72 research outputs found

    fixA, B and C genes are essential for symbiotic and free-living, microaerobic nitrogen fixation

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    AbstractSite-specific mutations were contructed within Bradyrhizobium japonicum fixA, B and C regions that had previously been identified by interspecies hybridization [(1985) Mol.Gen.Genet. 199, 315-322]. The corresponding mutants were not only Fix− in otherwise fully developed soybean root nodules but also Nif− in free-living microaerobic culture. Specific fixA, B and C probes hybridized to DNA of the aerobic diazotroph, Azotobacter vinelandii. We hypothesize, therefore, that fixA, B and C genes are of general importance for aerobic/microaerobic nitrogen fixation

    Disparate role of rhizobial ACC deaminase in root-nodule symbioses

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    The enzyme 1-aminocyclopropane-1-carboxylate (ACC) deaminase converts ACC, a precursor of the plant hormone ethylene, into ammonia and α-ketobutyrate. ACC deaminase is widespread among the rhizobia in which it might play a crucial role in protecting rhizobia against inhibitory effects of ethylene synthesized by the host plant in response to the nodulation process. The beneficial action of this enzyme was demonstrated in several rhizobia such as Mesorhizobium loti and Rhizobium leguminosarum where knock-out mutants of the ACC deaminase gene showed nodulation defects. The genome of the slow-growing rhizobial species Bradyrhizobium japonicum also carries an annotated gene for a putative ACC deaminase (blr0241). Here, we tested the possible importance of this enzyme in B. japonicum by constructing an insertion mutant of blr0241 and studying its phenotype. First, the activity of ACC deaminase itself was measured. Unlike the B. japonicum wild type, the blr0241 mutant did not show any enzymatic activity. By contrast, the mutant was not impaired in its ability to nodulate soybean, cowpea, siratro, and mungbean. Likewise, symbiotic nitrogen fixation activity remained unaffected. Furthermore, a co-inoculation assay with the B. japonicum wild type and the blr0241 mutant for soybean and siratro nodulation revealed that the mutant was not affected in its competitiveness for nodulation and nodule occupation. The results show that the role previously ascribed to ACC deaminase in the rhizobia cannot be generalized, and species-specific differences may exis

    Autoregulation of fixK 2 gene expression in Bradyrhizobium japonicum

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    Several essential Bradyrhizobium japonicum genes for a symbiotic, nitrogen-fixing root-nodule symbiosis are positively controlled under micro-oxic conditions by the FixLJ-FixK2 regulatory cascade. Negative control is exerted by reactive oxygen species at the level of the FixK2 protein. Furthermore, we noticed that fixK 2 gene expression is increased in a fixK 2 mutant, suggesting that FixK2 in the wild type has a negative effect, directly or indirectly, on its own expression. To possibly understand this effect, the transcription pattern of the fixLJ-bll2758-fixK 2 gene region was examined more closely. While fixK 2 gene transcription is activated by FixJ, the bll2758 gene is transcribed from its own promoter in a FixK2-dependent manner, and there is no read-through transcription from bll2758 into fixK 2. The bll2758-encoded protein is predicted to be a stand-alone receiver domain of a response regulator, making it a prime candidate for exerting an inhibitory role on the expression of fixK 2. Transcriptome profiling of a bll2758 knock-out mutant revealed, however, that neither fixK 2 itself nor any of the known FixJ- and FixK2-dependent target genes is significantly affected in their expression. This precludes a role of the bll2758 product as a so-called FixT-like protein in the inhibition of FixLJ function, as was proposed for Sinorhizobium meliloti and Caulobacter crescentus. Instead, we rationalize that other transcription factors, whose genes are activated by FixK2, might be involved in the negative autoregulation of fixK 2 gene expressio

    Periplasmic protein thiol:disulfide oxidoreductases of Escherichia coli

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    Disulfide bond formation is part of the folding pathway for many periplasmic and outer membrane proteins that contain structural disulfide bonds. In Escherichia coli, a broad variety of periplasmic protein thiol:disulfide oxidoreductases have been identified in recent years, which substantially contribute to this pathway. Like the well-known cytoplasmic thioredoxins and glutaredoxins, these periplasmic protein thiol:disulfide oxidoreductases contain the conserved C-X-X-C motif in their active site. Most of them have a domain that displays the thioredoxin-like fold. In contrast to the cytoplasmic system, which consists exclusively of reducing proteins, the periplasmic oxidoreductases have either an oxidising, a reducing or an isomerisation activity. Apart from understanding their physiological role, it is of interest to learn how these proteins interact with their target molecules and how they are recycled as electron donors or acceptors. This review reflects the recently made efforts to elucidate the sources of oxidising and reducing power in the periplasm as well as the different properties of certain periplasmic protein thiol:disulfide oxidoreductases of E. col

