Control of nitrogen fixation and ammonia excretion in Azorhizobium caulinodans

Due to the costly energy demands of nitrogen (N) fixation, diazotrophic bacteria have evolved complex regulatory networks that permit expression of the catalyst nitrogenase only under conditions of N starvation, whereas the same condition stimulates upregulation of high-affinity ammonia (NH₃) assimilation by glutamine synthetase (GS), preventing excess release of excess NH₃ for plants. Diazotrophic bacteria can be engineered to excrete NH₃ by interference with GS, however control is required to minimise growth penalties and prevent unintended provision of NH₃ to non-target plants. Here, we tested two strategies to control GS regulation and NH₃ excretion in our model cereal symbiont Azorhizobium caulinodans AcLP, a derivative of ORS571. We first attempted to recapitulate previous work where mutation of both P_II homologues glnB and glnK stimulated GS shutdown but found that one of these genes was essential for growth. Secondly, we expressed unidirectional adenylyl transferases (uATs) in a ΔglnE mutant of AcLP which permitted strong GS shutdown and excretion of NH₃ derived from N₂ fixation and completely alleviated negative feedback regulation on nitrogenase expression. We placed a uAT allele under control of the NifA-dependent promoter PnifH, permitting GS shutdown and NH₃ excretion specifically under microaerobic conditions, the same cue that initiates N₂ fixation, then deleted nifA and transferred a rhizopine nifA_L94Q/D95Q-rpoN controller plasmid into this strain, permitting coupled rhizopine-dependent activation of N₂ fixation and NH₃ excretion. This highly sophisticated and multi-layered control circuitry brings us a step closer to the development of a "synthetic symbioses” where N₂ fixation and NH₃ excretion could be specifically activated in diazotrophic bacteria colonising transgenic rhizopine producing cereals, targeting delivery of fixed N to the crop while preventing interaction with non-target plants.     

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.     

Role of the fixGHI region of Azorhizobium caulinodans in free-living and symbiotic nitrogen fixation

Abstract Rat intestinal epithelial cells were isolated and the activity of the calcium- and phospholipid-dependent protein kinase C (PKC) was investigated. The stimulation of activity by Escherichia coli heat stable enterotoxin (STa) was about 5-fold compared to control activity (16.91 ± 1.69 vs 93.56 ± 10.40 nmol/mg protein/min) and was dose dependent. Maximum enzyme activity was observed after incubation for 1 min with 6 ng of purified STa. The synergistic effects of calcium, phosphatidylserine and diolein on the enzyme activity were noted both in control and STa-treated cells. Staurosporine, a potent PKC inhibitor, significantly reduced the enzyme activity. Autoradiographic analysis of polyacrylamide gel electrophoresis revealed that pretreatment of the cells with STa also resulted in the phosphorylation of specific membrane proteins each with a molecular mass of 37 kDa, 100 kDa and 140 kDa. However, STa had no direct role on the enzyme activity. Our results, therefore, provide evidence for the involvement of PKC in STa-induced signal transduction in rat enterocytes.     

Characterization of the fixABC region of Azorhizobium caulinodans ORS571 and identification of a new nitrogen fixation gene

Summary The fast growing strain, Azorhizobium caulinodans ORS571, isolated from stem nodules of the tropical legume Sesbania rostrata, can grow in the free-living state at the expense of molecular nitrogen. Five point mutants impaired in nitrogen fixation in the free-living state have been complemented by a plasmid containing the cloned fix-ABC region of strain ORS571. Genetic analysis of the mutants showed that one was impaired in fixC, one in fixA and the three others in a new gene, located upstream from fixA and designated nifO. Site-directed Tn5 mutagenesis was performed to obtain Tn5 insertions in fixB and fixC. The four genes are required for nitrogen fixation both in the free-living state and under symbiotic conditions. The nucleotide sequence of nifO was established. The gene is transcribed independently of fixA and does not correspond to fixX, recently identified in Rhizobium meliloti and R. leguminosarum. Biochemical analysis of the five point mutants showed that they synthesized normal amounts of nitrogenase components. It is unlikely that fixA, fixC and nifO are involved in electron transport to nitrogenase. FixC could be required for the formation of a functional nitrogenase component 2.     

Role of PII proteins in nitrogen fixation control of Herbaspirillum seropedicae strain SmR1

Background  

The PII protein family comprises homotrimeric proteins which act as transducers of the cellular nitrogen and carbon status in prokaryotes and plants. In Herbaspirillum seropedicae, two PII-like proteins (GlnB and GlnK), encoded by the genes glnB and glnK, were identified. The glnB gene is monocistronic and its expression is constitutive, while glnK is located in the nlmAglnKamtB operon and is expressed under nitrogen-limiting conditions.     

