STRUCTURAL AND GENETIC STUDIES OF NITROGEN FIXATION (NIF) GENES IN SLOW-GROWING RHIZOBIA AND CHARACTERIZATION OF MUTANTS THAT FAIL TO FIX NITROGEN (NUCLEOTIDE SEQUENCE, PROMOTERS, TRANSCRIPTION INITIATION, FUSIONS, CONSERVATION)

The nucleotide sequence of the structural gene (nifD) coding for the alpha-subunit of dinitrogenase along with its flanking sequences has been determined in the slow-growing cowpea Rhizobium IRc78. The coding sequence consists of 1500 nucleotides, which corresponds to a predicted amino acid sequence of 500 residues and a molecular weight of 56,025. Cowpea Rhizobium nifD and nifK (encodes the beta-subunit of dinitrogenase) are linked, separated by 69 nucleotides. The structural genes of dinitrogenase (nifDK) are transcribed from a different promoter than the structural gene of dinitrogenase reductase (nifH). Transcription of nifDK initiates 41 nucleotides upstream of the start codon for the nifDK operon. Two transcription initiation sites, localized at 152 and 114 nucleotides upstream of the start codon, were determined for the nifH gene. The promoter and partial N-terminal nucleotide sequence, and transcription initiation of the nifH gene from stem Rhizobium BTAil were also determined. Unexpectedly, the nucleotide sequence displayed 100% homology to the corresponding sequence from cowpea Rhizobium IRc78 and the transcription initiation sites were localized to identical positions. Two nucleotide sequences, a hexamer (GGTTGC) and a pentamer (TGGCA), centered at approximately -15 and -25, respectively, are conserved in the nifD and nifH promoter regions and are not present in the 69-nucleotide nifDK junction. No sequence homology other than a possible ribosome binding site, TTGAA (or T)GGA, located 14 nucleotides upstream of the initiation codon was detected between the transcribed but untranslated leader regions of nifD and nifH.; The isolation and characterization of nitrogenase promoters and structural genes in slow-growing rhizobia has facilitated the translational fusion of nitrogenase promoter and N-terminal sequences to the lacZ gene. Insertion into a recombinational/insertional vector that is mobilizable to Rhizobium enabled the stable chromosomal integration of nif promoter-lacZ fusions in Bradyrhizobium japonicum I110 Fix('-) mutants. When assayed for the inability to activate beta-galactosidase expression no nif regulatory mutants were identified from among the general class of Fix('-) mutants. However, the level of beta-galactosidase activity present in strains containing the nifH promoter-lacZ fusion was two-fold higher than observed in strains containing the nifD promoter-lacZ fusion.