereas dinitrogenase activity remains essentially unaltered. Further analysis indicates that Delta rnf mutants accumulate an inactive and iron-deficient form of NifH because they have lower rates of incorporation of [4Fe-4S] into NifH. Delta rnf mutations also cause a noticeable decrease in aconitase activity; however, they do not produce general oxidative stress or modification of Fe metabolism in A. vinelandii. Our results suggest the existence of a redox regulatory mechanism in A. vinelandii that controls the rate of expression and maturation of nitrogenase by the activity of the Rnf protein complexes. rnf1 plays a major and more specific role in this scheme, but the additive effects of mutations in rnf1 and rnf2 indicate the existence of functional complementation between the two homologous systems.