Functional analysis of the fixNOQP region of Azorhizobium caulinodans.

The deduced amino acid sequences of four open reading frames identified upstream of the fixGHI region in Azorhizobium caulinodans are very similar to the putative terminal oxidase complex coded by the fixNOQP operons from Rhizobium meliloti and Bradyrhizobium japonicum. The expression of the A. caulinodans fixNOQP genes, which was maximal under microaerobiosis, was positively regulated by FixK and independent of NifA. In contrast to the Fix- phenotype of B. japonicum and R. meliloti fixN mutants, an A. caulinodans fixNO-deleted mutant strain retained 50% of the nitrogenase activity of the wild type in the symbiotic state. In addition, the nitrogenase activity was scarcely reduced under free-living conditions. Analysis of membrane fractions of A. caulinodans wild-type and mutant strains suggests that the fixNOQP region encodes two proteins with covalently bound hemes, tentatively assigned to fixO and fixP. Spectral analysis showed a large decrease in the c-type cytochrome content of the fixN mutant compared with the wild type. These results provide evidence for the involvement of FixNOQP proteins in a respiratory process. The partial impairment in nitrogen fixation of the fixN mutant in planta may be due to the activity of an alternative terminal oxidase compensating for the loss of the oxidase complex encoded by fixNOQP.     

Involvement of fixLJ in the regulation of nitrogen fixation in Azorhizobium caulinodans

A gene bank of Azorhizobium caulinodans DNA constructed in the bacteriophage lambda GEM11 was screened with Rhizobium meliloti fixL and fixJ genes as probes. One positive recombinant phage, ORS lambda L, was isolated. The nucleotide sequence of a 3.7 kb fragment was established. Two open reading frames of 1512bp and 613bp were identified as fixL and fixJ. Kanamycin cartridges were inserted into the cloned fixL and fixJ genes and recombined into the host genome. The resulting mutants were Nif- Fix-, suggesting that the two genes were required for symbiotic nitrogen fixation and for nitrogen fixation in the free-living state. Using pnifH-lacZ and pnifA-lacZ fusions, it was shown that the FixLJ products controlled the expression of nifH and nifA in bacteria grown in the free-living state.     

Regulatory role of Rhizobium etli CNPAF512 fnrN during symbiosis

The Rhizobium etli CNPAF512 fnrN gene was identified in the fixABCX rpoN(2) region. The corresponding protein contains the hallmark residues characteristic of proteins belonging to the class IB group of Fnr-related proteins. The expression of R. etli fnrN is highly induced under free-living microaerobic conditions and during symbiosis. This microaerobic and symbiotic induction of fnrN is not controlled by the sigma factor RpoN and the symbiotic regulator nifA or fixLJ, but it is due to positive autoregulation. Inoculation of Phaseolus vulgaris with an R. etli fnrN mutant strain resulted in a severe reduction in the bacteroid nitrogen fixation capacity compared to the wild-type capacity, confirming the importance of FnrN during symbiosis. The expression of the R. etli fixN, fixG, and arcA genes is strictly controlled by fnrN under free-living microaerobic conditions and in bacteroids during symbiosis with the host. However, there is an additional level of regulation of fixN and fixG under symbiotic conditions. A phylogenetic analysis of the available rhizobial FnrN and FixK proteins grouped the proteins in three different clusters.     

Emerging complexity in the denitrification regulatory network of Bradyrhizobium japonicum

Bradyrhizobium japonicum is a Gram-negative soil bacterium symbiotically associated with soya bean plants, which is also able to denitrify under free-living and symbiotic conditions. In B. japonicum, the napEDABC, nirK, norCBQD and nosRZDYFLX genes which encode reductases for nitrate, nitrite, nitric oxide and nitrous oxide respectively are required for denitrification. Similar to many other denitrifiers, expression of denitrification genes in B. japonicum requires both oxygen limitation and the presence of nitrate or a derived nitrogen oxide. In B. japonicum, a sophisticated regulatory network consisting of two linked regulatory cascades co-ordinates the expression of genes required for microaerobic respiration (the FixLJ/FixK2 cascade) and for nitrogen fixation (the RegSR/NifA cascade). The involvement of the FixLJ/FixK2 regulatory cascade in the microaerobic induction of the denitrification genes is well established. In addition, the FNR (fumarase and nitrate reduction regulator)/CRP(cAMP receptor protein)-type regulator NnrR expands the FixLJ/FixK2 regulatory cascade by an additional control level. A role for NifA is suggested in this process by recent experiments which have shown that it is required for full expression of denitrification genes in B. japonicum. The present review summarizes the current understanding of the regulatory network of denitrification in B. japonicum.     

