Conservation of structure and location of Rhizobium meliloti and Klebsiella pneumoniae nifB genes.

Using transposon Tn5-mediated mutagenesis, an essential Rhizobium meliloti nitrogen fixation (nif) gene was identified and located directly downstream of the regulatory gene nifA. Maxicell and DNA sequence analysis demonstrated that the new gene is transcribed in the same direction as nifA and codes for a 54-kilodalton protein. In Klebsiella pneumoniae, the nifBQ operon is located directly downstream of a gene which is structurally and functionally homologous to the R. meliloti nifA gene. The DNA sequences of the K. pneumoniae nifB and nifQ genes (which code for 51- and 20-kilodalton proteins, respectively) were determined. The DNA sequence of the newly identified R. meliloti gene was approximately 50% homologous to the K. pneumoniae nifB gene. R. meliloti does not contain a gene homologous to nifQ directly downstream of nifB. The R. meliloti nifB product shares approximately 40% amino acid homology with the K. pneumoniae nifB product, and 10 of the 12 cysteine residues of the R. meliloti nifB product are conserved with 10 of the 17 cysteine residues of the K. pneumoniae nifB product.     

Organization and partial sequence of a DNA region of the Rhizobium leguminosarum symbiotic plasmid pRL6JI containing the genes fixABC, nifA, nifB and a novel open reading frame.

By hybridization and heteroduplex studies the fixABC and nifA genes of the Rhizobium leguminosarum symbiotic plasmid pRL6JI have been identified. DNA sequencing of the region containing nifA showed an open reading frame of 1557 bp encoding a protein of 56, 178 D. Based on sequence homology, this ORF was confirmed to correspond to the nifA gene. Comparison of three nifA proteins (Klebsiella pneumoniae, Rhizobium meliloti, Rhizobium leguminosarum) revealed only a weak relationship in their N-terminal regions, whereas the C-terminal parts exhibited strong homology. Sequence analysis also showed that the R. leguminosarum nifA gene is followed by nifB and preceded by fixC with an open reading frame inserted in between. This novel ORF of 294 bp was found to be highly conserved also in R. meliloti. No known promoter and termination signals could be defined on the sequenced R. leguminosarum fragment.     

Nitrogen fixation (nif) genes of the cyanobacterium Anabaena species strain PCC 7120. The nifB-fdxN-nifS-nifU operon

A second nitrogen fixation (nif) operon in the cyanobacterium (blue-green alga) Anabaena (Nostoc) sp. strain PCC 7120 has been identified and sequenced. It is located just upstream of the nifHDK operon and consists of four genes in the order nifB, fdxN, nifS, and nifU. The three nif genes were identified on the basis of their similarity with the corresponding genes from other diazotrophs. The fourth gene, fdxN, codes for a bacterial type ferredoxin (Mulligan, M. E., Buikema, W. J., and Haselkorn, R. (1988) J. Bacteriol. 167, 4406-4410). The four genes are probably transcribed as a single operon, but are expressed at a lower level than the nifHDK operon, and only after a developmentally induced DNA rearrangement occurs that excises a 55-kilobase pair element from within the fdxN gene (Golden, J. W., Mulligan, M. E., and Haselkorn, R. (1987) Nature 327, 526-529; Golden, J. W., Carrasco, C. D., Mulligan, M. E., Schneider, G. J., and Haselkorn, R. (1988) J. Bacteriol. 170, 5034-5041). The promoter for the nifB operon was located by primer extension. Comparison of the nifB 5'-flanking sequence with the nifH 5'-flanking sequence did not reveal any consensus base pairs that would define a nif promoter for Anabaena. The operon contains two instances of 7-base pair directly repeated sequences: seven copies of the repeated sequence are found between the nifB and fdxN genes and six copies are found between the nifS and nifU genes. The function of these repeats is unknown.     

Transcription of the Azospirillum brasilense nifH gene is positively regulated by NifA and NtrA and is negatively controlled by the cellular nitrogen status.

The expression of a translational Azospirillum brasilense nifH-uidA fusion was studied in A. brasilense and in Rhizobium meliloti strains with mutations in nifA, ntrA and ntrC. Induction of the fusion was observed in the R. meliloti wild-type and NtrC- strains on incubation under microaerobic conditions but not in the NifA- and NtrA- strains, showing the absolute requirement of both sigma 54 and NifA for activation of the nifH promoter. Histochemical analysis of the root nodules elicited by R. meliloti wild-type showed expression of the fusion in the late symbiotic zone but not in the meristematic and the early symbiotic zones. No induction of the nifH-uidA fusion was observed in the R. meliloti wild-type or NifA- strains incubated aerobically in nitrogen-free medium, indicating that, in contrast to R. meliloti nifH, A. brasilense nifH cannot be activated directly by NtrC. Expression of the nifH gene in A. brasilense only occurs under nitrogen-limiting, microaerobic conditions, suggesting the presence of a nitrogen-dependent control system for nif gene expression.     

