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  • 1
    Electronic Resource
    Electronic Resource
    Springer
    Archives of microbiology 161 (1994), S. 99-99 
    ISSN: 1432-072X
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Type of Medium: Electronic Resource
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  • 2
    ISSN: 1432-072X
    Keywords: Bradyrhizobium japonicum ; Electron transfer flavoprotein ; etf Genes ; fix Genes ; Nitrogen fixation ; Phylogenetic tree ; Protein family
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A group of four co-regulated genes (fixA, fixB, fixC, fixX) essential for symbiotic nitrogen fixation has been described in several rhizobial species, includingBradyrhizobium japonicum. The complete nucleotide sequence of theB. japonicum fixA, fixB andfixC, genes is reported here. The derived amino acid sequences confirmed the previously noted sequence similarity between FixA and the β-subunit and between FixB and the α-subunit of mammalian andParacoccus denitrificans electron transfer flavoproteins (ETF). Since the classical role of ETF is in β-oxidation of fatty acids, a process unrelated to nitrogen fixation, we rationalized thatB. japonicum ought to contain bona fideetf genes in addition to theetf-like genesfixA andfixB. Therefore, we identified, cloned, sequenced, and transcriptionally analyzed theB. japonicum etfSL genes encoding the β-and α-subunits of ETF. TheetfSL genes, but not thefix genes, are transcribed in aerobically grown cells. An amino acid sequence comparison between all available ETFs and ETF-like proteins revealed the existence of two distinguishable subfamilies. Group I comprises housekeeping ETFs that link acyl-CoA dehydrogenase reactions with the respiratory chain, such as in the fatty acid degradation pathway.B. japonicum EtfS and EtfL clearly belong to this group. Group II contains ETF-like proteins that are synthesized only under certain specific growth conditions and receive electrons from the oxidation of specific substrates. The products of the anaerobically inducedfixA andfixB genes ofB. japonicum are members of that group.B. japonicum is the first example of an organism in which genes for proteins of both groups I and II of the ETF family have been identified.
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  • 3
    ISSN: 1432-072X
    Keywords: Key words Bradyrhizobium japonicum ; Heat shock ; DegP ; HtrA ; Periplasmic serine protease ; Small ; heat-shock protein ; Mutants
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A degP (htrA)-like gene of Bradyrhizobium japonicum was identified immediately downstream of two genes (hspB and hspC) coding for small heat-shock proteins. All three genes are oriented in the same direction and are separated by only 85 and 72 bp, and a heat-inducible transcript covering hspB, hspC, and degP was detected by RT-PCR. These results show that the genes are organized in an operon. Two mutants, a degP insertion mutant and a ΔhspBCdegP mutant, were constructed by marker replacement mutagenesis. Immunoblot analysis performed with a serum raised against the amino-terminal end of IbpA, an HspB homolog of Escherichia coli, identified three heat-inducible protein bands in B. japonicum extract, one of which was missing in the deletion mutant. None of the mutants showed an obvious defect during growth at different temperatures, after heat-shock treatment, or in the presence of solvents. Moreover, they were not affected in root-nodule symbiosis, indicating that the small heat-shock proteins HspB and HspC and the DegP homolog of B. japonicum are not required under a wide range of growth conditions.
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  • 4
    ISSN: 1432-072X
    Keywords: Key words Cpn60 ; groESL ; Heat shock protein ; Hsp60 ; NifA ; Nitrogen fixation ; Symbiosis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract At least five highly conserved, but disparately regulated groESL operons are present in Bradyrhizobium japonicum. Expression of groESL 3 is coregulated with symbiotic nitrogen fixation genes, implying a role of GroESL chaperonins in the nitrogen fixation process. Null mutants of individual groEL genes, however, were not impaired in symbiotic nitrogen fixation activity. By contrast, the groEL 3-plus-groEL 4 double mutant strain D4, which is mutated in those groEL genes that contribute most to the GroEL pool under symbiotic conditions, exhibited less than 5% Fix activity as compared to the wild-type. Expression of lacZ fusions made to several representative nif and fix genes was not, or only marginally, reduced in mutant D4, indicating that the requirement of chaperonins for nitrogen fixation does not occur at the level of RegSR-NifA-σ54- or FixLJ-FixK2-dependent gene regulation. Instead, immunoblot analyses revealed that the level of NifH and NifDK nitrogenase proteins was drastically decreased in extracts prepared from D4 bacteroids and from free-living cells grown anaerobically. Transcriptional fusions of the anaerobically induced groESL 3 promoter (P3) to all five B. japonicum groESL operons and also to groESL from Escherichia coli were integrated into the chromosome of mutant D4. Strains harboring P3 fused to groESL 1, groESL 2, groESL 5, or E. coli groESL partially complemented the symbiotic defect of mutant D4, whereas the wild-type phenotype was completely restored in strains complemented with P3 fused to groESL 3 (control) or groESL 4. Likewise, the growth defect of an E. coli groEL mutant could be corrected at least partially by individual B. japonicum groESL operons. In conclusion, both series of complementation analyses were not indicative of a strict substrate specificity of any of the B. japonicum groESL gene products, which is in good agreement with their high degree of sequence conservation.
