The napEDABC gene cluster that encodes the periplasmic nitrate reductase from Bradyrhizobium japonicum USDA110 has been isolated and characterized. napA encodes the catalytic subunit, and the napB and napC gene products are predicted to be a soluble dihaem c and a membrane-anchored tetrahaem c-type cytochrome, respectively. napE encodes a transmembrane protein of unknown function, and the napD gene product is a soluble protein which is assumed to play a role in the maturation of NapA. Western blots of the periplasmic fraction from wild-type cells grown anaerobically with nitrate revealed the presence of a protein band with a molecular size of about 90 kDa corresponding to NapA. A B. japonicum mutant carrying an insertion in the napA gene was unable to grow under nitrate-respiring conditions, lacked nitrate reductase activity, and did not show the 90 kDa protein band. Complementation of the mutant with a plasmid bearing the napEDABC genes restored both nitrate-dependent anaerobic growth of the cells and nitrate reductase activity. A membrane-bound and a periplasmic c-type cytochrome, with molecular masses of 25 kDa and 15 kDa, respectively, were not detected in the napA mutant strain incubated anaerobically with nitrate, which identifies those proteins as the NapC and the NapB components of the B. japonicum periplasmic nitrate reductase enzyme. These results suggest that the periplasmic nitrate reductase is the enzyme responsible for anaerobic growth of B. japonicum under nitrate-respiring conditions. The promoter region of the napEDABC genes has been characterized by primer extension. A major transcript initiates 66?5 bp downstream of the centre of a putative FNR-like binding site.

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.

No abstract

The napEDABC genes of Bradyrhizobium japonicum encode the periplasmic nitrate reductase, an Mo-containing enzyme which catalyses the reduction of nitrate to nitrite when oxygen concentrations are limiting. In this bacterium, another set of genes, modABC, code for a high affinity ABC-type Mo transport system. A B. japonicum modA mutant has been obtained that is not capable of growing anaerobically with nitrate and lacks nitrate reductase activity. Under nitrate respiring conditions, when Mo concentrations are limiting, the B. japonicum modA mutant lacked both the 90 kDa protein corresponding to the NapA component of the periplasmic nitrate reductase, and the membrane-bound 25 kDa c-type cytochrome NapC. Regulatory studies using a napE-lacZ fusion indicated that napE expression was highly reduced in the modA mutant background when the cells were incubated anaerobically with nitrate under Mo-deficient conditions.