Rhizobium sp. strain ORS571 ammonium assimilation and nitrogen fixation.

Among rhizobia studied, Rhizobium sp. strain ORS571 alone grew unambiguously on N2 as sole N source. In ORS571 , only the glutamine synthetase (GS)-glutamate synthase ( GOGAT ) pathway assimilated ammonium. However, ORS571 exhibited two unique physiological aspects of this pathway: ORS571 had only GS I, whereas all other Rhizobiaceae studied had both GS I and GS II, and both NADPH- and NADH-dependent GOGAT activities were present. ORS571 GS-affected and NADPH- GOGAT -affected mutant strains were defective in both ammonium assimilation (Asm-) and N2 fixation (Nif-) in culture and in planta ; NADH- GOGAT mutants were Asm- but Nif+. "Bacteroid" GS activity was essentially nil, suggesting symbiotic ammonium export. Physiological studies on effects of glutamine, ammonium, methionine sulfoximine, and diazo-oxo-norleucine on nitrogenase induction in culture implied a regulatory role for the intracellular glutamine pool.     

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 Tn5-induced NADPH-glutamate synthase (GOGAT-) mutants of Azorhizobium sesbaniae ORS571 and cloning of the corresponding glt locus

Summary Random Tn5 mutagenesis was used to isolate two independent Azorhizobium sesbaniae ORS571 mutants disturbed in ammonium assimilation (Asm^-). Both Asm^- mutant strains were shown to lack NADPH-glutamate synthase (NADPH-GOGAT) activity and to carry Tn5 insertions ca. 1.5 kb apart in the ORS571 chromosome. The Tn5-containing region of one of the GOGAT^- mutant strains was cloned in pACYC184 and used to identify the wild-type glt (GOGAT) locus in a phage clone bank of ORS571. The cloned region was shown to have DNA homology with the Escherichia coli glt locus and to complement the Asm^- phenotype of E. coli and ORS571 GOGAT^- strains. The ORS571 GOGAT^- mutations were found to interfere with free-living as well as symbiotic nitrogen fixation. Expression of ORS571 NADPH-GOGAT activity was shown to be independent of the nitrogen regulation (ntr) system.     

Characterization of three genomic loci encoding Rhizobium sp. strain ORS571 N2 fixation genes.

Sixty-five independent, N2 fixation-defective (Nif-) vector insertion (Vi) mutants were selected, cloned, and mapped to the ORS571 genome. The recombinant Nif::Vi plasmids obtained in this way were used as DNA hybridization probes to isolate homologous phages from a genomic library of ORS571 constructed in lambda EMBL3. Genomic maps were drawn for three ORS571 Nif gene loci. Forty-five Nif::Vi mutants in genomic Nif locus 1 defined two gene clusters separated by 8 kilobase pairs (kb) of DNA. In the first cluster, 36 Nif::Vi mutants mapped to a 7-kb DNA segment that showed DNA homology with Klebsiella pneumoniae nifHDKE and encoded at least two Nif operons. In the other cluster, nine Nif::Vi mutants mapped to a 1.5-kb DNA segment that showed homology with K. pneumoniae and Rhizobium meliloti nifA; this DNA segment encoded a separate Nif operon. Fifteen Nif::Vi mutants mapped to a 3.5-kb DNA segment defined as Nif locus 2 and showed DNA homology with the R. meliloti P2 fixABC operon. Nif locus 2 carries a second nifH (nifH2) gene. Four Nif::Vi mutants mapped to a 2-kb DNA segment defined as Nif locus 3 and showed DNA homology with K. pneumoniae nifB. DNA from lambda Nif phages comprising all three genomic Nif loci was subcloned in plasmid vectors able to stably replicate in ORS571. These plasmid subclones were introduced into ORS571 strains carrying physically mapped Nif::Vi insertions, and genetic complementations were conducted. With the exception of certain mutants mapping to the nifDK genes, all mutants could be complemented to Nif+ when they carried plasmid subclones of defined genomic DNA regions. Conversely, most nifDK mutants behaved as pseudodominant alleles.     

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.     