Context-dependent functions of the PII and GlnK signal transduction proteins in Escherichia coli

Two closely related signal transduction proteins, PII and GlnK, have distinct physiological roles in the regulation of nitrogen assimilation. Here, we examined the physiological roles of PII and GlnK when these proteins were expressed from various regulated or constitutive promoters. The results indicate that the distinct functions of PII and GlnK were correlated with the timing of expression and levels of accumulation of the two proteins. GlnK was functionally converted into PII when its expression was rendered constitutive and at the appropriate level, while PII was functionally converted into GlnK by engineering its expression from the nitrogen-regulated glnK promoter. Also, the physiological roles of both proteins were altered by engineering their expression from the nitrogen-regulated glnA promoter. We hypothesize that the use of two functionally identical PII-like proteins, which have distinct patterns of expression, may allow fine control of Ntr genes over a wide range of environmental conditions. In addition, we describe results suggesting that an additional, unknown mechanism may control the cellular level of GlnK.     

Cloning of Azorhizobium caulinodans nicotinate catabolism genes and characterization of their importance in N2 fixation.

Twenty Azorhizobium caulinodans vector insertion (Vi) mutants unable to catabolize nicotinate (Nic- phenotype) were identified and directly cloned as pVi plasmids. These pVi plasmids were used as DNA hybridization probes to isolate homologous wild-type sequences. From subsequent physical mapping experiments, the nic::Vi mutants defined four distinct loci. Two, possibly three, of these loci are physically linked. A. caulinodans nic loci II and III encode the structural genes for nicotinate catabolism; nic loci I and IV encode nicotinate-driven respiratory chain components. Recombinant lambda bacteriophages corresponding to three of these loci were subcloned in pRK293; resulting plasmids were used for complementation tests with resolved nic::IS50 derivatives of the nic::Vi mutants. When wild-type A. caulinodans was cultured in defined liquid medium under 3% O2, nicotinate catabolism stimulated N2 fixation 10-fold. In these exponentially growing cultures, the entire (300 microM) nicotinate supplement was exhausted within 10 h. While nic::Vi mutants retained the ability to fix some N2, they did so at rates only 10% of that of the wild type: nitrogenase activity by nic::Vi mutants was not stimulated by 300 microM added nicotinate. Higher-level (5 mM) nicotinate supplementation inhibited N2 fixation. Because 5 mM nicotinate repressed nitrogenase induction in all nic::Vi mutants as well, this repression was independent of nicotinate catabolism. During catabolism, nicotinate is first oxidized to 6-OH-nicotinate by a membrane-bound nicotinate hydroxylase which drives a respiratory chain to O2. In A. caulinodans wild-type cultures, added 300 microM 6-OH-nicotinate stimulated N2 fixation twofold better than did added 300 microM nicotinate. Likewise, nic::Vi mutant 61302, defective in nicotinate hydroxylase, fixed N2 at wild-type levels when supplemented with 300 microM 6-OH-nicotinate. Therefore, nicotinate catabolism stimulates N2 fixation not by nicotinate hydroxylase-driven respiration but rather by some subsequent aspect(s) of nicotinate catabolism.     

Isolation and characterization of hydrogenase-negative mutants of Azorhizobium caulinodans ORS571

Hydrogenase-negative (Hup^-) mutants of Azorhizobium caulinodans ORS571 were isolated by means of Tn5 mutagenesis. The colony test used for screening for Hup^- strains was based on the absence of reduction of triphenyltetrazolium chloride with hydrogen. Suspensions from cultures of the mutant strains grown under derepressing conditions did not use hydrogen with methylene blue or oxygen as the hydrogen acceptor. The mutants were shown to carry single Tn5 insertions at different locations in the A. caulinodans genome. Molar growth yields (corrected for poly--hydroxybutyrate formation) in chemostat cultures of the mutants were similar to those of the wild type. Molar growth yields of the mutants were not increased by passing additional hydrogen through chemostat cultures, which is in agreement with the hydrogenase-negative phenotype of the mutants. H₂/N₂ ratios (mol H₂ formed per mol N₂ fixed) were calculated from the hydrogen content of the effluent gas and the N-content of the bacterial dry weight. Low H₂/N₂ ratios (between 1.2 and 1.9) were found in both energy-limited (oxygen or succinate) cultures and in cultures limited by the supply of an anabolic substrate (Mg²⁺). ATP/2e values (mol ATP used at the transport of 2e to nitrogen or H⁺) were calculated from the H₂/N₂ ratios and the molar growth yields of nitrogen-fixing and ammonia-assimilating cultures. ATP/2e values were between 7 and 11. It was concluded that the calculated ATP/2e values comprise not only 4 mol ATP used at the transport of 2e through nitrogenase but also energy equivalents needed for reversed electron flow from NADH to the low-potential hydrogen donor used by nitrogenase.     

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.     

Isolation and Characterization of an Oxygen Sensitive Mutant of Azorhizobium caulinodans

An oxygen sensitive mutant of Azorhizobium caulinodans strain IRBG 46 was isolated by NTG mutagenesis. It was defective in N₂ fixation under 3% O₂ level, while under 1% O₂ it was almost as active as the parent strain IRBG 46. The mutant was also found to be a slow grower with reduced respiratory activity, low azide tolerance and no catalase activity. However, it did not differ from its parent strain with respect to nitrate respiration. Under symbiotic condition the mutant formed smaller, light green nodules as compared to bigger, dark green nodules formed by the wild type strain. The mutant was also defective in N₂ fixation under symbiotic condition. Complementation analysis showed that the mutation might be in either fixL or fixJ gene which are involved in O₂ regulation of nif/fix gene expression. A possible role of all these factors in conferring a highly O₂ tolerant nitrogen fixing system in the organism, has been discussed.     

