The Bradyrhizobium japonicum napEDABC genes are controlled by the FixLJ-FixK2-NnrR regulatory cascade

Abstract

Nitrate respiration by the N(2)-fixing symbiotic bacteria Bradyrhizobium japonicum USDA110 is mediated by a Nap (periplasmic nitrate reductase) encoded by the napEDABC genes. Expression of a transcriptional fusion of the nap promoter region to the reporter gene lacZ, P(napE)-lacZ, was very low in aerobically grown cells of USDA110, but expression was induced approx. 3-fold when the cells were cultured under microaerobic conditions, and 12-fold when nitrate was added to the microaerobic incubation medium. The P(napE)-lacZ fusion was not expressed in the fixL 7403, fixJ 7360 and fixK(2) 9043 mutant strains. Microaerobic induction of the P(napE)-lacZ fusion was retained in the nnrR 8678 mutant, but no increase in beta-galactosidase activity was observed upon nitrate addition. Western-blot and Methyl Viologen-dependent nitrate reductase activity assays showed that synthesis and activity of the catalytic NapA subunit in USDA110 was similar to that in the napC 0906 and nirK GRK308 mutant strains incubated microaerobically with nitrate. These results suggest that nitrate and nitrite, which are not reduced by the napC 0906 and nirK GRK308 mutant cells respectively, induced the synthesis and activity of NapA; conversely, formation of endogenous NO was not required for induction of Nap expression.