Rhizobial factors required for stem nodule maturation and maintenance in Sesbania rostrata-Azorhizobium caulinodans ORS571 symbiosis

The molecular and physiological mechanisms behind the maturation and maintenance of N(2)-fixing nodules during development of symbiosis between rhizobia and legumes still remain unclear, although the early events of symbiosis are relatively well understood. Azorhizobium caulinodans ORS571 is a microsymbiont of the tropical legume Sesbania rostrata, forming N(2)-fixing nodules not only on the roots but also on the stems. In this study, 10,080 transposon-inserted mutants of A. caulinodans ORS571 were individually inoculated onto the stems of S. rostrata, and those mutants that induced ineffective stem nodules, as displayed by halted development at various stages, were selected. From repeated observations on stem nodulation, 108 Tn5 mutants were selected and categorized into seven nodulation types based on size and N(2) fixation activity. Tn5 insertions of some mutants were found in the well-known nodulation, nitrogen fixation, and symbiosis-related genes, such as nod, nif, and fix, respectively, lipopolysaccharide synthesis-related genes, C(4) metabolism-related genes, and so on. However, other genes have not been reported to have roles in legume-rhizobium symbiosis. The list of newly identified symbiosis-related genes will present clues to aid in understanding the maturation and maintenance mechanisms of nodules.     

茎瘤固氮根瘤菌ORS571中motA基因的功能分析

[目的] MotA是细菌的鞭毛马达蛋白,是跨膜质子通道的重要组成结构之一,在调控鞭毛运动中具有至关重要的作用。本研究探究了Azorhizobium caulinodans ORS571中鞭毛马达基因motA对菌株表型和植物互作的影响。[方法] 通过同源重组原理和三亲接合转移方法构建突变菌株∆motA,测定野生型与突变体在菌体生长、运动、固氮、胞外多糖合成、生物膜形成及根系定殖能力的差异。[结果] 与野生型相比,突变体菌体生长没有明显差异,但其运动能力完全丧失,固氮、胞外多糖合成、生物膜形成及根系定殖能力减弱。[结论] MotA鞭毛马达蛋白对A.caulinodans ORS571的运动、固氮、胞外多糖合成、生物膜形成及根系定殖能力均有调控作用。     

Identification of cyclic intermediates in Azorhizobium caulinodans nicotinate catabolism.

In wild-type Azorhizobium caulinodans ORS571, nicotinate served both as anabolic substrate for NAD+ production and as catabolic substrate for use as the N source. Catabolic enzyme activities were greatest from cultures grown with nicotinate as the N source and least when cultures were grown with ammonium as the N source. Vector insertion mutants unable to catabolize nicotinate (nic::Vi mutants) still required micromolar quantities of this compound for growth. Therefore, A. caulinodans wild type is NAD+ auxotrophic. As the first two intermediates in A. caulinodans nicotinate catabolism, two cyclic compounds, 6-hydroxynicotinate and 1,4,5,6-tetrahydro-6-oxonicotinate, were identified. These compounds were purified from the growth medium of strain 61009 (a nic::Vi mutant) by high-performance liquid chromatography; their identities were subsequently confirmed by UV absorbance, nuclear magnetic resonance, and mass spectra. The conversion of 1 mol of nicotinate to 6-hydroxynicotinate consumed 0.5 mol of O2. From 18O isotopic incorporation experiments, water was the hydroxyl-equivalent source. A nicotinate hydroxylase activity proved to be cell wall-membrane associated; this activity served as direct electron donor (not indirect via NADP+) to O2 via membrane electron transport. These catabolic reactions have not previously been witnessed together in the same organism. A. caulinodans nicotinate catabolism seems coupled to N2 fixation, although the explicit mechanism of this coupling remains to be determined.     

茎瘤固氮根瘤菌（Azorhizobium caulinodans）ORS571产生的植物激素

茎瘤固氮根瘤基ＯＲＳ５７１菌株在离体培养条件下，能利用色氨酸合成吲哚乙酸（ＩＡＡ）。随着菌龄的老化，合成的ＩＡＡ量也增加。除ＩＡＡ外，该菌株还产生类ＧＡ物质。本研究未检出细胞分裂素（Ｃｙｔｏｋｉｎｉｎ）类物质。     

An Azorhizobium caulinodans ORS571 locus involved in lipopolysaccharide production and nodule formation on Sesbania rostrata stems and roots.

Azorhizobium caulinodans ORS571 is able to nodulate roots and stems of the tropical legume Sesbania rostrata. An ORS571 Tn5 insertion mutant, strain ORS571-X15, had a rough colony morphology, was nonmotile, and showed clumping behavior on various media. When this pleiotropic mutant was inoculated on roots or stems of the host, no nodules developed (Nod-). Compared with the wild type, strain ORS571-X15 produced lipopolysaccharides (LPS) with an altered ladder pattern on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels, suggestive of a different O-antigen structure with a lower degree of polymerization. A cosmid clone, pRG20, that fully complemented all phenotypes of ORS571-X15 was isolated. With a 6-kb EcoRI subfragment of pRG20, clumping was relieved and nodulation was almost completely restored, but the strain was still nonmotile. LPS preparations from these complemented strains resembled the wild-type LPS, although minor quantitative and qualitative differences were evident. The sequence of the locus hit by the Tn5 in ORS571-X15 (the oac locus) revealed a striking homology with the rfb locus of Salmonella typhimurium, which is involved in O-antigen biosynthesis. The Tn5 insertion position was mapped to the oac3 gene, homologous to rfbA, encoding dTDP-D-glucose synthase. Biochemical assaying showed that ORS571-X15 is indeed defective in dTDP-D-glucose synthase activity, essential for the production of particular deoxyhexoses. Therefore, it was proposed that the O antigen of the mutant strain is devoid of such sugars.     

