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.     

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.     

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.     

Azorhizobium caulinodans uses both cytochrome bd (quinol) and cytochrome cbb3 (cytochrome c) terminal oxidases for symbiotic N2 fixation.

Azorhizobium caulinodans employs both cytochrome bd (cytbd; quinol oxidase) and cytcbb3 (cytc oxidase) as terminal oxidases in environments with very low O2 concentrations. To investigate physiological roles of these two terminal oxidases both in microaerobic culture and in symbiosis, knockout mutants were constructed. As evidenced by visible absorbance spectra taken from mutant bacteria carrying perfect gene replacements, both the cytbd- and cytcbb3- mutations were null alleles. In aerobic culture under 2% O2 atmosphere, Azorhizobium cytbd- and cytcbb3- single mutants both fixed N2 at 70 to 90% of wild-type rates; in root nodule symbiosis, both single mutants fixed N2 at 50% of wild-type rates. In contrast, Azorhizobium cytbd- cytcbb3-double mutants, which carry both null alleles, completely lacked symbiotic N2 fixation activity. Therefore, both Azorhizobium cytbd and cytcbb3 oxidases drive respiration in environments with nanomolar O2 concentrations during symbiotic N2 fixation. In culture under a 2% O2 atmosphere, Azorhizobium cytbd- cytcbb3- double mutants fixed N2 at 70% of wild-type rates, presumably reflecting cytaa3 and cytbo (and other) terminal oxidase activities. In microaerobic continuous cultures in rich medium, Azorhizobium cytbd- and cytcbb3- single mutants were compared for their ability to deplete a limiting-O2 sparge; cytbd oxidase activity maintained dissolved O2 at 3.6 microM steady state, whereas cytcbb3 oxidase activity depleted O2 to submicromolar levels. Growth rates reflected this difference; cytcbb3 oxidase activity disproportionately supported microaerobic growth. Paradoxically, in O2 limited continuous culture, Azorhizobium cytbd oxidase is inactive below 3.6 microM dissolved O2 whereas in Sesbania rostrata symbiotic nodules, in which physiological, dissolved O2 is maintained at 10 to 20 nM, both Azorhizobium cytbd and cytcbb3 seem to contribute equally as respiratory terminal oxidases.     

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.     

Regulation of nitrogen fixation in Azospirillum brasilense Sp7: Involvement of nifA, glnA and glnB gene products

The expression of nifA-, niH- and nifB-lacZ fusions was examined in different mutants of Azospirillum brasilense. Mutations in nifA, glnA and glnB severely impaired the expression of nifH- and nifB-lacZ fusions. By contrast, a nifA-lacZ fusion was not affected in a nifA or a glnB background and was only partially impaired in glnA mutants. It is proposed that in A. brasilense, the PII protein and glutamine synthetase are involved in a post-translational modification of NifA.     

毛萼田菁茎瘤菌(Azorhizobium caulinodans)吸氢活性缺陷株的筛选及特性

利用转座子Tn5随机插入突变法,从毛萼田菁茎瘤菌（Azorhizobiumcaulinodans）中筛选得到两株吸氢活性缺陷突变株（Hup-）R－49和R－309,其固氮酶活性也大大降低,约为野生型固氮酶活性的6％～8％。以带有Bradyrhizobiumjaponicum的5．9kb的hup基因的质粒pHR11为探针与A．caulinodansORS571和B.japonicum122DES（Hup＋）的总DNA进行分子杂交,放射自显影表明,A．coulinodans基因组含有与B.japonicum的hup基因同源的DNA片段,将携带B．japonicumhup基因的嵌合质粒pHR11与A.caulinodansHup-突变株进行体内遗传互补,接合转移子吸氢酶活性可以恢复至野生型的60％,同时固氮酶的活性亦有所提高。     

Characterization of the cycHJKL genes involved in cytochrome c biogenesis and symbiotic nitrogen fixation in Rhizobium leguminosarum.

