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  • 1
    Electronic Resource
    Electronic Resource
    Isolation and characterization of hydrogenase-negative mutants of Azorhizobium caulinodans ORS571 (1988)
    Springer
    Archives of microbiology 150 (1988), S. 595-599 
    Details
    ISSN: 1432-072X
    Keywords: Azorhizobium caulinodans ORS571 ; Hydrogenase ; Nitrogen fixation ; Chemostat cultures ; H2/N2 ratio ; ATP/2e value
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract 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-β-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.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00408256
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  • 2
    Electronic Resource
    Electronic Resource
    The Azorhizobium caulinodans nitrogen-fixation regulatory gene, nifA, is controlled by the cellular nitrogen and oxygen status (1989)
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 3 (1989), S. 0 
    Details
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: The nucleotrde sequence of the Azorhizobium caulinodans ORS571 nlfA 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 nifAlntr-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.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1365-2958.1989.tb00231.x
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  • 3
    Electronic Resource
    Electronic Resource
    Rep-PCR mediated genomic fingerprinting of rhizobia and computer-assisted phylogenetic pattern analysis (1996)
    Springer
    World journal of microbiology and biotechnology 12 (1996), S. 163-174 
    Details
    ISSN: 1573-0972
    Keywords: Azorhizobium caulinodans ; DNA fingerprinting ; genomic structure ; nodules ; PCR ; phylogeny ; repetitive DNA sequences ; Rhizobium loti ; Rhizobium meliloti ; strain identification
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract A rapid and reproducible method has been developed for genomic fingerprinting of rhizobia and other soil microbes interacting with plants. The method is based on the use of oligonucleotide primers, corresponding to conserved motifs in naturally occurring interspersed repetitive DNA elements in bacteria (rep-elements), and the polymerase chain reaction (rep-PCR). Rep-PCR results in the amplification of inter-element genomic DNA fragments of characteristic lengths and thereby generates a genomic fingerprint. These fingerprints resemble UPC bar code patterns, and can be used to identify bacteria at the sub-species and strain level, as well as for phylogenetic analyses. Here we show that highly characteristic and very reproducible rep-PCR generated genomic fingerprints can be obtained not only from purified genomic DNA, but also directly from rhizobial cells derived from liquid cultures or from colonies on plates, as well as from nodule tissue. We examine the effect of growth phase of the bacterial cells, serial subculturing and other parameters on the reproducibility of the rep-PCR fingerprinting protocol. Moreover, we describe the results of mixing experiments designed to determine if individual genomic fingerprints can be recognized in mixtures of strains. Lastly, we review the use of computer-based fragment detection and phylogentic analysis packages to analyse rep-PCR generated genomic fingerprints of a collection of Rhizobium loti and Bradyrhizobium strains nodulating different Lotus spp.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00364681
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  • 4
    Electronic Resource
    Electronic Resource
    Book reviews (1992)
    Springer
    Theoretical and applied genetics 84 (1992), S. 256-258 
    Details
    ISSN: 1432-2242
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00224007
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  • 5
    Electronic Resource
    Electronic Resource
    Regulation of plant genes specifically induced in nitrogen-fixing nodules: role of cis-acting elements and trans-acting factors in leghemoglobin gene expression (1989)
    Springer
    Plant molecular biology 13 (1989), S. 319-325 
    Details
    ISSN: 1573-5028
    Keywords: nodulins ; leghmoglobin ; gene regulation ; cis-acting elements ; trans-acting factors ; transgenic plants
