Structural genes of dinitrogenase and dinitrogenase reductase are transcribed from two separate promoters in the broad host range cowpea Rhizobium strain IRc78.

The nucleotide sequence of the structural gene (nifD) coding for the alpha-subunit of dinitrogenase along with its flanking sequences has been determined in 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. Nucleotide homology to nifD from the blue-green alga, Anabaena, and Parasponia Rhizobium, are 63% and 90%, respectively. Cowpea Rhizobium IRc78 nifD and nifK (encodes the beta-subunit of dinitrogenase) genes are linked, separated by 69 nucleotides. In contrast to fast-growing rhizobia, 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 operon. Two nucleotide sequences, a hexamer (G-G-T-T-G-C) and a pentamer (T-G-G-C-A), 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, T-T-G-A-[unk]-G-G-A, located 14 nucleotides upstream of the initiation codon was detected between the transcribed but untranslated leader regions of nifD and nifH.

Nitrogenase promoter-lacZ fusion studies of essential nitrogen fixation genes in Bradyrhizobium japonicum I110.

DNA fragments containing either the nifD or nifH promoter and 5' structural gene sequences from Bradyrhizobium japonicum I110 were fused in frame to the lacZ gene. Stable integration of these nif promoter-lacZ fusions by homologous double reciprocal crossover into a symbiotically nonessential region of the B. japonicum chromosome provided an easy assay for the effects of potential nif regulatory mutants. The level of beta-galactosidase activity expressed from these two nif promoter-lacZ fusions was assayed in bacteroids of B. japonicum I110 wild type and Fix mutants generated by transposon Tn5 mutagenesis and identified in the accompanying paper. No nif-positive regulatory mutants were identified from among an array of Fix- mutants in which Tn5 was inserted 9 kilobase pairs upstream of the nifDK operon and within the 18-kilobase-pair region separating the nifDK and nifH operons. This result indicates that there are no genes in these regions involved in the regulation of nitrogenase structural gene expression. Interestingly, the level of beta-galactosidase activity expressed from the nifH promoter was twice that expressed from the nifD promoter, suggesting that the normal cellular level of the nifH gene product in bacteroids is in a 2:1 ratio with the nifD gene product instead of in the 1:1 stoichiometry of the nitrogenase enzyme complex.

A plasmid sequence from Rhizobium leguminosarum 300 contains homology to sequences near the octopine TL-DNA right border.

The DNA sequence from a Rhizobium leguminosarum 300 (RL300) plasmid that contains homology to the Tc-DNA of Agrobacterium tumefaciens is described. The RL300 sequence has 78% homology to a 359 bp sequence in the Tc-DNA of pTi15955. The RL300 homology starts approximately 100 bp from the 24 bp border sequence of the TL-DNA and ends approximately 3 bp from an IS66 homolog in the Tc-DNA. An unusual feature of the RL300 homology is the presence of 81 bp direct repeats with Tc-DNA homology, separated by 201 bp. One end of each direct repeat has a 12 bp palindrome. Four cloned sequences of RL300 with homology to the T DNA region were hybridized to plasmid lysates of RL300 derivatives to determine the source of each plasmid. The sequenced homolog, originally on pRH228, was isolated from pRL7JI; the other 3 homologs were isolated from the transmissable plasmids pRL7JI (pRH235) and pRL8JI (pRH235 and pRH236).

Expression of beta-galactosidase controlled by a nitrogenase promoter in stem nodules of Aeschynomene scabra.

A 365-base-pair (bp) DNA fragment, containing the promoter region of the nitrogenase reductase (nifH) gene from stem Rhizobium BTAi1, has been isolated and sequenced. The transcription initiation sites were localized at positions 152 (major initiation) and 114 (minor initiation) nucleotides upstream of the translation initiation codon. The 200-bp nucleotide sequence upstream of the nifH structural gene shows substantial homology to the corresponding nifH regions of cowpea Rhizobium (100%), Parasponia Rhizobium (89%), and Rhizobium japonicum (88%). The nifH promoter region of stem Rhizobium BTAi1 was fused to the lacZ gene of Escherichia coli. The fusion and a 1.6-kilobase DNA specifying neomycin phosphotransferase were inserted into a 3,4-kilobase fragment of stem Rhizobium chromosome, and the resulting construct was placed on a mobilizable vector, pREV1000. Stem Rhizobium transconjugants resistant to kanamycin were found to contain the nifH promoter region-lacZ fusion linked to the neomycin phosphotransferase gene at the site of chromosomal homology. Analysis of the DNA from stable transconjugants showed integration of a single copy of these sequences into the chromosome by a double-reciprocal crossover event. The transconjugants formed nitrogen-fixing nodules, indicating that the insertion occurred in a "nonessential" region of the stem Rhizobium chromosome. Transconjugant strain BTAi1000 grows on beta-galactosidase indicator plates under aerobic conditions as white colonies, whereas under microaerobic conditions (97% N(2)/3% O(2)), which derepress nitrogenase, the colonies turn blue within 15-24 hr. beta-Galactosidase activity in derepressed cultures of BTAi1000 showed a 200-fold increase in comparison to the wild-type strain, whereas stem nodules formed by BTAi1000 exhibited 15- to 20-fold higher beta-galactosidase values than wild-type nodules. Nitrogenase promoter-dependent expression of beta-galactosidase in stem nodules was inhibited by fixed nitrogen, suggesting that the nifH promoter-lacZ fusion is controlled coordinately in trans with the native nif region.