The nifA gene product from Rhizobium leguminosarum biovar trifolii lacks the N-terminal domain found in other NifA proteins

The nifA gene has been identified between the fixX and nifB genes in the clover microsymbiont Rhizobium leguminosarum biovar trifolii (R.I. bv. trifolii) strain ANU843. Expression of the nifA gene is induced in the symbiotic state and site-directed mutagenesis experiments indicate that nifA expression is essential for symbiotic nitrogen fixation. Interestingly, the predicted R.I. bv. trifolii NifA protein lacks an N-terminal domain that is present in the homologous proteins from R.I. bv. viciae, Rhizobium meliloti, Bradyrhizobium japonicum, Klebsiella pneumoniae and all other documented NifA proteins. This indicates that this N-terminal domain is not essential for NifA function in R.I. bv. trifolii.     

Rhizobium meliloti nifN (fixF) gene is part of an operon regulated by a nifA-dependent promoter and codes for a polypeptide homologous to the nifK gene product.

An essential gene for symbiotic nitrogen fixation (fixF) is located near the common nodulation region of Rhizobium meliloti. A DNA fragment carrying fixF was characterized by hybridization with Klebsiella pneumoniae nif DNA and by nucleotide sequence analysis. The fixF gene was found to be related to K. pneumoniae nifN and was therefore renamed as the R. meliloti nifN gene. Upstream of the nifN coding region a second open reading frame was identified coding for a putative polypeptide of 110 amino acids (ORF110). By fragment-specific Tn5 mutagenesis it was shown that the nifN gene and ORF110 form an operon. The control region of this operon contains a nif promoter and also the putative nifA-binding sequence. For the deduced amino acid sequence of the nifN gene product a striking homology to the R. meliloti nifK protein was found. One cysteine residue and its adjacent amino acid sequence, which are highly conserved in the R. meliloti nifK, R. meliloti nifN, and K. pneumoniae nifN proteins, may play a role in binding the FeMo cofactor.     

Sequence and structural organization of a nif A-like gene and part of a nifB-like gene of Herbaspirillum seropedicae strain Z78.

The deduced amino acid sequence derived from the sequence of a fragment of DNA from the free-living diazotroph Herbaspirillum seropedicae was aligned to the homologous protein sequences encoded by the nifA genes from Azorhizobium caulinodans, Rhizobium leguminosarum, Rhizobium meliloti and Klebsiella pneumoniae. High similarity was found in the central domain and in the C-terminal region. The H. seropedicae putative NifA sequence was also found to contain an interdomain linker similar to that conserved among rhizobial NifA proteins, but not K. pneumoniae or Azotobacter vinelandii. Analysis of the regulatory sequences found 5' from nifA indicated that the expression of this gene in H. seropedicae is likely to be controlled by NifA, NtrC and RpoN, as judged by the presence of specific NifA- and NtrC-binding sites and characteristic -24/-12 promoters. Possible additional regulatory features included an 'anaerobox' and a site for integration host factor. The N-terminus of another open reading frame was found 3' from nifA and tentatively identified as nifB by amino acid sequence comparison. The putative nifB promoter sequence suggests that expression of H. seropedicae nifB may be activated by NifA and dependent on RpoN.     

Characterization of nifB, nifS, and nifU genes in the cyanobacterium Anabaena variabilis: NifB is required for the vanadium-dependent nitrogenase.

Anabaena variabilis ATCC 29413 is a heterotrophic, nitrogen-fixing cyanobacterium containing both a Mo-dependent nitrogenase encoded by the nif genes and V-dependent nitrogenase encoded by the vnf genes. The nifB, nifS, and nifU genes of A. variabilis were cloned, mapped, and partially sequenced. The fdxN gene was between nifB and nifS. Growth and acetylene reduction assays using wild-type and mutant strains indicated that the nifB product (NifB) was required for nitrogen fixation not only by the enzyme encoded by the nif genes but also by the enzyme encoded by the vnf genes. Neither NifS nor NifU was essential for nitrogen fixation in A. variabilis.     

Rhizobium meliloti ntrA (rpoN) gene is required for diverse metabolic functions.

We report the identification and cloning of an ntrA-like (glnF rpoN) gene of Rhizobium meliloti and show that the R. meliloti ntrA product (NtrA) is required for C4-dicarboxylate transport as well as for nitrate assimilation and symbiotic nitrogen fixation. DNA sequence analysis showed that R. meliloti NtrA is 38% homologous with Klebsiella pneumoniae NtrA. Subcloning and complementation analysis suggested that the R. meliloti ntrA promoter lies within 125 base pairs of the initiation codon and may be constitutively expressed.     

The nifA gene of Rhizobium meliloti is oxygen regulated.

Experiments using plasmid-borne gene fusions and direct RNA measurements have revealed that expression from the nifA gene is induced in Rhizobium meliloti when the external oxygen concentration is reduced to microaerobic levels. Induction occurs in the absence of alfalfa and in the presence of fixed nitrogen and does not require ntrC. The production of functional nifA gene product (NifA) can be demonstrated by its ability to activate the nitrogenase promoter P1. Aerobic induction of nifA can also occur during nitrogen starvation at low pH, but in this case induction is dependent on ntrC and does not lead to P1 activation. The data indicate that reduced oxygen tension is potentially a major trigger for symbiotic activation of nitrogen fixation in Rhizobium species.