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  • 5
    Electronic Resource
    Electronic Resource
    Springer
    Archives of microbiology 135 (1983), S. 103-109 
    ISSN: 1432-072X
    Keywords: RNA polymerase ; Transcription ; Nitrogen fixation ; Symbiosis ; Rhizobium
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract DNA-dependend RNA polymerase (EC 2.7.7.6) from Rhizobium japonicum was purified. The subunit structure was found to be ββ′α2σ, with the following apparent molecular weights determined by electrophoresis: M r (β and β') 150,000 each, M r (σ) 96,000, M r (α) 40,000, M r (holoenzyme) 490,000, M r (core enzyme) 380,000. The recovery of σ was 28%. RNA polymerase from aerobically grown R. japonicum cells and from nitrogen-fixing cells have the same electrophoretic properties suggesting that no chemical modification of the enzyme occurs when cells undergo this metabolic differentiation. The enzyme is Mg2+-dependent, rifampicin-sensitive, and has optimal activity at alkaline pH (8–10) and at 35–40° C. It binds strongly to bacteriophage T7 promoters, weakly to antibiotic resistance genes, and not at all to cloned R. japonicum nif DNA. Preliminary in vitro transcription experiments, including nif DNA as template, revealed that additional factors may be required for selective transcription from promoters.
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  • 6
    ISSN: 1432-072X
    Keywords: Bradyrhizobium japonicum ; NifA activity ; nifA mRNA half-lives ; Oxygen shift ; Post-transcriptional control
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Previous work had shown that Bradyrhizobium japonicum nifA-dependent nif gene activation was inhibited by oxygen via a post-transcriptional mechanism. In the present report we demonstrate that this inhibition occurs at the NifA protein level and that it is irreversible. To narrow down the level of control the influence of oxygen on nifA mRNA stability and NifA protein activity was analyzed. The half-lives of B. japonicum and Klebsiella pneumoniae (control) nifA mRNAs derived from constitutively expressed nifA genes did not differ significantly under aerobic and anaerobic conditions which makes it unlikely that oxygen exerts its effect by selectively destabilizing B. japonicum nifA mRNA. By making use of its ability to activate in Escherichia coli a B. japonicum nifD' — 'lacZ fusion, the NifA protein was assayed by the determination of lacZ mRNA and β-galactosidase synthesis. Oxygen shift experiments clearly demonstrated that B. japonicum NifA activity (but not that of K. pneumoniae) was drastically reduced within minutes upon a shift to aerobiosis and that the inactivation was irreversible.
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  • 7
    ISSN: 1432-072X
    Keywords: Bradyrhizobium ; Gene cloning ; Heme ; Marker exchange mutagenesis ; Nitrogen fixation ; Respiration ; Symbiosis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Random and site-directed Tn5-induced mutagenesis of Bradyrhizobium japonicum yielded two mutations, one in strain 2960 and the other in strain 2606::Tn5-20, which mapped close to each other but in separate genes. The corresponding wild-type genes were cloned, and their approximate location on the cloned DNA was determined. Mutant 2960 was Fix- and formed green nodules on soybean, whereas strain 2606::Tn5-20 had ca. 4% of wild-type Fix activity and formed white nodules. Cytochrome oxidase assays (Nadi tests) showed a negative reaction with both mutants, indicating a functional deficiency of cytochrome c or its terminal oxidase or both. However, the mutants grew well under aerobic conditions on minimal media with different carbon sources. Furthermore, mutant 2960 had a reduced activity in hydrogen uptake, was unable to grow anaerobically with nitrate as the terminal electron acceptor and 2960-infected soybean nodules contained little, if any, functional leghemoglobin. Southern blot analysis showed that a B. japonicum heme biosynthesis mutant [strain LO505: O'Brian MR, Kirshbom PM, Maier RJ (1987) Proc Natl Acad Sci USA 84: 8390–8393] had its mutation close to the Tn5 insertion site of our mutant 2606::Tn5-20. This finding, combined with the observed phenotypes, suggested that the genes affected in mutants 2960 and 2606::Tn5-20 were involved in some steps of heme biosynthesis thus explaining the pleiotropic respiratory deficiencies of the mutants. Similar to strain LO505, the mutant 2606::Tn5-20 (but not 2960) was defective in the activity of protoporphyrinogen IX oxidase which catalyzes the penultimate step in the heme biosynthesis pathway. This suggests that one of the two cloned genes may code for this enzyme.
