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Nitrogen-fixing rhizobial strains isolated from common bean seeds: phylogeny, physiology, and genome analysis.

ABSTRACT: Rhizobial bacteria are commonly found in soil but also establish symbiotic relationships with legumes, inhabiting the root nodules, where they fix nitrogen. Endophytic rhizobia have also been reported in the roots and stems of legumes and other plants. We isolated several rhizobial strains from the nodules of noninoculated bean plants and looked for their provenance in the interiors of the seeds. Nine isolates were obtained, covering most known bean symbiont species, which belong to the Rhizobium and Sinorhizobium groups. The strains showed several large plasmids, except for a Sinorhizobium americanum isolate. Two strains, one Rhizobium phaseoli and one S. americanum strain, were thoroughly characterized. Optimal symbiotic performance was observed for both of these strains. The S. americanum strain showed biotin prototrophy when subcultured, as well as high pyruvate dehydrogenase (PDH) activity, both of which are key factors in maintaining optimal growth. The R. phaseoli strain was a biotin auxotroph, did not grow when subcultured, accumulated a large amount of poly-?-hydroxybutyrate, and exhibited low PDH activity. The physiology and genomes of these strains showed features that may have resulted from their lifestyle inside the seeds: stress sensitivity, a ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) complex, a homocitrate synthase (usually present only in free-living diazotrophs), a hydrogenase uptake cluster, and the presence of prophages. We propose that colonization by rhizobia and their presence in Phaseolus seeds may be part of a persistence mechanism that helps to retain and disperse rhizobial strains.

SUBMITTER: Mora Y

PROVIDER: S-EPMC4178601 | biostudies-literature | 2014 Sep

REPOSITORIES: biostudies-literature

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Nitrogen-fixing rhizobial strains isolated from common bean seeds: phylogeny, physiology, and genome analysis.

Mora Yolanda Y Díaz Rafael R Vargas-Lagunas Carmen C Peralta Humberto H Guerrero Gabriela G Aguilar Alejandro A Encarnación Sergio S Girard Lourdes L Mora Jaime J

Applied and environmental microbiology 20140707 18

Rhizobial bacteria are commonly found in soil but also establish symbiotic relationships with legumes, inhabiting the root nodules, where they fix nitrogen. Endophytic rhizobia have also been reported in the roots and stems of legumes and other plants. We isolated several rhizobial strains from the nodules of noninoculated bean plants and looked for their provenance in the interiors of the seeds. Nine isolates were obtained, covering most known bean symbiont species, which belong to the Rhizobiu ...[more]

PMID: 25002426

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Project description:BackgroundRhizobia are soil bacteria that establish symbiotic relationships with legumes and fix nitrogen in root nodules. We recently reported that several nitrogen-fixing rhizobial strains, belonging to Rhizobium phaseoli, R. trifolii, R. grahamii and Sinorhizobium americanum, were able to colonize Phaseolus vulgaris (common bean) seeds. To gain further insight into the traits that support this ability, we analyzed the genomic sequences and proteomes of R. phaseoli (CCGM1) and S. americanum (CCGM7) strains from seeds and compared them with those of the closely related strains CIAT652 and CFNEI73, respectively, isolated only from nodules.ResultsIn a fine structural study of the S. americanum genomes, the chromosomes, megaplasmids and symbiotic plasmids were highly conserved and syntenic, with the exception of the smaller plasmid, which appeared unrelated. The symbiotic tract of CCGM7 appeared more disperse, possibly due to the action of transposases. The chromosomes of seed strains had less transposases and strain-specific genes. The seed strains CCGM1 and CCGM7 shared about half of their genomes with their closest strains (3353 and 3472 orthologs respectively), but a large fraction of the rest also had homology with other rhizobia. They contained 315 and 204 strain-specific genes, respectively, particularly abundant in the functions of transcription, motility, energy generation and cofactor biosynthesis. The proteomes of seed and nodule strains were obtained and showed a particular profile for each of the strains. About 82 % of the proteins in the comparisons appeared similar. Forty of the most abundant proteins in each strain were identified; these proteins in seed strains were involved in stress responses and coenzyme and cofactor biosynthesis and in the nodule strains mainly in central processes. Only 3 % of the abundant proteins had hypothetical functions.ConclusionsFunctions that were enriched in the genomes and proteomes of seed strains possibly participate in the successful occupancy of the new niche. The genome of the strains had features possibly related to their presence in the seeds. This study helps to understand traits of rhizobia involved in seed adaptation.

