The Rhodobacter capsulatus glnB gene is regulated by NtrC at tandem rpoN-independent promoters.

The protein encoded by glnB of Rhodobacter capsulatus is part of a nitrogen-sensing cascade which regulates the expression of nitrogen fixation genes (nif). The expression of glnB was studied by using lacZ fusions, primer extension analysis, and in vitro DNase I footprinting. Our results suggest that glnB is transcribed from two promoters, one of which requires the R. capsulatus ntrC gene but is rpoN independent. Another promoter upstream of glnB is repressed by NtrC; purified R. capsulatus NtrC binds to sites that overlap this distal promoter region.     

Symbiotic nitrogen fixation by a nifA deletion mutant of Rhizobium meliloti: the role of an unusual ntrC allele.

In the N2-fixing alfalfa symbiont Rhizobium meliloti, the three sigma 54 (NTRA)-dependent positively acting regulatory proteins NIFA, NTRC, and DCTD are required for activation of promoters involved in N2 fixation (pnifHDKE and pfixABCX), nitrogen assimilation (pglnII), and C4-dicarboxylate transport (pdctA), respectively. Here, we describe an allele of ntrC which results in the constitutive activation of the above NTRC-, NIFA-, and DCTD-regulated promoters. The expression and activation of wild-type NTRC occur in response to nitrogen availability, whereas in cells carrying the ntrC283 allele, the NTRC283 protein appears constitutively active and is constitutively expressed. The ntrC283 allele was shown to carry a single mutation resulting in the replacement of an Asp by a Tyr residue in the helix-turn-helix motif of ntrC283. Introduction of the ntrC283 allele into a nifA deletion mutant restores the N2-fixation ability to 70 to 80% of the wild-type level. Thus, the nifA gene is dispensable for symbiotic N2 fixation.     

The NifA-RpoN Regulon of Mesorhizobium loti Strain R7A and Its Symbiotic Activation by a Novel LacI/GalR-Family Regulator

Mesorhizobium loti is the microsymbiont of Lotus species, including the model legume L. japonicus. M. loti differs from other rhizobia in that it contains two copies of the key nitrogen fixation regulatory gene nifA, nifA1 and nifA2, both of which are located on the symbiosis island ICEMlSym^R7A. M. loti R7A also contains two rpoN genes, rpoN1 located on the chromosome outside of ICEMlSym^R7A and rpoN2 that is located on ICEMlSym^R7A. The aims of the current work were to establish how nifA expression was activated in M. loti and to characterise the NifA-RpoN regulon. The nifA2 and rpoN2 genes were essential for nitrogen fixation whereas nifA1 and rpoN1 were dispensable. Expression of nifA2 was activated, possibly in response to an inositol derivative, by a novel regulator of the LacI/GalR family encoded by the fixV gene located upstream of nifA2. Other than the well-characterized nif/fix genes, most NifA2-regulated genes were not required for nitrogen fixation although they were strongly expressed in nodules. The NifA-regulated nifZ and fixU genes, along with nifQ which was not NifA-regulated, were required in M. loti for a fully effective symbiosis although they are not present in some other rhizobia. The NifA-regulated gene msi158 that encodes a porin was also required for a fully effective symbiosis. Several metabolic genes that lacked NifA-regulated promoters were strongly expressed in nodules in a NifA2-dependent manner but again mutants did not have an overt symbiotic phenotype. In summary, many genes encoded on ICEMlSym^R7A were strongly expressed in nodules but not free-living rhizobia, but were not essential for symbiotic nitrogen fixation. It seems likely that some of these genes have functional homologues elsewhere in the genome and that bacteroid metabolism may be sufficiently plastic to adapt to loss of certain enzymatic functions.     

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.     

