Regulation Of Nitrogen Fixation Genes Research Articles

Advances in the mechanism of cereal/legume intercropping promotion of symbiotic nitrogen fixation

Biological nitrogen fixation by rhizobia in symbiotic association with legumes is a valuable N source for agroecosystems, and thus it is crucial for increased sustainability of agricultural production. Previous studies found that legume and cereal intercropping can promote symbiotic nitrogen fixation mainly due to rhizosphere nitrogen depletion by the cereal and that soil mineral nitrogen is absorbed more by cereals than legumes, which forces the legume to increase its reliance on symbiotic nitrogen fixation. However, in addition to the nitrogen depletion theory in the rhizosphere, stoichiometry (such as nitrogen and phosphorus coupling) and molecular regulation mechanisms are also important in understanding legume nitrogen fixation. The legume usually maintains a stable stoichiometric relationship between nitrogen and phosphorus. Symbiotic nitrogen fixation of legumes tends to have a high demand for phosphorus, mainly because nitrogen fixation by legumes is a high energy-consuming process, and the required energy is directly provided by ATP. Therefore, when the phosphorus obtained by the intercropped legume is insufficient to meet the increase in symbiotic nitrogen fixation, legume growth is limited by phosphorus. Under phosphorus limitation, the phosphorus uptake strategy of the legume can be stimulated, i.e. by increasing the infection of arbuscular mycorrhizal fungi (AMF) and secretion of root exudates. The AMF mycelia of legumes can extend beyond the rhizosphere, linking the roots to the surrounding soil microhabitats and expanding the area of phosphorus absorbed by the roots. On the other hand, mobilizing rhizosphere insoluble phosphorus through root exudates is also an important pathway for improving phosphorus utilization. Increased soil phosphorus availability promotes plant growth and increased allocation of carbon (C) sources to roots and nodules, resulting in a larger root system or greater nodule formation, finally higher symbiotic nitrogen fixation. In addition, the formation of root nodules is influenced by rhizosphere talk between the cereal and legume, which are directly related to the level of nitrogen. A recent study found that under low N concentration conditions, cereal root exudates stimulated flavonoid biosynthesis and secretion partly through upregulating expression of the Chalcone-flavanone isomerase gene ( CFI ) in legumes. Meanwhile, an auxin-responsive GH3 family gene ( GH3.1 ) and the nodulation gene ENODL2 displayed significant increases, suggesting that nodulation continues under the regulation of auxin signaling and the nodulin-like protein. At nodule maturity, cereal root exudates continue to up-regulate key nodulation genes, such as NODL4 , ENOD93 and the N fixation gene FixI3 , finally promoting symbiotic nitrogen fixation. Moreover, a high concentration of soil mineral N significantly suppressed expression of the early nodulin gene NIN in Lotus corniculatus , which inhibited nodulation and symbiotic nitrogen fixation. In the cereal/legume intercropping system, rhizosphere nitrogen depletion can relieve the inhibition of nitrate on the expression of key nodulation and N fixation genes, thus promoting nodulation and symbiotic nitrogen fixation. In conclusion, intercropping can promote nitrogen uptake and optimize nitrogen use efficiency through the coupling effects of rhizosphere nitrogen depletion, phosphorus mobilization and the regulation of nitrogen fixation genes. This review will help improve our understanding of the mechanism of symbiotic nitrogen fixation in the cereal/legume intercropping system.

Interaction of an IHF-like protein with the Rhizobium etli nifA promoter

The nifA gene fulfills an essential role in the regulation of nitrogen fixation genes in Rhizobium etli. Transcription analysis of the nifA gene, assessed using promoter deletions, indicated an oxygen-independent expression, threefold higher during symbiosis as compared with free-living conditions. Electrophoretic mobility shift assays using those nifA promoter deletion fragments, which were actively transcribed, demonstrated the specific interaction with R. etli cellular protein(s) resulting in the formation of two DNA-protein complexes. An interacting protein was purified by liquid chromatography on Heparin Sepharose and Mono S columns. The purified 12 kDa R. etli protein cross-reacted with antibodies directed against Escherichia coli integration host factor (IHF). Furthermore, purified E. coli IHF was able to specifically bind to the R. etli nifA promoter region. These results point to an as yet undisclosed function of IHF in the regulation of R. etli nifA expression.