    The Bradyrhizobium japonicum phoB gene is required for phosphate-limited growth but not for symbiotic nitrogen fixation

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    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 plan

    Host-specific symbiotic requirement of BdeAB, a RegR-controlled RND-type efflux system in Bradyrhizobium japonicum

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    Multidrug efflux systems not only cause resistance against antibiotics and toxic compounds but also mediate successful host colonization by certain plant-associated bacteria. The genome of the nitrogen-fixing soybean symbiont Bradyrhizobium japonicum encodes 24 members of the family of resistance/nodulation/cell division (RND) multidrug efflux systems, of which BdeAB is genetically controlled by the RegSR two-component regulatory system. Phylogenetic analysis of the membrane components of these 24 RND-type transporters revealed that BdeB is more closely related to functionally characterized orthologs in other bacteria, including those associated with plants, than to any of the other 23 paralogs in B. japonicum. A mutant with a deletion of the bdeAB genes was more susceptible to inhibition by the aminoglycosides kanamycin and gentamicin than the wild type, and had a strongly decreased symbiotic nitrogen-fixation activity on soybean, but not on the alternative host plants mungbean and cowpea, and only very marginally on siratro. The host-specific role of a multidrug efflux pump is a novel feature in the rhizobia-legume symbioses. Consistent with the RegSR dependency of bdeAB, a B. japonicum regR mutant was found to have a greater sensitivity against the two tested antibiotics and a symbiotic defect that is most pronounced for soybea

    Dissection of the Bradyrhizobium japonicum NifA+σ54 regulon, and identification of a ferredoxin gene ( fdxN ) for symbiotic nitrogen fixation

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    Hierarchically organized regulatory proteins form a complex network for expression control of symbiotic and accessory genes in the nitrogen-fixing soybean symbiont Bradyrhizobium japonicum. A genome-wide survey of regulatory interactions was made possible with the design of a custom-made gene chip. Here, we report the first use of the microarray in a comprehensive and complete characterization of the B. japonicum NifA+σ54 regulon which forms an important node in the entire network. Comparative transcript profiles of anaerobically grown wild-type, nifA, and rpoN 1/2 mutant cells were complemented with a position-specific frequency matrix-based search for NifA- and σ54-binding sites plus a simple operon definition. One of the newly identified NifA+σ54-dependent genes, fdxN, encodes a ferredoxin required for efficient symbiotic nitrogen fixation, which makes it a candidate for being a direct electron donor to nitrogenase. The fdxN gene has an unconventional, albeit functional σ54 promoter with the dinucleotide GA instead of the consensus GC motif at position −12. A GC-containing mutant promoter and the atypical GA-containing promoter of the wild type were disparately activated. Expression analyses were also carried out with two other NifA+σ54 targets (ectC; ahpC). Incidentally, the tiling-like design of the microarray has helped to arrive at completely revised annotations of the ectC- and ahpC-upstream DNA regions, which are now compatible with promoter locations. Taken together, the approaches used here led to a substantial expansion of the NifA+σ54 regulon size, culminating in a total of 65 genes for nitrogen fixation and diverse other processe

    Biological and Pharmacokinetic Studies with β-Peptides

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    Interactions and cleavage reactions of β-amino acids and β-oligopeptides (up to nine residues, carrying the side chains of Ala, Val, Leu, Ile, Phe, Ser, Lys, and Hop) with biological systems, such as the most potent peptidases (pronase, proteinase K, 20S proteasome), microorganisms (Pseudomonas aeruginosa and Pseudomonas putida), and mammalian blood (intravenous application to rats) have been investigated and compared with ?-peptides. The results are: i) the three peptidases do not cleave β-peptides at all (within 24 h), and they are not inhibited by a β-peptide; ii) except for certain 3-aminobutanoic-acid (β-HAla) derivatives, neither free, nor N-acetyl-β-amino acids, nor β-peptides (offered as sole N and C source) lead to growth of the two bacteria tested; iii) two water-soluble β-heptapeptides (with Lys side chains) were shown to have elimination half-lives t1/2(β) of 3 and 10 h at 100- and 30-ng/ml levels, respectively, in the rodent blood – much larger than those of α-peptides. Thus, the preliminary results described here confirm the much greater stability of β-peptides, as compared to α-peptides, towards metabolization processes, but they also suggest that there may be interactions (by hitherto unknown mechanisms) between the worlds of α- and β-peptides
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