Rhizobium leguminosarum bv. viciae contains a second fnr/fixK-like gene and an unusual fixL homologue

Genes of Rhizobium leguminosarum bv. viciae VF39 coding for the regulatory elements NifA, FixL and FixK were isolated, sequenced and genetically analysed. The fixK–fixL region is located upstream of the fixNOQP operon on the non-nodulation plasmid pRleVF39c. The deduced amino acid sequence of FixL revealed an unusual structure in that it contains a receiver module (homologous to the N-terminal domain of response regulators) fused to its transmitter domain. An oxygen-sensing haem-binding domain, found in other FixL proteins, is conserved in R. leguminosarum bv. viciae FixL. R. leguminosarum bv. viciae possesses a second fnr -like gene, designated fixK , whose encoded gene product is very similar to Rhizobium meliloti and Azorhizobium caulinodans FixK. Individual R. leguminosarum bv. viciae fixK and fixL insertion mutants displayed a Fix⁺ phenotype. A reduced nitrogen-fixation activity was found for a R. leguminosarum bv. viciae fnrN -deletion mutant, whereas no nitrogen-fixation activity was detectable for a fixK / fnrN double mutant. The R. leguminosarum bv. viciae nifA gene is expressed independently of FixL and FixK under aerobic and microaerobic conditions, whereas fixL gene expression is induced under microaerobiosis. Another orf was identified downstream of fixK–fixL and encodes a product which has homology to pseudoazurins from different species. Mutation of this azu gene showed that it is dispensable for nitrogen fixation.     

A mutant of Escherichia coli capable of utilizing 3-deoxy-d-manno-2-octulosonic acid as sole carbon source

Abstract The hypothesis that rifampicin resistance mutations (possibly leading to altered RNA polymerases) have a pleiotropic effect on symbiotic nitrogen fixation was tested using the Rhizobium japonicum -soybean symbiosis. A total of 20 spontaneous rifampicin-resistant mutants of R. japonicum strain 110 were analyzed biochemically. RNA polymerase assays revealed that the enzyme from 15 mutants was indeed rifampicin-insensitive. Two of these mutants were found to possess an enzyme with an electrophoretically altered β subunit. All rifampicin-resistant mutants were able to form nodules on soybeans and fix nitrogen symbiotically; free-living nitrogen fixation under microaerophilic culture conditions was also unaffected.     

Agrobacterium tumefaciens Is a Diazotrophic Bacterium

This is the first report that Agrobacterium tumefaciens can fix nitrogen in a free-living condition as shown by its abilities to grow on nitrogen-free medium, reduce acetylene to ethylene, and incorporate ¹⁵N supplied as ¹⁵N₂. As with most other well-characterized diazotrophic bacteria, the presence of NH₄⁺ in the medium and aerobic conditions repress nitrogen fixation by A. tumefaciens. The system requires molybdenum. No evidence for nodulation was found with pea, peanut, or soybean plants. Further understanding of the nitrogen-fixing ability of this bacterium, which has always been considered a pathogen, should cast new light on the evolution of a pathogenic versus symbiotic relationship.     

