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  • 1
    ISSN: 1432-072X
    Keywords: Bacillus thuringiensis ; Plasmid pC194 ; Transformation ; Plasmid transfer ; Protoplasts
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The Staphylococcus aureus plasmid pC194 which codes for resistance to chloramphenicol was introduced into six Bacillus thuringiensis strains representing five varieties by protoplast transformation. Six other varieties could not be transformed. pC194 could be identified in transformed strains as autonomous plasmid. The transformed clones contained in addition a new extrachromosomal element of somewhat lower electrophoretic mobility hybridizing with pC194, and pC194 in multimeric forms. pC194 was also transferred from one B. thuringiensis variety to another and from Bacillus thuringiensis to Bacillus subtilis and vice versa by a conjugation-like process, requiring close cell-to-cell contact.
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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 wordsBradyrhizobium 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, including Bradyrhizobium japonicum. The complete nucleotide sequence of the B. japonicum fixA, fixB and fixC, 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 and Paracoccus 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 that B. japonicum ought to contain bona fide etf genes in addition to the etf-like genes fixA and fixB. Therefore, we identified, cloned, sequenced, and transcriptionally analyzed the B. japonicum etfSL genes encoding the β- and α-subunits of ETF. The etfSL genes, but not the fix 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 induced fixA and fixB genes of B. 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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  • 4
    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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  • 5
    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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  • 6
    ISSN: 1574-6968
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: We identified by cloning and DNA sequence analysis the phosphate regulatory gene phoB of Bradyrhizobium japonicum. The deduced gene product displayed pronounced similarity to the PhoB protein of Sinorhizobium meliloti (71.4% identical amino acids), Escherichia coli (50.2%) and other bacterial species. Insertion of a kanamycin resistance cassette into phoB led to impaired growth of the B. japonicum mutant in media containing approximately 25 μM phosphate or less. A standard plant infection test using wild-type and phoB-defective B. japonicum strains showed that the phoB mutation had no effect on the symbiotic properties of B. japonicum with its soybean host plant.
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  • 7
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Science, Ltd
    Molecular microbiology 41 (2001), S. 0 
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: In Bradyrhizobium japonicum, the nitrogen-fixing symbiont of soybeans, we have identified a haem uptake system, Hmu, that comprises a cluster of nine open reading frames. Predicted products of these genes include: HmuR, a TonB-dependent haem receptor in the outer membrane; HmuT, a periplasmic haem-binding protein; and HmuUV, an ABC transporter in the inner membrane. Furthermore, we identified homologues of ExbBD and TonB, that are required for energy transduction from the inner to the outer membrane. Mutant analysis and complementation tests indicated that HmuR and the ExbBD–TonB system, but not the HmuTUV transporter, are essential for haem uptake or haem acquisition from haemoglobin and leghaemoglobin. The TonB system seems to be specific for haem uptake as it is dispensable for siderophore uptake. Therefore, we propose the existence of a second TonB homologue functioning in the uptake of Fe-chelates. When tested on soybean host plants, hmuT-hmuR and exbD-tonB mutants exhibited wild-type symbiotic properties. Thus, haem uptake is not essential for symbiotic nitrogen fixation but it may enable B. japonicum to have access to alternative iron sources in its non-symbiotic state. Transcript analysis and expression studies with lacZ fusions showed that expression of hmuT and hmuR is induced under low iron supply. The same was observed in fur and irr mutant backgrounds although maximal induction levels were decreased. We conclude either that both regulators, Fur and Irr, independently mediate transcriptional control by iron or that a yet unknown iron regulatory system activates gene expression under iron deprivation. An A/T-rich cis-acting element, located in the promoter region of the divergently transcribed hmuTUV and hmuR genes, is possibly required for this type of iron control.