Mutants of Rhizobium leguminosarum bv. viciae unable to respire via the cytochrome aa3 pathway were identified by the inability to oxidize N,N'-dimethyl-p-phenylenediamine. Two mutants which were complemented by cosmid pIJ1942 from an R. leguminosarum clone bank were identified. Although pea nodules induced by these mutants contained many bacteroids, no symbiotic nitrogen fixation was detected. Heme staining of cellular proteins revealed that all cytochrome c-type heme proteins were absent. These mutants lacked spectroscopically detectable cytochrome c, but cytochromes aa3 and d were present, the latter at a higher-than-normal level. DNA sequence analysis of complementing plasmids revealed four apparently cotranscribed open reading frames (cycH, cycJ, cycK, and cycL). CycH, CycJ, CycK, and CycL are homologous to Bradyrhizobium japonicum and Rhizobium meliloti proteins thought to be involved in the attachment of heme to cytochrome c apoproteins; CycK and CycL are also homologous to the Rhodobacter capsulatus ccl1 and ccl2 gene products and the Escherichia coli nrfE and nrfF gene products involved in the assembly of c-type cytochromes. The absence of cytochrome c heme proteins in these R. leguminosarum mutants is consistent with the view that the cycHJKL operon could be involved in the attachment of heme to apocytochrome c.     

NAD(P)+-malic enzyme mutants of Sinorhizobium sp. strain NGR234, but not Azorhizobium caulinodans ORS571, maintain symbiotic N2 fixation capabilities

C(4)-dicarboxylic acids appear to be metabolized via the tricarboxylic acid (TCA) cycle in N(2)-fixing bacteria (bacteroids) within legume nodules. In Sinorhizobium meliloti bacteroids from alfalfa, NAD(+)-malic enzyme (DME) is required for N(2) fixation, and this activity is thought to be required for the anaplerotic synthesis of pyruvate. In contrast, in the pea symbiont Rhizobium leguminosarum, pyruvate synthesis occurs via either DME or a pathway catalyzed by phosphoenolpyruvate carboxykinase (PCK) and pyruvate kinase (PYK). Here we report that dme mutants of the broad-host-range Sinorhizobium sp. strain NGR234 formed nodules whose level of N(2) fixation varied from 27 to 83% (plant dry weight) of the wild-type level, depending on the host plant inoculated. NGR234 bacteroids had significant PCK activity, and while single pckA and single dme mutants fixed N(2) at reduced rates, a pckA dme double mutant had no N(2)-fixing activity (Fix(-)). Thus, NGR234 bacteroids appear to synthesize pyruvate from TCA cycle intermediates via DME or PCK pathways. These NGR234 data, together with other reports, suggested that the completely Fix(-) phenotype of S. meliloti dme mutants may be specific to the alfalfa-S. meliloti symbiosis. We therefore examined the ME-like genes azc3656 and azc0119 from Azorhizobium caulinodans, as azc3656 mutants were previously shown to form Fix(-) nodules on the tropical legume Sesbania rostrata. We found that purified AZC3656 protein is an NAD(P)(+)-malic enzyme whose activity is inhibited by acetyl-coenzyme A (acetyl-CoA) and stimulated by succinate and fumarate. Thus, whereas DME is required for symbiotic N(2) fixation in A. caulinodans and S. meliloti, in other rhizobia this activity can be bypassed via another pathway(s).     

Characterisation of the glnK-amtB operon and the involvement of AmtB in methylammonium uptake in Azorhizobium caulinodans

This work reports the characterisation of the Azorhizobium caulinodans amtB gene, the deduced protein sequence of which shares similarity to those of several ammonium transporters. amtB is located downstream from glnK, a glnB-like gene. It is cotranscribed with glnK from an NtrC- and σ⁵⁴-dependent promoter. glnK and amtB insertion mutant strains have been isolated. Methylammonium uptake was assayed in these strains and in other mutant strains in which the regulation of nitrogen metabolism is impaired. Our data suggest that the AmtB protein is an ammonium transporter, which is mainly regulated by NtrC in response to nitrogen availability.     