Nicotinic acid requirement and degradation by Sesbania rhizobium strain ORS571

Abstract Sesbania rhizobium strain ORS571, which grows in the free-living state at the expense of N₂ or ammonia, requires nicotinic acid as a growth factor and also uses this vitamin as a nitrogen source in culture medium devoid of ammonia. Consequently, under nitrogen-fixing conditions, Sesbania rhizobium requires about 10-times more nicotinic acid than when grown in the presence of ammonia. The physiological implications of this property are discussed.     

Identification of a new inducible nodulation gene in Azorhizobium caulinodans.

The narrow host range bacterial strain Azorhizobium caulinodans ORS571 induces the formation of nitrogen-fixing nodules on the root and stem of the tropical legume Sesbania rostrata. Here, a new flavonoid-inducible locus of ORS571 is described, locus 4. The locus was identified and isolated via the occurrence of particular sequences, the gamma and delta elements. These elements are reiterated in the ORS571 genome, linked to symbiotic loci. Sequencing of locus 4 showed the presence of an open reading frame (ORF6) that is flanked downstream by a gamma element and upstream by a delta element. The gamma element is approximately 180 bp in size, and shows homology to the insertion element ISRm3, an insertion sequence belonging to a distinct class of IS elements. The delta element is about 300 bp in size and has homology with repeated sequences found in other Rhizobiaceae. The ORF6 gene product shows a low, but significant homology to the mouse mastocytoma antigen P35B (Szikora et al., EMBO J. 9: 1041-1050, 1990) and to a class of NAD/NADP-binding sugar epimerase/dehydrogenases (Pissowotzki et al., Mol. Gen. Genet. 231: 113-213, 1991). Immediately upstream from ORF6, a nod box-related sequence is present, the arrangement of which is fully consistent with a recently presented model for the nod box structure (Goethals et al., Proc. Natl. Acad. Sci. USA 89: 1646-1650, 1992). Insertional inactivation of ORF6 did not affect the nodulation and fixation performance on S. rostrata. However, on S. formosa roots the nodulation kinetics of such a mutant was clearly affected (about 5 days delay). We propose to call this new symbiotic gene nolK.     

The xylem of rice (Oryza sativa) is colonized by Azorhizobium caulinodans

Following inoculation with Azorhizobium caulinodans ORS571 (pXLGD4), lateral root development of rice and colonization of lateral root cracks by bacteria were shown to be stimulated by the flavonoid naringenin. Rice seedlings growing aseptically in the presence of naringenin were inoculated with ORS571 (pXLGD4), carrying the lacZ reporter gene. By microscopic analysis of sections of inoculated rice roots, it has been demonstrated that the xylem of rice roots can be colonized by Azorhizobium caulinodans. We discuss whether this colonization of the xylem of rice roots by azorhizobia could provide a suitable niche for endophytic nitrogen fixation.     

Stable isotope probing with 15N2 reveals novel noncultivated diazotrophs in soil

Biological nitrogen fixation is a fundamental component of the nitrogen cycle and is the dominant natural process through which fixed nitrogen is made available to the biosphere. While the process of nitrogen fixation has been studied extensively with a limited set of cultivated isolates, examinations of nifH gene diversity in natural systems reveal the existence of a wide range of noncultivated diazotrophs. These noncultivated diazotrophs remain uncharacterized, as do their contributions to nitrogen fixation in natural systems. We have employed a novel 15N2-DNA stable isotope probing (5N2-DNA-SIP) method to identify free-living diazotrophs in soil that are responsible for nitrogen fixation in situ. Analyses of 16S rRNA genes from 15N-labeled DNA provide evidence for nitrogen fixation by three microbial groups, one of which belongs to the Rhizobiales while the other two represent deeply divergent lineages of noncultivated bacteria within the Betaproteobacteria and Actinobacteria, respectively. Analysis of nifH genes from 15N-labeled DNA also revealed three microbial groups, one of which was associated with Alphaproteobacteria while the others were associated with two noncultivated groups that are deeply divergent within nifH cluster I. These results reveal that noncultivated free-living diazotrophs can mediate nitrogen fixation in soils and that 15N2-DNA-SIP can be used to gain access to DNA from these organisms. In addition, this research provides the first evidence for nitrogen fixation by Actinobacteria outside of the order Actinomycetales.     