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Transgenic alfalfa plants harboring a gene fusion between the soybean leghemoglobin (lbc3) promoter region and the chloramphenicol acetyl transferase (cat) gene were used to determine the influence of rhizobial mutants on lb gene expression in nodules. The promoter region of the Sesbania rostrata glb3 (Srglb3) leghemoglobin gene was examined for the presence of conserved motifs homologous to binding site 1 and 2 of the soybean lbc3 promoter region, found to interact with a trans-acting factor present in soybean nodule nuclear extracts (Jensen EO, Marcker KA, Schell J, de Bruijn FJ, EMBO J 7: 1265–1271, 1988). Subfragments of the S. rostrata glb3 (Srglb3) promoter region were examined for binding to trans-acting factors from nodule nuclear extracts. In addition to the binding sites previously identified (Metz BA, Welters P, Hoffmann HJ, Jensen EO, Schell J, de Bruijn FJ, Mol Gen Genet 214: 181–191), several other sites were found to interact with trans-acting factors. In most cases the same trans-acting factor(s) were shown to be involved. One fragment (202) was found to bind specifically to a different factor (protein) which was extremely heat-resistant (100°C). The appearance of this factor was shown to be developmentally regulated since the expected protein-DNA complexes were first observed around 12 days after infection, concomitant with the production of leghemoglobin proteins. Fragments of the Srglb3 5′ upstream region were fused to the β-glucuronidase reporter gene with its own CAAT and TATA box region or those of the cauliflower mosaic virus 35S and nopaline synthase (nos) promoters. These constructs were used to generate transgenic Lotus corniculatus plants and their expression was measured in different plant tissues. The Srglb3 CAAT and TATA box region was found to be required for nodule-specific expression and several upstream enhancer-type regions were identified.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00025320
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  • 6
    Electronic Resource
    Electronic Resource
    Potential and pitfalls of trying to extend symbiotic interactions of nitrogen-fixing organisms to presently non-nodulated plants, such as rice (1995)
    Springer
    Plant and soil 174 (1995), S. 225-240 
    Details
    ISSN: 1573-5036
    Keywords: endophytic microbes ; endo-symbiosis ; Rhizobium ; rice ; root hypertrophies ; ultrastructure
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Abstract It has been a long-standing goal in the field of biological nitrogen fixation to extend nitrogen-fixing symbioses to presently non-nodulated cereal plants, such as rice. A number of researchers have recently described the induction of “nodule-like” structures on the roots of cereals primarily by rhizobia, in either the presence or absence of plant cell-wall-degrading enzymes or plant hormones. We briefly review this research and discuss the potential problems associated with the introduction of nitrogen-fixing microbes in novel physiological environments, such as rice roots. The results of experiments carried out in China on the induction of “nodule-like” structures on rice roots by rhizobia are highlighted. In addition, we present preliminary results of a series of experiments designed to repeat and evaluate these results using a variety of microscopic techniques and molecular genetic approaches.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00032249
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  • 7
    Electronic Resource
    Electronic Resource
    Isolation and characterization of Tn5-induced NADPH-glutamate synthase (GOGAT-) mutants of Azorhizobium sesbaniae ORS571 and cloning of the corresponding glt locus (1987)
    Springer
    Molecular genetics and genomics 210 (1987), S. 195-202 
    Details
    ISSN: 1617-4623
    Keywords: Transposon Tn5 mutagenesis ; Azorhizobium sesbaniae ORS571 ; Nitrogen fixation/assimilation ; Glutamate synthase
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: 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.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00325684
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  • 8
    Electronic Resource
    Electronic Resource
    Characterization of a novel Azorhizobium caulinodans ORS571 two-component regulatory system, NtrY/NtrX, involved in nitrogen fixation and metabolism (1991)
    Springer
    Molecular genetics and genomics 231 (1991), S. 124-138 
    Details
    ISSN: 1617-4623
    Keywords: Azorhizobium caulinodans ORS571 ; nifA regulation ; Nitrogen regulation ; Two-component regulatory system ; Nitrogen fixation and metabolism