Bacterial Regulatory Networks Include Direct Contact of Response Regulator Proteins: Interaction of RegA and NtrX in Rhodobacter capsulatus

The formation of photosynthetic complexes in facultatively photosynthetic bacteria is controlled by the oxygen tension in the environment. In Rhodobacter capsulatus the two-component system RegB/RegA plays a major role in the redox control of photosynthesis genes but also controls other redox-dependent systems. The response regulator RegA is phosphorylated under low oxygen tension and activates the puf and puc operons, which encode pigment binding proteins, by binding to their promoter regions. Data from a yeast two-hybrid analysis as well as an in vitroanalysis indicate that RegA interacts with the NtrX protein, the response regulator of the NtrY/NtrX two-component system which is believed to be involved in regulation of nitrogen fixation genes. Our further analysis revealed that NtrX is indeed involved in the regulation of the puf and puc operons. Furthermore, we showed that an altered NtrX protein, which is predicted to adopt the conformation of phosphorylated NtrX protein, binds within the puf promoter region close to the RegA binding sites. We conclude that a direct interaction of two response regulators connects the regulatory systems for redox control and nitrogen control.

Recent developments in the structural organization and regulation of nitrogen fixation genes in Herbaspirillum seropedicae

Herbaspirillum seropedicae is a nitrogen-fixing bacterium found in association with economically important gramineae. Regulation of nitrogen fixation involves the transcriptional activator NifA protein. The regulation of NifA protein and its truncated mutant proteins is described and compared with that of other nitrogen fixation bacteria. Nitrogen fixation control in H. seropedicae, of the β-subgroup of Proteobacteria, has regulatory features in common with Klebsiella pneumoniae, of the γ-subgroup, at the level of nifA expression and with rhizobia and Azospirillum brasilense, of the α-subgroup, at the level of control of NifA by oxygen.

Mechanism of coordinated synthesis of the antagonistic regulatory proteins NifL and NifA of Klebsiella pneumoniae.

The nifLA operon of Klebsiella pneumoniae codes for the two antagonistic regulatory proteins which control expression of all other nitrogen fixation genes. NifA is a transcriptional activator, and NifL inhibits NifA. The importance of a correct NifL-NifA stoichiometry for efficient regulation of nitrogen fixation genes has been investigated by constructing a strain with an altered nifL-nifA gene dosage ratio, resulting from the integration of an extra copy of nifA. Results showed that a balanced synthesis of both gene products is essential for correct regulation. Effects of mutations provoking translation termination of nifL upstream or downstream of its natural stop codon, combined with overproduction of both proteins when the genes are transcribed and translated from signals of the phi10 gene of the phage T7, showed that, in addition to the previously reported transcriptional polarity, there is translational coupling between nifL and nifA. In spite of the apparently efficient ribosome binding site of nifA, its rate of independent translation is very low. This is due to a secondary structure masking the Shine-Dalgarno sequence of nifA, which could be melted by ribosomes translating nifL. Mutational analysis confirmed the functional significance of the secondary structure in preventing independent translation of nifA. Translational coupling between the two cistrons is proposed as an efficient mechanism to prevent production of an excess of NifA, which would affect the normal regulation of nitrogen fixation genes.

Transcription termination within the regulatorynifLA operon ofKlebsiella pneumoniae

The effect of premature stop codons in thenifL gene on the expression ofnifA-lacZ operon and protein fusions inKlebsiella pneumoniae was analysed in detail. Our results revealed transcriptional polarity in this operon. By dissecting the operon, intragenic regions containing Rho-dependent transcription terminators have been identified. As shown for other Rhodependent terminators, their cytosine content is much higher than the incidence of guanines. However, other regions of the operon that have this feature did not show termination activity, suggesting that, contrary to previous reports, a correlation between these parameters cannot readily be established. Some of our results also suggested that, in addition to polarity, other mechanisms may prevent expression ofnifA when translation ofnifL is altered. Their importance for efficient regulation of nitrogen fixation genes is discussed.

