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. Bacteria can populate diverse environments, from soil to water, even in the most extreme sites on the earth. These organisms can also be found on and inside other organisms, such as fungi, animals, and plants. The microbiome is a recently developed concept used to define the prokaryotic populations in close relationship with more-complex organisms (1, 2). Bacteria can flourish as endophytes inside plant roots, stems, leaves, and seeds (3) in crops (e.g., wheat, rice, maize, sorghum, and sugarcane), legumes (e.g., clover, common bean, and alfalfa), trees (e.g., Populus), grasses (e.g., switchgrass), and model plants (e.g., Arabidopsis) (4). Most common endophytic bacterial species have been described as members of the phyla Firmicutes and Proteobacteria (5).Legumes are economically valuable plants in agriculture and establish symbiotic relationships with rhizobial bacteria (6). These bacteria contribute to the formation of plant root nodules, colonizing them and fixing atmospheric nitrogen. This phenomenon has been studied intensively (7). Endophytic rhizobia, in tissues other than nodules, have also been isolated from clover and pea (e.g., Rhizobium phaseoli and Rhizobium leguminosarum bv. trifolii) (8). Several nonrhizobial bacterial species have been isolated from Phaseolus tissues (roots, stems, and seeds); these include Acinetobacter, Bacillus, Enterococcus, Nocardioides, Paracoccus, Phyll...

Genomic studies of nitrogen-fixing rhizobial strains from Phaseolus vulgaris seeds and nodules

“…As described previously, the first non efficient strain tested was an Agrobacterium tumefaciens devoid of pTi and carrying instead a pSym derived from R. etli strain CFN42 [12]. Using these procedures, ten strains were isolated that showed plasmid profiles not observed previously.…”

“…However, it was easy to observe the plasmids of other strains of S. americanum, CCBAU051121 and CCBAU051127 [30]. In our previous report, we did not observe plasmids in strain CCGM7 [12]. However, given the genome assembly and the report that CFNEI73 contained three plasmids [15], additional efforts were made to detect its plasmids.…”

Ribosomal protein biomarkers provide root nodule bacterial identification by MALDI-TOF MS

“…Over the years, genome data has become essential for understanding the molecular basis of plant symbioses (Mora et al 2014), and some of the mechanisms involved in the evolution and speciation of rhizobia . Our results now illustrate how such genomic datasets can become crucial elements for establishing new technological developments able to facilitate large-scale studies of rhizobial populations in fields and natural environments.…”

Analysis of genome sequence and symbiotic ability of rhizobial strains isolated from seeds of common bean (Phaseolus vulgaris)

“…of genesNo. of RNAs%G + C contentGenBank accession no. Rhizobium phaseoli  CCGM1 a 6.87139129,0095564286161.1JFGP00000000 CCGM27.0353353,3231968345761.3MZZP00000000 CCGM86.4752204,7111262475861.5MZZZ00000000 CCGM96.92116137,9961367365261.2NAAA00000000 CIAT6526.44––4 b 61096161.3CP001074–77Related to Rhizobium leguminosarum  CCGM47.1242355,929867985961.5NAON00000000 CCGM56.9147318,8001266176061.4NAOO00000000 CCGM67.4069356,3553671395861.2NAOP00000000 WSM23046.88––5 2 64156061.2CP001191–95 Rhizobium grahamii  CCGM37.0866288,5763068395359.5NAAC00000000 CCGE5027.1580343,6688068445459.4AEYE00000000 a Published previously in [8]. b Complete genomes: chromosome and plasmids…”