Biogenesis of iron-sulfur proteins in eukaryotes: a novel task of mitochondria that is inherited from bacteria.
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Fe/S clusters are co-factors of numerous proteins with important functions in metabolism, electron transport and regulation of gene expression. Presumably, Fe/S proteins have occurred early in evolution and are present in cells of virtually all species. Biosynthesis of these proteins is a complex process involving numerous components. In mitochondria, this process is accomplished by the so-called ISC (iron-sulfur cluster assembly) machinery which is derived from the bacterial ancestor of the organelles and is conserved from lower to higher eukaryotes. The mitochondrial ISC machinery is responsible for biogenesis iron-sulfur proteins both within and outside the organelle. Maturation of the latter proteins involves the ABC transporter Atm1p which presumably exports iron-sulfur clusters from the organelle. This review summarizes recent developments in our understanding of the biogenesis of iron-sulfur proteins both within bacteria and eukaryotes. (+info)
Generation of new hydrogen-recycling Rhizobiaceae strains by introduction of a novel hup minitransposon.
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Hydrogen evolution by nitrogenase is a source of inefficiency for the nitrogen fixation process by the Rhizobium-legume symbiosis. To develop a strategy to generate rhizobial strains with H(2)-recycling ability, we have constructed a Tn5 derivative minitransposon (TnHB100) that contains the ca. 18-kb H(2) uptake (hup) gene cluster from Rhizobium leguminosarum bv. viciae UPM791. Bacteroids from TnHB100-containing strains of R. leguminosarum bv. viciae PRE, Bradyrhizobium japonicum, R. etli, and Mesorhizobium loti expressed high levels of hydrogenase activity that resulted in full recycling of the hydrogen evolved by nitrogenase in nodules. Efficient processing of the hydrogenase large subunit (HupL) in these strains was shown by immunoblot analysis of bacteroid extracts. In contrast, Sinorhizobium meliloti, M. ciceri, and R. leguminosarum bv. viciae UML2 strains showed poor expression of the hup system that resulted in H(2)-evolving nodules. For the latter group of strains, no immunoreactive material was detected in bacteroid extracts using anti-HupL antiserum, suggesting a low level of transcription of hup genes or HupL instability. A general procedure for the characterization of the minitransposon insertion site and removal of antibiotic resistance gene included in TnHB100 has been developed and used to generate engineered strains suitable for field release. (+info)
Accumulation of ENOD2-like transcripts in non-nodulating woody papilionoid legumes.
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Japanese pagodatree (Styphnolobium japonicum [L.] Schott) and American yellowwood (Cladrastis kentukea Dum.-Cours.) Rudd are the first woody, non-nodulating papilionoid legumes shown to possess putative early nodulin 2 (ENOD2) genes. ENOD2 cDNAs from Japanese pagodatree (807 bp) and American yellowwood (735 bp) have 75% to 79% sequence identity to ENOD2 sequences and encode deduced proteins that possess conserved ENOD2 pentapeptides (PPHEK and PPEYQ). Lower percentages of glucose and higher percentages of histidine and valine suggest that SjENOD2 and CkENOD2 are different from other ENOD2s. Hybridization analyses indicate the clones represent ENOD2 gene families of two to four genes in Japanese pagodatree and American yellowwood genomes, and ENOD2-like transcripts were detected in stems and flowers, as well as roots. Only roots of control species that nodulate, Maackia amurensis Rupr. & Maxim. and alfalfa (Medicago sativa), produced pseudonodules after treatment with zeatin or 2,3,5-triiodobenzoic acid, an auxin transport inhibitor. Accumulation of MaENOD2 transcripts was enhanced during the first 10 d of treatment, but 2,3,5-triiodobenzoic acid and zeatin enhanced transcript accumulation after 30 d in roots of Japanese pagodatree and American yellowwood. Characteristics that distinguish ENOD2 gene families in basal, non-nodulating woody legumes from other ENOD2 genes may provide new information about the function of these genes during symbiotic and non-symbiotic organ development. (+info)
Characterization and expression analysis of the yellow lupin (Lupinus luteus L.) gene coding for nodule specific proline-rich protein.
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The LlPRP2 gene coding for a proline-rich protein shows a high level of similarity to, as well as significant differences from the family of ENOD2 nodule-specific genes. Several sequence motifs with putative regulatory function were identified in the 5' and 3' noncoding regions of the LlPRP2 gene. Northern blot analysis revealed that the expression of the LlPRP2 gene begins 9 days after inoculation of yellow lupin roots with Bradyrhizobium sp. (Lupinus); the expression is restricted to symbiotic nodules and is not detected in other tissues or organs. Detailed hybridization analysis showed that, when expression is activated, the LlPRP2 transcript is modified so as to produce at least three bands and a continuous distribution of decay intermediates. The modification of the LlPRP2 transcript probably involves degradation from the 5'- and/or 3'-ends of the RNA molecules. Southern blot analysis indicates that only one gene is present in the yellow lupin genome. The presence of genes homologous to the LlPRP2 gene was confirmed for three cultivars of yellow lupin and for Lupinus angustifolius. However, LlPRP2 homologues were not detected in Lupinus albus cv. Bac, indicating that this plant may lack the ENOD2 sequence. (+info)
Saprophytic intracellular rhizobia in alfalfa nodules.
