Recognition of -24/-12-type promoters by RNA polymerase requires a special sigma factor, sigma 54 (RpoN NtrA GlnF). In the nitrogen-fixing soybean symbiont Bradyrhizobium japonicum, two functional, highly conserved rpoN genes (rpoN1 and rpoN2) were identified and sequenced. The two predicted B. japonicum RpoN protein sequences were 87% identical, and both showed different levels of homology to the RpoN proteins of other bacteria. Downstream of rpoN2 (but not of rpoN1), two additional open reading frames were identified that corresponded to open reading frames located at similar positions in Klebsiella pneumoniae and Pseudomonas putida. Both B. japonicum rpoN genes complemented the succinate- and nitrate-negative phenotypes of a Rhizobium meliloti rpoN mutant. B. japonicum strains carrying single or double rpoN mutations were still able to utilize C4-dicarboxylates as a carbon source and histidine, proline, or arginine as a nitrogen source, whereas the ability to assimilate nitrate required expression of at least one of the two rpN genes. In symbiosis both rpoN genes could replace each other functionally. The rpoN1/2 double mutant induced about twice as many nodules on soybeans as did the wild type, and these nodules lacked nitrogen fixation activity completely. Transcription of a nifH'-'lacZ fusion was not activated in the rpoN1/2 mutant background, whereas expression of a fixR'-'lacZ fusion in this mutant was affected only marginally. By using rpoN'-'lacZ fusions, rpoN1 expression was shown to be activated at least sevenfold in microaerobiosis as compared with that in aerobiosis, and this type of regulation involved fixLJ. Expression of rpoN2 was observed under all conditions tested and was increased fivefold in an rpoN2 mutant. The data suggested that the rpoN1 gene was regulated in response to oxygen, whereas the rpoN2 gene was negatively autoregulated.