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Azorhizobium caulinodans ORS571 nifA regulation is partially mediated by the nitrogen regulatory gene ntrC. However, the residual nifA expression in ntrC mutant strains is still modulated by the cellular nitrogen and oxygen status. A second ntrC-homologous region, linked to ntrC, was identified and characterized by site-directed insertion mutagenesis and DNA sequencing. Tn5 insertions in this region cause pleiotropic defects in nitrogen metabolism and affect free-living as well as symbiotic nitrogen fixation. DNA sequencing and complementation studies revealed the existence of a bicistronic operon (ntrYX). NtrY is likely to represent the transmembrane ‘sensor’ protein element in a two-component regulatory system. NtrX shares a high degree of homology with NtrC proteins of other organisms and probably constitutes the regulator protein element. The regulation of the ntrYX and ntrC loci and the effects of ntrYX, ntrY and ntrX mutations on nifA expression were examined using β-galactosidase gene fusions. NtrY/NtrX were found to modulate nifA expression and ntrYX transcription was shown to be partially under the control of NtrC.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00293830
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  • 9
    Electronic Resource
    Electronic Resource
    Cloning and analysis of Agrobacterium tumefaciens C58 loci involved in glutamine biosynthesis: Neither the glnA (GSI) nor the glnII (GSII) gene plays a special role in virulence (1988)
    Springer
    Molecular genetics and genomics 212 (1988), S. 38-47 
    Details
    ISSN: 1617-4623
    Keywords: Agrobacterium tumefaciens C58 ; Glutamine synthetase ; Glutamine auxotroph ; Virulence ; Nitrogen assimilation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Using heterologous complementation of a glutamine synthetase deficient (glnA; GS-) Escherichia coli mutant strain and heterologous DNA hybridization probes from Rhizobium meliloti and Bradyrhizobium japonicum, three distinct Agrobacterium tumefaciens loci involved in glutamine biosynthesis were identified. These loci correspond to the glnA (GSI), glnII (GSII) and a third previously unidentified locus, which is capable of complementing an E. coli glnA mutant, but may be cryptic in A. tumefaciens. The gene products encoded by the cloned glnA and glnII loci were identified using maxicells. Single insertion mutations in the glnA (GSI) and glnII (GSII) genes and a glnA glnII double mutant were constructed using gene replacement techniques. These mutant strains were examined for GSI and II activities, for growth on a variety of nitrogen (N) sources and for virulence properties on Kalanchoë plants. Neither glnA (GSI) nor glnII (GSII) were found to be essential for tumour induction on Kalanchoë nor for opine catabolism.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00322442
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  • 10
    Electronic Resource
    Electronic Resource
    The cloning and transposon Tn5 mutagenesis of the glnA region of Klebsiella pneumoniae: Identification of glnR, a gene involved in the regulation of the nif and hut operons (1981)
    Springer
    Molecular genetics and genomics 183 (1981), S. 289-297 
    Details
    ISSN: 1617-4623
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary A 15 kilobase HindIII fragment of Klebsiella pneumoniae DNA containing the glnA gene was cloned into the plasmid vector pACYC184. The resulting plasmid, pFB51, complements glnA - mutations in Escherichia coli and K. pneumoniae. pFB51 also complements the GlnR phenotype of a Klebsiella pneumoniae gln regulatory mutant (KP5060) defined by the restoration of Hut+ and Nif+ (histidine utilization and nitrogen fixation) phenotypes to this strain. Three recombinant plasmids containing subsegments of the 15 kb HindIII fragment were derived from pFB51. Plasmid pFB514 which contains a spontaneous 4 kb delection of K. pneumoniae DNA from pFB51 is more stable than pFB51 and is still able to complement glnA - mutations and the GlnR- phenotype of KP5060. Plasmids pFB53 and pFB54, which contain a 6.5 kb SalI DNA fragment from pFB51 recloned into pACYC184 in opposite orientations, complement glnA - mutations but not the GlnR- phenotype of KP5060. Plasmids pFB514 and pFB53 were mutagenized by transposon Tn5 resulting in a total of 92 Tn5 insertions in the cloned fragments. Utilizing these insertion mutations, a correlated physical and genetic map was constructed by determining the physical location of each Tn5 insertion and by analyzing the ability of each Tn5 mutated plasmid to complement a glnA - strain of E. coli and a glnA - GlnR- strain of K. pneumoniae. Two classes of Tn5 insertions with an altered Gln phenotpye were obtained. One cluster of insertions spanning a 1.3 kb region abolished complementation of the glnA - mutations. A second 2 kb cluster of Tn5 insertions, immediately adjacent to the first cluster, abolished the ability of pFB514 plasmid to complement the GlnR- phenotype, while glnA - complementation was unaffected. We propose that the second cluster of Tn5 insertions define a DNA region coding for a positive regulatory factor for nitrogen fixation (nif) and histidine utilization (hut) genes (glnR).
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00270631
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