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  • 8
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Phosphatidycholine (PC), the major membrane phospholipid in eukaryotes, is found in only some bacteria including members of the family Rhizobiaceae. For this reason, it has long been speculated that rhizobial PC might be required for a successful interaction of rhizobia with their legume host plants in order to allow the formation of nitrogen-fixing root nodules. A major pathway for PC formation in prokaryotes involves a threefold methylation of the precursor phosphatidylethanolamine (PE). Here, we report on the isolation of a Bradyrhizobium japonicum gene (pmtA) encoding the phospholipid N-methyltransferase PmtA. Upon expression of the bradyrhizobial pmtA gene in Escherichia coli, predominantly monomethylphosphatidylethanolamine was formed from PE. PmtA-deficient B. japonicum mutants still produced low levels of PC by a second methylation pathway. The amount of PC formed in such mutants (6% of total phospholipids) was greatly decreased compared with the wild type (52% of total phospholipids). Root nodules of soybean plants infected with B. japonicum pmtA mutants showed a nitrogen fixation activity of only 18% of the wild-type level. The interior colour of the nodules was beige instead of red, suggesting decreased amounts of leghaemoglobin. Moreover, ultrastructure analysis of these nodules demonstrated a greatly reduced number of bacteroids within infected plant cells. These data suggest that the biosynthesis of wild-type amounts of PC are required to allow for an efficient symbiotic interaction of B. japonicum with its soybean host plant.
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  • 9
    Electronic Resource
    Electronic Resource
    Oxford BSL : Blackwell Science Ltd
    Molecular microbiology 19 (1996), S. 0 
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Heat-shock regulation was detected for three out of the five members of the groESL multigene family in Bradyrhizobium japonicum. The results uncovered the simultaneous presence of two distinct heat-shock control systems which so far have not been reported to co-exist in a single prokaryotic organism. The first system concerns groESL1 whose transcription is controlled in a σ32-dependent manner similar to that known from work done with Escherichia coli. Heat-shock control of groESL4 is mediated by the second system, which is characterized by an inverted-repeat DNA structure originally described as a heat-shock regulatory element (CIRCE) in Bacillus subtilis. This element represses expression of groESL4 under non-stress conditions, as inferred from the increased expression of a groESL4′–′lacZ fusion suffering a 4 bp deletion within the CIRCE element. The two control systems clearly differ with respect to the temperature dependence and the kinetics of the heat-shock response, and they also respond differently to the stress signal elicited by incorporation of the amino acid analogue p-F-phenylalanine into cellular protein. Knock-out mutations in groEL4 resulted in an increased expression of groESL4, suggesting that repression via CIRCE depends, itself, upon the cellular level of GroEL4 protein.
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  • 10
    Electronic Resource
    Electronic Resource
    Oxford BSL : Blackwell Science Ltd
    Molecular microbiology 24 (1997), S. 0 
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Bradyrhizobium japonicum possesses a subclass of heat-shock genes whose members are transcribed from a σ32 consensus promoter. Having identified previously one gene (rpoH1) encoding a σ32-like RNA polymerase transcription factor, we report here the characterization of two additional rpoH-like genes (rpoH2 and rpoH3). B. japonicum thus represents the first example of an organism possessing an rpoH multigene family. All three rpoH genes encode functional proteins that are able to initiate transcription from the Escherichia coli groE promoter. Each rpoH gene is apparently regulated by a different mechanism. Although both rpoH1 and rpoH2 are transcribed from σ70-type promoters, transcription of the rpoH1 operon was found to be heat inducible by an unknown mechanism, whereas the level of rpoH2 mRNA decreased after heat shock. At extreme temperatures (48°C), rpoH2 was transcribed from a second promoter that resembled the E. coliσE-type promoter. The rpoH3 gene was found to be associated with two upstream genes, ragA and ragB, coding for a classical two-component regulatory system. Transcription initiated from a promoter that mapped in front of the putative response regulator gene ragA, suggesting that ragA, ragB and rpoH3 are organized in an operon. The ragA promoter was similar to a σ32 consensus promoter. The three B. japonicum rpoH genes also varied in their significance to support growth of the organism. While the rpoH2 gene could not be eliminated by mutation, knock-out mutants of rpoH1 and/or rpoH3 were readily obtained and shown to be indistinguishable from the wild type under aerobic growth conditions or during root-nodule symbiosis. We conclude that rpoH2 is essential for the synthesis of cellular proteins under physiological growth conditions, whereas rpoH1, and probably also rpoH3, are involved in their synthesis during the stress response.
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