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Bradyrhizobium japonicum utilizes cytochrome cbb3 oxidase encoded by the fixNOQP operon to support microaerobic respiration under free-living and symbiotic conditions. It has been previously shown that, under denitrifying conditions, inactivation of the cycA gene encoding cytochrome c550, the electron donor to the Cu-containing nitrite reductase, reduces cbb3 expression. In order to establish the role of c550 in electron transport to the cbb3 oxidase, in this work, we have analyzed cbb3 expression and activity in the cycA mutant grown under microaerobic or denitrifying conditions. Under denitrifying conditions, mutation of cycA had a negative effect on cytochrome c oxidase activity, heme c (FixP and FixO) and heme b cytochromes as well as expression of a fixP'–' lacZ fusion. Similarly, cbb3 oxidase was expressed very weakly in a napC mutant lacking the c-type cytochrome, which transfers electrons to the NapAB structural subunit of the periplasmic nitrate reductase. These results suggest that a change in the electron flow through the denitrification pathway may affect the cellular redox state, leading to alterations in cbb3 expression. In fact, levels of fixP'–' lacZ expression were largely dependent on the oxidized or reduced nature of the carbon source in the medium. Maximal expression observed in cells grown under denitrifying conditions with an oxidized carbon source required the regulatory protein RegR. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]