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  • 8
    ISSN: 1432-072X
    Keywords: Bradyrhizobium japonicum ; Branched respiratory chain ; Cytochrome oxidase genes ; Heme/copper oxidases
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Bradyrhizobium japonicum possesses a mitochondria-like respiratory chain terminating with an aa 3-type cytochrome c oxidase. The gene for subunit I of this enzyme (coxA) had been identified and cloned previously via heterologous hybridization using a Paracoccus denitrificans DNA probe. In the course of these studies, another B. japonicum DNA region was discovered which apparently encoded a second terminal oxidase that was different from cytochrome aa 3 but also belonged to the superfamily of heme/copper oxidases. Nucleotide sequence analysis revealed a cluster of at least four genes, coxMNOP, organized most probably in an operon. The predicted coxM gene product shared significant similarity with subunit II of cytochrome c oxidases from other organisms: in particular, all of the proposed CuA ligands were conserved as well as three of the four acidic amino acid residues that might be involved in the binding of cytochrome c. The coxN gene encoded a polypeptide with about 40% sequence identity with subunit I representatives including the previously found CoxA protein: the six presumed histidine ligands of the prosthetic groups (two hemes and CuB) were strictly conserved. A remarkable feature of the DNA seqence was the presence of two genes, coxO and coxP, whose products were both homologous to subunit III proteins. A B.japonicum coxN mutant strain was created by marker exchange mutagenesis which, however, exhibited no obvious defects in free-living, aerobic growth or in root nodule symbiosis with soybean. This shows that the coxMNOP genes are not essential for respiration in the N2 fixing bacteroid.
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  • 9
    ISSN: 1432-072X
    Keywords: Nitrogen fixation ; Gene expression ; Regulation ; Messenger RNA ; Transcription ; Klebsiella pneumoniae
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Nitrogenase messenger RNA synthesis in Klebsiella pneumoniae was determined by labelling cells with (3H)uracil and isolating total RnA, which was then hybridized to filterbound recombinant plasmid pSA30 DNA carrying the nitrogenase structural genes nifH, D, and K. Derepression of nitrogenase mRNA starts 1.5 h before the onset of nitrogenase activity (as measured by acetylene reduction). Exposure of nif-derepressed cultures to either NH 4 + , air, or high temperatures (39° C) results in a rapid decrease of the synthesis rates both of nitrogenase mRNA and nitrogenase polypeptides. Nitrogenase mRNA is remarkably stable. After blocking transcription with rifampicin, hybridizable and actively translatable nitrogenase mRNA survives with an average half-life of 18 min. Half-lives are considerably shorter when rifampicin-inhibited cultures are simultaneously shifted to conditions which are non-permissive for nitrogenase synthesis, pointing to some posttranscriptional influence on nitrogenase mRNA stability. In all experiments performed there was no evidence for uncoupling of nitrogenase mRNA synthesis from nitrogenase mRNA translation, indicating that nitrogenase synthesis is regulated solely by transcriptional control.
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  • 10
    Electronic Resource
    Electronic Resource
    Springer
    Molecular genetics and genomics 193 (1984), S. 46-52 
    ISSN: 1617-4623
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary The slow-growing soybean symbiont, Rhizobium japonicum, has not readily been accessible so far to classical mutational analysis of genes responsible for symbiotic nitrogen fixation. We have overcome part of this problem by the successful application of a site-directed mutagenesis technique to this organism. The following steps are involved: (i) local Tn5 mutagenesis, in E. coli, of cloned R. japonicum DNA (e.g. the nifDK operon); (ii) conjugational transfer of the mutated DNA into R. japonicum using vectors which are unable to replicate there; (iii) selection of R. japonicum exconjugants which have exchanged their wild-type genomic DNA region for the Tn5-containing fragment by homologous recombination. While using this technique it appeared mandatory to distinguish double-crossover-events (true replacements) from single-crossover events (replicon fusions or cointegrations). Only the true replacement mutants were genetically stable; their phenotypes were determined with respect to nodulation (Nod) and nitrogen fixation (Fix) by plant infection tests. Tn5 mutations within nifD and nifK caused a Nod+ Fix- phenotype, whereas mutants with insertions in the immediate vicinity on either side of nifDK were found to be Nod+ Fix+, suggesting that genes flanking nifDK may not be involved in the nitrogen fixing symbiosis. Nodule reisolates were found to carry Tn5 at their original locations.
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