Frías José E, Flores Enrique
Instituto de Bioquímica Vegetal y Fotosíntesis, Consejo Superior de Investigaciones Científicas and Universidad de Sevilla, Seville, Spain.
Instituto de Bioquímica Vegetal y Fotosíntesis, Consejo Superior de Investigaciones Científicas and Universidad de Sevilla, Seville, Spain
J Bacteriol. 2015 Jul;197(14):2442-52. doi: 10.1128/JB.00198-15. Epub 2015 May 11.
Nitrate is widely used as a nitrogen source by cyanobacteria, in which the nitrate assimilation structural genes frequently constitute the so-called nirA operon. This operon contains the genes encoding nitrite reductase (nirA), a nitrate/nitrite transporter (frequently an ABC-type transporter; nrtABCD), and nitrate reductase (narB). In the model filamentous cyanobacterium Anabaena sp. strain PCC 7120, which can fix N2 in specialized cells termed heterocysts, the nirA operon is expressed at high levels only in media containing nitrate or nitrite and lacking ammonium, a preferred nitrogen source. Here we examined the genes downstream of the nirA operon in Anabaena and found that a small open reading frame of unknown function, alr0613, can be cotranscribed with the operon. The next gene in the genome, alr0614 (narM), showed an expression pattern similar to that of the nirA operon, implying correlated expression of narM and the operon. A mutant of narM with an insertion mutation failed to produce nitrate reductase activity, consistent with the idea that NarM is required for the maturation of NarB. Both narM and narB mutants were impaired in the nitrate-dependent induction of the nirA operon, suggesting that nitrite is an inducer of the operon in Anabaena. It has previously been shown that the nitrite reductase protein NirA requires NirB, a protein likely involved in protein-protein interactions, to attain maximum activity. Bacterial two-hybrid analysis confirmed possible NirA-NirB and NarB-NarM interactions, suggesting that the development of both nitrite reductase and nitrate reductase activities in cyanobacteria involves physical interaction of the corresponding enzymes with their cognate partners, NirB and NarM, respectively.
Nitrate is an important source of nitrogen for many microorganisms that is utilized through the nitrate assimilation system, which includes nitrate/nitrite membrane transporters and the nitrate and nitrite reductases. Many cyanobacteria assimilate nitrate, but regulation of the nitrate assimilation system varies in different cyanobacterial groups. In the N2-fixing, heterocyst-forming cyanobacteria, the nirA operon, which includes the structural genes for the nitrate assimilation system, is expressed in the presence of nitrate or nitrite if ammonium is not available to the cells. Here we studied the genes required for production of an active nitrate reductase, providing information on the nitrate-dependent induction of the operon, and found evidence for possible protein-protein interactions in the maturation of nitrate reductase and nitrite reductase.
硝酸盐被蓝细菌广泛用作氮源，其中硝酸盐同化结构基因经常构成所谓的nirA操纵子。该操纵子包含编码亚硝酸还原酶（nirA）、硝酸盐/亚硝酸盐转运蛋白（通常是一种ABC型转运蛋白；nrtABCD）和硝酸还原酶（narB）的基因。在模式丝状蓝细菌鱼腥藻属菌株PCC 7120中，它可以在称为异形胞的特殊细胞中固定N2，nirA操纵子仅在含有硝酸盐或亚硝酸盐且缺乏铵（一种优质氮源）的培养基中高水平表达。在这里，我们研究了鱼腥藻中nirA操纵子下游的基因，发现一个功能未知的小开放阅读框alr0613可以与该操纵子共转录。基因组中的下一个基因alr0614（narM）表现出与nirA操纵子相似的表达模式，这意味着narM和该操纵子存在相关表达。一个具有插入突变的narM突变体未能产生硝酸还原酶活性，这与NarM是NarB成熟所必需的观点一致。narM和narB突变体在nirA操纵子的硝酸盐依赖性诱导方面均受损，这表明亚硝酸盐是鱼腥藻中该操纵子的诱导物。此前已表明，亚硝酸还原酶蛋白NirA需要NirB（一种可能参与蛋白质-蛋白质相互作用的蛋白）才能达到最大活性。细菌双杂交分析证实了可能存在的NirA-NirB和NarB-NarM相互作用，这表明蓝细菌中亚硝酸还原酶和硝酸还原酶活性的发展分别涉及相应酶与其同源伴侣NirB和NarM的物理相互作用。
硝酸盐是许多微生物的重要氮源，它们通过硝酸盐同化系统利用硝酸盐，该系统包括硝酸盐/亚硝酸盐膜转运蛋白以及硝酸盐和亚硝酸盐还原酶。许多蓝细菌同化硝酸盐，但不同蓝细菌群体中硝酸盐同化系统的调节有所不同。在能够固定N2的、形成异形胞的蓝细菌中，如果细胞无法获得铵，则在存在硝酸盐或亚硝酸盐的情况下，包含硝酸盐同化系统结构基因的nirA操纵子会表达。在这里，我们研究了产生活性硝酸还原酶所需的基因，提供了有关该操纵子的硝酸盐依赖性诱导的信息，并发现了硝酸还原酶和亚硝酸还原酶成熟过程中可能存在蛋白质-蛋白质相互作用的证据。