Utilization of nitrogen compounds and ammonia assimilation by chromatiaceae

Chromatium vinosum strain D, Thiocapsa roseopersicina strain 6311 and Ectothiorhodospira mobilis strain 8112 were grown anaerobically in the light with various single nitrogen sources. When substituted for NH₄Cl only glutamine and casamino acids supported good growth of all strains tested. Peptone and urea were utilized by C. vinosum and T. roseopersicina, glutamate, asparagine and nitrate only by C. vinosum. The strains were able to grow with molecular nitrogen; complete inhibition of this growth was observed in the presence of alanine with E. mobilis, and of alanine or asparagine with T. roseopersicina.Glutamate dehydrogenase, requiring either NADH or NADPH, NADH-linked glutamate synthase, and glutamine synthetase were demonstrate in the above organisms grown on NH₄Cl.     

Epiphytic Occurrence of Azorhizobium caulinodans and Other Rhizobia on Host and Nonhost Legumes

[This corrects the article on p. 2407 in vol. 55.].     

Epiphytic Occurrence of Azorhizobium caulinodans and Other Rhizobia on Host and Nonhost Legumes

A large population of Azorhizobium caulinodans was present on Sesbania rostrata; up to 5 × 10⁻⁵ cm⁻² were found on leaves and fewer were found on flowers. Although A. caulinodans was also present on the leaves of Sesbania aculeata (nonhost), the populations were much smaller than that observed on S. rostrata. The population of S. aculeata rhizobia on host leaves was less than 30 cm⁻², and their presence on host flowers was sporadic. Aeschynomene afraspera and Aeschynomene aspera rhizobia, which are profusely stem nodulating, were found on the leaves of host and nonhost plants and on the flowers of host plants, but, Aeschynomene pratensis and Aeschynomene sensitiva rhizobia were not found on the leaves and flowers of host plants.     

Molecular and functional characterization of the Azorhizobium caulinodans ORS571 hydrogenase gene cluster

In this work, we report the cloning and sequencing of the Azorhizobium caulinodans ORS571 hydrogenase gene cluster. Sequence analysis revealed the presence of 20 open reading frames hupTUVhypFhupSLCDFGHJK hypABhupRhypCDEhupE. The physical and genetic organization of A. caulinodans ORS571 hydrogenase system suggests a close relatedness to that of Rhodobacter capsulatus. In contrast to the latter species, a gene homologous to Rhizobium leguminosarum hupE was identified downstream of the hyp operon. A hupSL mutation drastically reduced the high levels of hydrogenase activity induced by the A. caulinodans ORS571 wild-type strain in symbiosis with Sesbania rostrata plants. However, no significant effects on dry weight and nitrogen content of S. rostrata plants inoculated with the hupSL mutant were observed in plant growth experiments.     

The mechanism of ammonia assimilation in nitrogen fixing bacteria

Summary Enzymatic and genetic evidence are presented for a new pathway of ammonia assimilation in nitrogen fixing bacteria: ammonium glutamine glutamate. This route to the important glutamate-glutamine family of amino acids differs from the conventional pathway, ammonium glutamate glutamine, in several respects. Glutamate synthetase [(glutamine amide-2-oxoglutarate aminotransferase) (oxidoreductase)], which is clearly distinct from glutamate dehydrogenase, catalyzes the reduced pyridine nucleotide dependent amination of -ketoglutarate with glutamine as amino donor yielding two molecules of glutamate as product. The enzyme is completely inhibited by the glutamine analogue DON, whereas glutamate dehydrogenase is not affected by this inhibitor; the glutamate synthetase reaction is irreversible. Glutamate synthetase is widely distributed in bacteria; the pyridine nucleotide coenzyme specificity of the enzyme varies in many of these species.The activities of key enzymes are modulated by environmental nitrogenous sources; for example, extracts of N₂-grown cells of Klebsiella pneumoniae form glutamate almost exclusively by this new route and contain only trace amounts of glutamate dehydrogenase activity whereas NH₃-grown cells possess both pathways. Also, the biosynthetically active form of glutamine synthetase with a low K _m for ammonium predominates in the N₂-grown cell.Several mutant strains of K. pneumoniae have been isolated which fail to fix nitrogen or to grow in an ammonium limited environment. Extracts of these strains prepared from cells grown on higher levels of ammonium have low levels of glutamate synthetase activity and contain the biosynthetically inactive species of glutamine synthetase along with high levels of glutamate dehydrogenase. These mutants missing the new assimilatory pathway have serious defects in their metabolism of many inorganic and organic nitrogen sources; utilization of at least 20 different compounds is effected. We conclude that the new ammonia assimilatory route plays an important role in nitrogenous metabolism and is essential for nitrogen fixation.Abbreviation DON 6-diazo-5-oxo-l-norleucine