Resumen de Desnitrificación en Bradyrhizobium japonicum. Implicación de las proteínas reguladoras FIXK2 y Nifa
Bradyrhizobium japonicum cytochrome c550, encoded by cycA, has been previously suggested to play a role in denitrification, the respiratory reduction of nitrate to dinitrogen. However, the exact role of this cytochrome in the denitrification process is unknown. This study shows that cytochrome c550 is involved in electron transfer to the copper-containing nitrite reductase of B. japonicum, as revealed by the inability of a cycA mutant strain to consume nitrite and, consequently, to grow under denitrifying conditions with nitrite as the electron acceptor. Mutation of cycA had no apparent effect on methylviologen-dependent nitrite reductase activity. However, succinate-dependent nitrite reduction was largely inhibited, suggesting that c550 is the in vivo electron donor to copper-containing nitrite reductase. In addition, this study demonstrates that a cytochrome c550 mutation has a negative effect on expression of the periplasmic nitrate reductase. This phenotype can be rescued by extending the growth period of the cells. A model is proposed whereby a mutation in cycA reduces expression of the cbb3-type oxidase, affecting oxygen consumption rate by the cells and consequently preventing maximal expression of the periplasmic nitrate reductase during the first days of the growth period.
Bradyrhizobium japonicum utilizes cytochrome cbb3 oxidase encoded by the fixNOQP operon to support microaerobic respiration under free-living and symbiotic conditions. This bacterium is also able to grow anaerobically with nitrate as electron acceptor. Under denitrifying conditions, where the cbb3 oxidase is also fully expressed, inactivation of the cycA gene, encoding the cytochrome c550, reduced O2 consumption rates and expression of the heme-stained FixP and FixO components of the cbb3 oxidase. In order to establish the role of cytochrome c550 in electron transport to the cbb3 oxidase, in this work, we have analyzed cytochrome c oxidase activity, and cbb3 expression in the cycA mutant incubated under low oxygen conditions without nitrate, where only the cbb3 pathway is induced, and with nitrate, were cbb3 and denitrification pathways are induced. Only when cells were incubated under denitrifying conditions, levels of cytochrome c oxidase activity, as well as, expression of a fixP¿-¿lacZ translational fusion, were highly reduced in the cycA mutant. Similarly, cbb3 oxidase was expressed very weakly in a napC mutant lacking the membrane-bound c-type cytochrome which transfers electrons to the NapAB structural subunit of the periplasmic nitrate reductase. The presence of a highly reduced carbon substrate or myxothiazol, a specific inhibitor of the cytochrome bc1 complex, inhibited ß-galactosidase activity in the wild type cells incubated anaerobically with nitrate. These results suggest that a change in the flow of electrons through the denitrification network may affect the cellular redox state, leading to alterations in cbb3 expression. We propose a redox-dependent regulation of the cbb3 cytochrome oxidase under denitrifying conditions.
Denitrification in Bradyrhizobium japonicum is catalyzed by the products of napEDABC, nirK, norCBQD and nos nosRZDYFLX genes which encode reductases for nitrate, nitrite, nitric oxide and nitrous oxide, respectively. Microaerobic induction of the denitrification genes depends on the fixLJ and fixK2 genes, whose products form the FixLJ-FixK2 regulatory cascade. In B. japonicum, a second oxygen-responsive regulatory cascade mediated by the nitrogen fixation regulatory protein, NifA, has been described. In this study, we have shown that cells of a B. japonicum nifA mutant are impaired in growth under anaerobic conditions with nitrate. Mutation of nifA also led to a decreased expression of napEDABC and nirK genes as well as to an impairment of MV+-dependent nitrate and nitrite reductase activities, after incubation under anaerobic conditions with nitrate. Furthermore, expression of the membrane-bound c-type cytochrome NapC is highly reduced in the nifA mutant strain. The promoter region of the B. japonicum nirK gene has been characterized by primer extension. A major transcript initiates 40.5 bp downstream of the axis of symmetry of a putative FixK2 binding site, which overlaps with a putative NifA-like binding site. Levels of the nirK transcripts are highly reduced in the nifA mutant strain. Taken together, these results demostrate that NifA is required for maximal expression of the napEDABC and nirK denitrification genes in cells incubated under low oxygen conditions and in the presence of nitrate. We propose a novel involvement of the nitrogen fixation NifA regulatory protein on the expression of the B. japonicum denitrification genes.