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In indeterminate alfalfa nodules, the establishment of the senescent zone IV, in which both symbionts undergo simultaneous degeneration, has been considered, until now, as the end point of the symbiotic interaction. However, we now describe an additional zone, zone V, proximal to the senescent zone IV and present in alfalfa nodules more than 6 weeks old. In zone V, a new round of bacterial release occurs from remaining infection threads, leading to the reinvasion of plant cells that have completely senesced. These intracellular rhizobia are rod shaped and do not display the ultrastructural differentiation features of bacteroids observed in the more distal zones of the nodule. Interestingly, we have found that oxygen is available in zone V at a concentration compatible with both bacterial development and nitrogen fixation gene expression in newly released rhizobia. However, this expression is not correlated with acetylene reduction. Moreover, the pattern of nifH expression in this zone, as well as new data relating to expression in zone II, strongly suggest that nifH transcription in the nodule is under the control of a negative regulator in addition to oxygen. Our results support the conclusion that zone V is an ecological niche where intracellular rhizobia take advantage of the interaction for their exclusive benefit and live as parallel saprophytic partners. The demonstration of such an advantage for rhizobia in nodules was the missing evidence that Rhizobium-legume interactions are indeed symbiotic and, in particular, suggests that benefits to the two partners are associated with different developmental stages within the nodule. (+info)
The dnaJ (hsp40) locus in Rhizobium leguminosarum bv. phaseoli is required for the establishment of an effective symbiosis with Phaseolus vulgaris.
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P121R25 is a Tn5-induced mutant of the effective Rhizobium leguminosarum bv. phaseoli strain P121R that is unable to use glutamate as the sole carbon and nitrogen source and is defective in symbiotic nitrogen fixation. Enzymatic analysis showed that three enzymes implicated in glutamate metabolism (glutamate dehydrogenase, 2-oxoglutarate dehydrogenase, and glutamate synthase) were affected by this mutation. Sequencing of the chromosomal locus bordering the Tn5 in P121R25 indicated the presence of the dnaK and dnaJ genes in an arrangement similar to that described in R. leguminosarum bv. viciae (GenBank accession number Y14649). The mutation was located in the dnaJ (hsp40) gene. (+info)
The Rhizobium leguminosarum bv. viciae glnD gene, encoding a uridylyltransferase/uridylyl-removing enzyme, is expressed in the root nodule but is not essential for nitrogen fixation.
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A Rhizobium leguminosarum bv. viciae VF39 gene (glnD) encoding the uridylyltransferase/uridylyl-removing enzyme, which constitutes the sensory component of the nitrogen regulation (ntr) system, was identified, cloned and characterized. The deduced amino acid sequence contains the conserved active site motif of the nucleotidyltransferase superfamily and is highly homologous to the glnD gene products of other bacterial species. Downstream of the VF39 glnD resides an open reading frame with similarity to the Salmonella typhimurium virulence factor gene mviN. Mutation of the glnD gene abolished the ability to use nitrate as a sole nitrogen source but not glutamine. In addition, neither uridylylation of P(II) nor induction of the ntr-regulated glnII gene (encoding glutamine synthetase II) under ammonium deficiency could be observed in mutant strains. This strongly suggests that glnD mutants harbour a permanently deuridylylated P(II) protein and as a consequence are unable to activate transcription from NtrC-dependent promoters. The glnD gene itself is expressed constitutively, irrespective of the nitrogen content of the medium. A functional GlnD protein is not essential for nitrogen fixation in R. leguminosarum bv. viciae, but in situ detection of glnD expression in the symbiotic and infection zone of the root nodule and quantitative measurements suggest that at least part of the ntr system functions in symbiosis. The results also indicate that the N-terminal part of GlnD is essential for the cell, as deletions in the 5'-region of the gene appear to be lethal and mutations possibly affecting the expression of the first half of the protein have a significant effect on the vitality of the mutant strain. (+info)
Genetic analysis of calcium spiking responses in nodulation mutants of Medicago truncatula.
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The symbiotic interaction between Medicago truncatula and Sinorhizobium meliloti results in the formation of nitrogen-fixing nodules on the roots of the host plant. The early stages of nodule formation are induced by bacteria via lipochitooligosaccharide signals known as Nod factors (NFs). These NFs are structurally specific for bacterium-host pairs and are sufficient to cause a range of early responses involved in the host developmental program. Early events in the signal transduction of NFs are not well defined. We have previously reported that Medicago sativa root hairs exposed to NF display sharp oscillations of cytoplasmic calcium ion concentration (calcium spiking). To assess the possible role of calcium spiking in the nodulation response, we analyzed M. truncatula mutants in five complementation groups. Each of the plant mutants is completely Nod- and is blocked at early stages of the symbiosis. We defined two genes, DMI1 and DMI2, required in common for early steps of infection and nodulation and for calcium spiking. Another mutant, altered in the DMI3 gene, has a similar mutant phenotype to dmi1 and dmi2 mutants but displays normal calcium spiking. The calcium behavior thus implies that the DMI3 gene acts either downstream of calcium spiking or downstream of a common branch point for the calcium response and the later nodulation responses. Two additional mutants, altered in the NSP and HCL genes, which show root hair branching in response to NF, are normal for calcium spiking. This system provides an opportunity to use genetics to study ligand-stimulated calcium spiking as a signal transduction event. (+info)