Azorhizobium caulinodans ORS571 colonizes the xylem of Arabidopsis thaliana

Improved conditions were used for the aseptic growth of Arabidopsis thaliana to investigate whether xylem colonization of A. thaliana by Azorhizobium caulinodans ORS571 might occur. When seedlings were inoculated with ORS571 (pXLGD4) tagged with the lacZ reporter gene, nearly all of the plants showed blue regions of ORS571 colonization at lateral root cracks (LRC). The flavonoids naringenin and liquiritigenin significantly stimulated colonization of LRC by ORS571. Blue bands of ORS571 (pXLGD4) bacteria were observed histochemically in the xylem of intact roots of inoculated plants. Detailed microscopic analysis of sections of primary and lateral roots from inoculated A. thaliana confirmed xylem colonization. Xylem colonization also occurred with an ORS571 nodC mutant deficient in nodulation factors. There was no significant difference in the percentage of plants with xylem colonization or in the mean length of xylem colonized per plant between plants inoculated with either ORS571 (pXLGD4) or ORS571::nodC (pXLGD4), with or without naringenin.     

Cloning and characterization of nifA and ntrC genes of the stem nodulating bacterium ORS571, the nitrogen fixing symbiont of Sesbania rostrata: Regulation of nitrogen fixation (nif) genes in the free living versus symbiotic state

Summary A cosmid bank of ORS571, a diazotrophic bacterium capable of inducing aerial stem and root nodules on Sesbania rostrata, was constructed in the vector pLAFR1. A DNA probe carrying the Klebsiella pneumoniae nifA gene was used to identify nifA-and ntrC-like regions of ORS571 in the cosmid bank by colony hybridization. Cosmids carrying these regions were mapped by restriction endonuclease analysis, Southern blotting and transposon Tn5 mutagenesis. Selected Tn5 insertion mutations in the nifA/ntrC homologous regions were used for gene-replacement experiments and the resulting ORS571 mutants were examined for Nif, Fix and Ntr phenotypes. Two clearly distinct regulatory loci were thus identified and named nifA and ntrC. Plasmids carrying gene fusions of the ORS571 nifH and nifD genes to lacZ were constructed and the regulation of the ORS571 nifHDK promoter, and of the Rhizobium meliloti nifHDK promoter, was studied under varying physiological conditions in ORS571, ORS571 nifA::Tn5 and ORS571 nitrC::Tn5 strains. A model for the role of nifA and ntrC in the regulation of ORS571 nif and other nitrogen assimilation genes is proposed.     

Variations in Outer-membrane Characteristics of Two Stem-nodulating Bacteria of <Emphasis Type="Italic">Sesbania rostrata</Emphasis> and its Role in Tolerance Towards Diverse Stress

Outer-membrane characteristics may determine the survivability of rhizobia under diverse abiotic and biotic stresses. Therefore, the role of lipopolysaccharides (LPS) and membrane proteins of two stem-nodulating bacteria of Sesbania rostrata (Azorhizobium caulinodans ORS571 and Rhizobium sp. WE7) in determining tolerance towards abiotic and biotic stresses (hydrophobics and phages) was investigated. Outer-membrane characteristics (LPS and membrane–protein profiles) of ORS571, WE7 and thirteen standard strains were distinct. ORS571 and WE7 also showed susceptibility towards morphologically distinct phages, i.e., ACSR16 (short-tailed) and WESR29 (long-tailed), respectively. ORS571 and WE7 were tolerant to hydrophobic compounds (triton X-100, rifampicin, crystal violet and deoxycholate). To ascertain the role of outer membrane characteristics in stress tolerance, phage-resistant transconjugant mutants of ORS571 (ORS571-M8 and ORS571-M20) and WE7 (WE7-M9) were developed. LPS- and membrane–protein profiles of mutants differed from that of respective wild types (ORS571 and WE7). In in vitro assay, phages got adsorbed onto purified LPS-membrane protein fractions of wild types. Phages did not adsorb onto membrane fraction of mutants and standard strains. Mutant with reduced expression of LPS (ORS571-M20 and WE7-M9) showed reduced tolerance towards hydrophobics. However, the tolerance was unaffected in mutant (ORS571-M8) where expression of LPS was not reduced but pattern was different. The tolerance level of mutants towards hydrophobics varied with the expression of LPS, whereas the specificity towards phages is correlated with the specific LPS pattern.