The Azorhizobium caulinodans nitrogen-fixation regulatory gene, nifA, is controlled by the cellular nitrogen and oxygen status

The nucleotide sequence of the Azorhizobium caulinodans ORS571 nifA locus was determined and the deduced NifA amino acid sequence compared with that of NifA from other nitrogen-fixing species. Highly conserved domains, including helix-turn-helix and ATP-binding motifs, and specific conserved residues, such as a cluster of cysteines, were identified. The nifA 5' upstream region was found to contain DNA sequence motifs highly homologous to promoter elements involved in nifA/ntr-mediated control and a consensus element found in the 5' upstream region of the Bradyrhizobium japonicum 5-aminolevulinic acid synthase (hemA) gene and of Escherichia coli genes activated during anaerobiosis via the fnr (fumarate nitrate reduction) control system. A nifA-lac fusion was constructed using miniMu-lac and its activity measured in different genetic backgrounds and under various physiological conditions (in culture and in planta). NifA expression was found to be negatively autoregulated, repressed by rich nitrogen sources and high oxygen concentrations, and controlled (partially) by the ntrC gene, both in culture and in planta. DNA supercoiling was also implicated in nifA regulation, since DNA gyrase inhibitors severely repressed nifA-lac expression.     

The pleiotropic nature of symbiotic regulatory mutants: Bradyrhizobium japonicum nifA gene is involved in control of nif gene expression and formation of determinate symbiosis

In the slow-growing soybean symbiont, Bradyrhizobium japonicum (strain 110), a nifA-like regulatory gene was located immediately upstream of the previously mapped fixA gene. By interspecies hybridization and partial DNA sequencing the gene was found to be homologous to nifA from Klebsiella pneumoniae and Rhizobium meliloti, and to a lesser extent, also to ntrC from K. pneumoniae. The B. japonicum nifA gene product was shown to activate B. japonicum and K. pneumoniae nif promoters (using nif::lacZ translational fusions) both in Escherichia coli and B. japonicum backgrounds. In the heterologous E. coli system activation was shown to be dependent on the ntrA gene product. Site-directed insertion and deletion/replacement mutagenesis revealed that nifA is probably the promoter-distal cistron within an operon. NifA- mutants were Fix- and pleiotropic: (i) they were defective in the synthesis of several proteins including the nifH gene product (nitrogenase Fe protein); the same proteins had been known to be repressed under aerobic growth of B. japonicum but derepressed at low O2 tension; (ii) the mutants had an altered nodulation phenotype inducing numerous, small, widely distributed soybean nodules in which the bacteroids were subject to severe degradation. These results show that nifA not only controls nitrogenase genes but also one or more genes involved in the establishment of a determinate, nitrogen-fixing root nodule symbiosis.     

Nitrogen fixation specific regulatory genes of Klebsiella pneumoniae and Rhizobium meliloti share homology with the general nitrogen regulatory gene ntrC of K. pneumoniae.

We have determined the complete nucleotide sequences of three functionally related nitrogen assimilation regulatory genes from Klebsiella pneumoniae and Rhizobium meliloti. These genes are: 1) The K. pneumoniae general nitrogen assimilation regulatory gene ntrC (formerly called glnG), 2) the K. pneumoniae nif-specific regulatory gene nifA, and 3) an R. meliloti nif-specific regulatory gene that appears to be functionally analogous to the K. pneumoniae nifA gene. In addition to the DNA sequence data, gel-purified K. pneumoniae nifA protein was used to determine the amino acid composition of the nifA protein. The K. pneumoniae ntrC and nifA genes code for proteins of 52,259 and 53,319 d respectively. The R. meliloti nifA gene codes for a 59,968 d protein. A central region within each polypeptide, consisting of approximately 200 amino acids, is between 52% and 58% conserved among the three proteins. Neither the amino termini nor the carboxy termini show any conserved sequences. Together with data that shows that the three regulatory proteins activate promoters that share a common consensus sequence in the -10 (5'-TTGCA-3') and -23 (5'-CTGG-3') regions, the sequence data presented here suggest a common evolutionary origin for the three regulatory genes.