We report the discovery of a Bradyrhizobium

The cloning, sequencing and mutational analysis of the Bradyrhizobium japonicum symbiotic nitrogen fixation genes fixL and fixJ are reported here. The two genes were adjacent and probably formed an operon, fixLJ. The predicted FixL and FixJ proteins, members of the two-component sensor/regulator family, were homologous over almost their entire lengths to the corresponding Rhizobium meliloti proteins (approx. 50% identity). Downstream of the B. japonicum fixJ gene was found an open reading frame with 138 codons (ORF138) whose product shared 36% homology with the N-terminal part of FixJ. Deletion and insertion mutations within fixL and fixJ led to a loss of approximately 90% wild-type symbiotic nitrogen fixation (Fix) activity, whereas an ORF138 mutant was Fix+. In fixL, fixJ and ORF138 mutant backgrounds, the aerobic expression of the fixR-nifA operon was not affected. NifA itself did not regulate the expression of the fixJ gene. Thus, the B. japonicum FixL and FixJ proteins were neither involved in the regulation of aerobic nifA gene expression nor in the anaerobic NifA-dependent autoregulation of the fixRnifA operon, rather they appeared to control symbiotically important genes other than those whose expression was dependent on the NifA protein. The fixL and fixJ mutant strains were unable to grow anaerobically with nitrate as the terminal electron acceptor. Therefore, some of the FixJ-dependent genes in B. japonicum may be concerned with anaerobic respiration.

The Bradyrhizobium japonicum N2 fixation regulatory gene, nifA, was sequenced and its transcription start site determined. Between the start of transcription and the nifA gene an open reading frame of 278 codons was found and named fixR. A deletion in fixR which allowed transcription into nifA resulted in a 50% reduced Fix activity. The fixRnifA operon was expressed in soybean root nodules, in cultures grown anaerobically with nitrate as terminal electron acceptor, in microaerobic cultures, and in aerobic cultures. The transcription start site (+1) was preceded by a characteristic nif(-24/-12)-type promoter consensus sequence. Double base-pair exchanges in the -12 but not in the -24 region resulted in a 'promoter-down' phenotype. A promoter-upstream DNA region between -50 and -148 was essential for maximal promoter activity. Expression from the promoter was not dependent on nifA. We conclude that the fixRnifA promoter is positively controlled, and that it requires a newly postulated transcriptional factor in order to become activated.

We describe the cloning, sequencing, regulation, and mutational analysis of a Bradyrhizobium japonicum fixK-like gene whose product belongs to the family of Fnr-Crp-related regulatory proteins. The predicted 237-amino-acid FixK protein was found to share between 28 and 38% sequence identity with the Escherichia coli Fnr protein, other bacterial Fnr-like proteins (FnrN, Anr, and HlyX), and two rhizobial FixK proteins.