Transcription termination within the regulatory

The effect of premature stop codons in the nifL gene on the expression of nifA-lacZ operon and protein fusions in Klebsiella pneumoniae was analysed in detail. Our results revealed transcriptional polarity in this operon. By dissecting the operon, intragenic regions containing Rho-dependent transcription terminators have been identified. As shown for other Rho-dependent terminators, their cytosine content is much higher than the incidence of guanines. However, other regions of the operon that have this feature did not show termination activity, suggesting that, contrary to previous reports, a correlation between these parameters cannot readily be established. Some of our results alos suggested that, in addition to polarity, other mechanisms may prevent expression of nifA when translation of nifL is altered. Their importance for efficient regulation of nitrogen fixation genes is discussed.

The role of uridylyltransferase in the control of Klebsiella pneumoniae nif gene regulation.

The glnD gene in enteric bacteria encodes a uridylyltransferase/uridylyl-removing enzyme which acts as the primary nitrogen sensor in the nitrogen regulation (Ntr) system. We have investigated the role of this enzyme in transcriptional regulation of nitrogen fixation genes in Klebsiella pneumoniae by cloning glnD from this organism and constructing a null mutant by insertional inactivation of the chromosomal gene using the omega interposon. K. pneumoniae glnD encodes a 102.3 kDa polypeptide which is highly homologous to the predicted products of both Escherichia coli glnD and Azotobacter vinelandii nfrX. The glnD-omega mutant was unable to uridylylate PII and was altered in adenylylation/deadenylylation of glutamine synthetase. Uridylyltransferase was required for derepression of ntr-regulated promoters such as glnAp2 and pnifL but was not involved in the nif-specific response to changes in nitrogen status mediated by the nifL product. We conclude that a separate, as yet uncharacterised, nitrogen control system may be responsible for nitrogen sensing by NifL.

The oxygen sensor FixL of Rhizobium meliloti is a membrane protein containing four possible transmembrane segments

Regulation of nitrogen fixation genes in Rhizobium meliloti is mediated by two proteins, FixL and FixJ, in response to oxygen availability. FixL is an oxygen-binding hemoprotein with kinase and phosphatase activities that is thought to sense oxygen levels directly and to transmit this signal to FixJ via phosphorylation-dephosphorylation reactions. FixJ controls the expression of other regulatory genes, including nifA, that regulate the transcription of genes required for symbiotic nitrogen fixation. We have been studying the structural and functional features of FixL that are required for oxygen sensing. We constructed mutant derivatives and confirmed that FixL consists of 505 amino acids instead of 464, as originally reported. Hydropathy plots of the full-length protein, together with TnphoA insertional analysis, lead us to propose that FixL is likely to be a polytopic integral membrane protein containing four membrane-spanning segments. We have also constructed an N-terminal deletion of the FixL protein whose in vivo activity indicates that the hydrophobic membrane-spanning regions are not absolutely required for oxygen sensing in vivo. We also report that FixL shares homology in its N terminus with other sensor proteins, including KinA from Bacillus subtilis and NtrB from Bradyrhizobium parasponia. The region of homology comprises a 70-amino-acid residue stretch that is also conserved in two oxygenases, P-450 and isopenicillin synthase.

The regulation of nitrogen utilization in enteric bacteria

The regulation of nitrogen utilization in enteric bacteria

Transcriptional regulation of nitrogen fixation genes by DNA supercoiling

DNA gyrase (ATP dependent topoisomerase type II, EC 5.99.1.3) was found to be essential for the expression of the Klebsiella pneumoniae nitrogen fixation gene cluster carried by plasmid pRD1 in Escherichia coli. In the absence of DNA gyrase activity, nitrogen fixation activity could be restored by providing a constitutively expressed nifA function in trans. Our results suggest that nif gene regulation by oxygen may be mediated through the alteration of the superhelical status of the promoter of the nifLA regulatory operon, in addition to the action of the nifL gene product.