Bacterial RuBisCO is required for efficient Bradyrhizobium/Aeschynomene symbiosis

Rhizobia and legume plants establish symbiotic associations resulting in the formation of organs specialized in nitrogen fixation. In such organs, termed nodules, bacteria differentiate into bacteroids which convert atmospheric nitrogen and supply the plant with organic nitrogen. As a counterpart, bacteroids receive carbon substrates from the plant. This rather simple model of metabolite exchange underlies symbiosis but does not describe the complexity of bacteroids' central metabolism. A previous study using the tropical symbiotic model Aeschynomene indica/photosynthetic Bradyrhizobium sp. ORS278 suggested a role of the bacterial Calvin cycle during the symbiotic process. Herein we investigated the role of two RuBisCO gene clusters of Bradyrhizobium sp. ORS278 during symbiosis. Using gene reporter fusion strains, we showed that cbbL1 but not the paralogous cbbL2 is expressed during symbiosis. Congruently, CbbL1 was detected in bacteroids by proteome analysis. The importance of CbbL1 for symbiotic nitrogen fixation was proven by a reverse genetic approach. Interestingly, despite its symbiotic nitrogen fixation defect, the cbbL1 mutant was not affected in nitrogen fixation activity under free living state. This study demonstrates a critical role for bacterial RuBisCO during a rhizobia/legume symbiotic interaction.     

An Fnr-like protein encoded in Rhizobium leguminosarum biovar viciae shows structural and functional homology to Rhizobium meliloti FixK

Summary A 1.9 kb DNA region of Rhizobium leguminosarum biovar viciae strain VF39 capable of promoting microaerobic and symbiotic induction of the Rhizobium meliloti fixN gene was identified by heterologous complementation. Sequence analysis of this DNA region revealed the presence of two complete open reading frames, orf240 and orf114. The deduced amino acid sequence of orf240 showed significant homology to Escherichia coli Fnr and R. meliloti FixK. The major difference between ORF240 and FixK is the presence of 21 N-terminal amino acids in ORF240 that have no counterpart in FixK. A similar protein domain is also present in E. coli Fnr and is essential for the oxygen-regulated activity of this protein. Analysis of the nucleotide sequence upstream of orf240 revealed a motif similar to the NtrA-dependent promoter consensus sequence, as well as two DNA regions resembling the Fnr consensus binding sequence. A Tn5-generated mutant in orf240 lost the ability to induce the R. meliloti fixN-lacZ fusion. Interestingly, this mutant was still capable of nitrogen fixation but showed reduced nitrogenase activity.     

Nitrogen metabolism in Sinorhizobium meliloti-alfalfa symbiosis: dissecting the role of GlnD and PII proteins

To contribute nitrogen for plant growth and establish an effective symbiosis with alfalfa, Sinorhizobium meliloti Rm1021 needs normal operation of the GlnD protein, a bifunctional uridylyltransferase/uridylyl-cleavage enzyme that measures cellular nitrogen status and initiates a nitrogen stress response (NSR). However, the only two known targets of GlnD modification in Rm1021, the PII proteins GlnB and GlnK, are not necessary for effectiveness. We introduced a Tyr→Phe variant of GlnB, which cannot be uridylylated, into a glnBglnK background to approximate the expected state in a glnD-sm2 mutant, and this strain was effective. These results suggested that unmodified PII does not inhibit effectiveness. We also generated a glnBglnK-glnD triple mutant and used this and other mutants to dissect the role of these proteins in regulating the free-living NSR and nitrogen metabolism in symbiosis. The glnD-sm2 mutation was dominant to the glnBglnK mutations in symbiosis but recessive in some free-living phenotypes. The data show that the GlnD protein has a role in free-living growth and in symbiotic nitrogen exchange that does not depend on the PII proteins, suggesting that S. meliloti GlnD can communicate with the cell by alternate mechanisms.     

非共生生物固氮微生物分子生态学研究进展

氮是限制生态系统生产力的主要元素,生物固氮是自然生态系统中氮的主要来源.生物固氮包括共生、联合和自生固氮3种类型,其中联合固氮和自生固氮统称为非共生固氮.相对于共生固氮而言,非共生固氮速率虽然较低,但其不需要与其他生物形成共生体系就可以生存并进行固氮,在时空分布上更加广泛,因此对生态系统氮循环特别是素输入具有重要贡献.本文对近年有关非共生固氮微生物的多样性、土壤和叶际固氮微生物的分布特征及影响因素等研究进展进行了综述,并在此基础上阐述了现有研究中存在的问题和发展前景.