Project description:BackgroundRhizobia are alpha-proteobacteria commonly found in soil and root nodules of legumes. It was recently reported that nitrogen-fixing rhizobia also inhabit legume seeds. In this study, we examined whole-genome sequences of seven strains of rhizobia isolated from seeds of common bean (Phaseolus vulgaris).ResultsRhizobial strains included in this study belonged to three different species, including Rhizobium phaseoli, R. leguminosarum, and R. grahamii. Genome sequence analyses revealed that six of the strains formed three pairs of highly related strains. Both strains comprising a pair shared all but one plasmid. In two out of three pairs, one of the member strains was effective in nodulation and nitrogen fixation, whereas the other was ineffective. The genome of the ineffective strain in each pair lacked several genes responsible for symbiosis, including nod, nif, and fix genes, whereas that of the effective strain harbored the corresponding genes in clusters, suggesting that recombination events provoked gene loss in ineffective strains. Comparisons of genomic sequences between seed strains and nodule strains of the same species showed high conservation of chromosomal sequences and lower conservation of plasmid sequences. Approximately 70% of all genes were shared among the strains of each species. However, paralogs were more abundant in seed strains than in nodule strains. Functional analysis showed that seed strains were particularly enriched in genes involved in the transport and metabolism of amino acids and carbohydrates, biosynthesis of cofactors and in transposons and prophages. Genomes of seed strains harbored several intact prophages, one of which was inserted at exactly the same genomic position in three strains of R. phaseoli and R. leguminosarum. The R. grahamii strain carried a prophage similar to a gene transfer agent (GTA); this represents the first GTA reported for this genus.ConclusionsSeeds represent a niche for bacteria; their access by rhizobia possibly triggered the infection of phages, recombination, loss or gain of plasmids, and loss of symbiosis genes. This process probably represents ongoing evolution that will eventually convert these strains into obligate endophytes.

Project description:Plants MADS-domain/AGL proteins constitute a large transcription factor (TF) family that controls the development of almost every plant organ. We performed a phylogeny of (ca. 500) MADS-domain proteins from Arabidopsis and four legume species. We identified clades with Arabidopsis MADS-domain proteins known to participate in root development that grouped legume MADS-proteins with similar high expression in roots and nodules. In this work, we analyzed the role of AGL transcription factors in the common bean (Phaseolus vulgaris) - Rhizobium etli N-fixing symbiosis. Sixteen P. vulgaris AGL genes (PvAGL), out of 93 family members, are expressed - at different levels - in roots and nodules. From there, we selected the PvAGL gene denominated PvFUL-like for overexpression or silencing in composite plants, with transgenic roots and nodules, that were used for phenotypic analysis upon inoculation with Rhizobium etli. Because of sequence identity in the DNA sequence used for RNAi-FUL-like construct, roots, and nodules expressing this construct -referred to as RNAi_AGL- showed lower expression of other five PvAGL genes highly expressed in roots/nodules. Contrasting with PvFUL-like overexpressing plants, rhizobia-inoculated plants expressing the RNAi_AGL silencing construct presented affection in the generation and growth of transgenic roots from composite plants, both under non-inoculated or rhizobia-inoculated condition. Furthermore, the rhizobia-inoculated plants showed decreased rhizobial infection concomitant with the lower expression level of early symbiotic genes and increased number of small, ineffective nodules that indicate an alteration in the autoregulation of the nodulation symbiotic process. We propose that the positive effects of PvAGL TF in the rhizobia symbiotic processes result from its potential interplay with NIN, the master symbiotic TF regulator, that showed a CArG-box consensus DNA sequence recognized for DNA binding of AGL TF and presented an increased or decreased expression level in roots from non-inoculated plants transformed with OE_FUL or RNAi_AGL construct, respectively. Our work contributes to defining novel transcriptional regulators for the common bean - rhizobia N-fixing symbiosis, a relevant process for sustainable agriculture.

Project description:Aluminum (Al) toxicity is widespread in acidic soils where the common bean (Phaseolus vulgaris), the most important legume for human consumption, is produced and it is a limiting factor for crop production and symbiotic nitrogen fixation. We characterized the nodule responses of common bean plants inoculated with Rhizobioum tropici CIAT899 and the root responses of nitrate-fertilized plants exposed to excess Al in low pH, for long or short periods. A 43-50% reduction in nitrogenase activity indicates that Al toxicity (Alt) highly affected nitrogen fixation in common bean. Bean roots and nodules showed characteristic symptoms for Alt. In mature nodules Al accumulation and lipoperoxidation were observed in the infected zone, while callose deposition and cell death occurred mainly in the nodule cortex. Regulatory mechanisms of plant responses to metal toxicity involve microRNAs (miRNAs) along other regulators. Using a miRNA-macroarray hybridization approach we identified 28 (14 up-regulated) Alt nodule-responsive miRNAs. We validated (quantitative reverse transcriptase-PCR) the expression of eight nodule responsive miRNAs in roots and in nodules exposed to high Al for long or short periods. The inverse correlation between the target and miRNA expression ratio (stress:control) was observed in every case. Generally, miRNAs showed a higher earlier response in roots than in nodules. Some of the common bean Alt-responsive miRNAs identified has also been reported as differentially expressed in other plant species subjected to similar stress condition. miRNA/target nodes analyzed in this work are known to be involved in relevant signaling pathways, thus we propose that the participation of miR164/NAC1 (NAM/ATAF/CUC transcription factor) and miR393/TIR1 (TRANSPORT INHIBITOR RESPONSE 1-like protein) in auxin and of miR170/SCL (SCARECROW-like protein transcription factor) in gibberellin signaling is relevant for common bean response/adaptation to Al stress. Our data provide a foundation for evaluating the individual roles of miRNAs in the response of common bean nodules to Alt.

Dataset Information

Nitrogen-fixing rhizobial strains isolated from common bean seeds: phylogeny, physiology, and genome analysis.

Publications

Nitrogen-fixing rhizobial strains isolated from common bean seeds: phylogeny, physiology, and genome analysis.

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