Fucosylation and arabinosylation of Nod factors in Azorhizobium caulinodans: involvement of nolKnodZ as well as noeC and/or downstream genes

The DNA region downstream of the nodABCSUIJ operon of Azorhizobium caulinodans was further characterized and two new genes, nodZ and noeC were identified in the same operon. The A. caulinodans wild-type strain produces a population of Nod factors that, at the reducing end, are either unmodified or carry a D -arabinosyl and/or an L -fucosyl branch. Nod factors produced by Tn 5 -insertion mutants in nodZ noeC , and the separate nolK locus, were analysed by thin-layer chromatography and mass spectrometry. Fucosylation of Nod factors depended on both nodZ and nolK . Arabinosylation depended on noeC and/or downstream genes. Protein extracts of A. caulinodans contained an enzymatic activity for fucose transfer from GDP-fucose to chitooligosaccharides and to Nod factors. By mutant analysis and expression of nodZ in Escherichia coli , the fucosyltransferase activity was ascribed to the protein encoded by nodZ . In addition, a Nod factor fucosyltransferase activity, independent of nodZ or other known nod genes, was detected in A. caulinodans . Finally, on the basis of sequence similarity of the nolK gene product, and mass spectrometric analysis of Nod factors produced by a nolK mutant, we propose that this gene is involved in the synthesis of GDP-fucose.     

Reiteration of genes involved in symbiotic nitrogen fixation by fast-growing Rhizobium japonicum.

By using cloned Rhizobium meliloti nodulation (nod) genes and nitrogen fixation (nif) genes, we found that the genes for both nodulation and nitrogen fixation were on a plasmid present in fast-growing Rhizobium japonicum strains. Two EcoRI restriction fragments from a plasmid of fast-growing R. japonicum hybridized with nif structural genes of R. meliloti, and three EcoRI restriction fragments hybridized with the nod clone of R. meliloti. Cross-hybridization between the hybridizing fragments revealed a reiteration of nod and nif DNA sequences in fast-growing R. japonicum. Both nif structural genes D and H were present on 4.2- and 4.9-kilobase EcoRI fragments, whereas nifK was present only on the 4.2-kilobase EcoR2 fragment. These results suggest that the nif gene organizations in fast-growing and in slow-growing R. japonicum strains are different.     

Control of the expression of bacterial genes involved in symbiotic nitrogen fixation

Several genera of N₂-fixing bacteria establish symbiotic associations with plants. Among these, the genus Rhizobium has the most significant contribution, in terms of yield, in many important crop plants. The establishment of the Rhizobium-legume symbiosis is a very complex process involving many genes which need to be co-ordinately regulated. In the first instance, plant signal molecules, known to be flavonoids, trigger the expression of host-specific genes in the bacterial partner through the action of the regulatory NodD protein. In response to these signals, Rhizobium bacteria synthesize lipo-oligosaccharide molecules which in turn cause cell differentiation and nodule development. Once the nodule has formed, Rhizobium cells differentiate into bacteroids and another set of genes is activated. These genes, designated nif and fix, are responsible for N₂ fixation. In this system, several regulatory proteins are involved in a complex manner, the most important being NifA and a two component (FixK and FixL) regulatory system. Our knowledge about the establishment of these symbioses has advanced recently, although there are many questions yet to be solved.     

Rhizobium meliloti fixGHI sequence predicts involvement of a specific cation pump in symbiotic nitrogen fixation.

We present genetic and structural analyses of a fix operon conserved among rhizobia, fixGHI from Rhizobium meliloti. The nucleotide sequence of the operon suggests it may contain a fourth gene, fixS. Adjacent open reading frames of this operon showed an overlap between TGA stop codons and ATG start codons in the form of an ATGA motif suggestive of translational coupling. All four predicted gene products contained probable transmembrane sequences. FixG contained two cysteine clusters typical of iron-sulfur centers and is predicted to be involved in a redox process. FixI was found to be homologous with P-type ATPases, particularly with K+ pumps from Escherichia coli and Streptococcus faecalis but also with eucaryotic Ca2+, Na+/K+, H+/K+, and H+ pumps, which implies that FixI is a pump of a specific cation involved in symbiotic nitrogen fixation. Since prototrophic growth of fixI mutants appeared to be unimpaired, the predicted FixI cation pump probably has a specifically symbiotic function. We suggest that the four proteins FixG, FixH, FixI, and FixS may participate in a membrane-bound complex coupling the FixI cation pump with a redox process catalyzed by FixG.