ABSTRACT
RhizoBindingSites is a de novo depurified database of conserved DNA motifs potentially involved in the transcriptional regulation of the Rhizobium, Sinorhizobium, Bradyrhizobium, Azorhizobium, and Mesorhizobium genera covering 9 representative symbiotic species, deduced from the upstream regulatory sequences of orthologous genes (O-matrices) from the Rhizobiales taxon. The sites collected with O-matrices per gene per genome from RhizoBindingSites were used to deduce matrices using the dyad-Regulatory Sequence Analysis Tool (RSAT) method, giving rise to novel S-matrices for the construction of the RizoBindingSites v2.0 database. A comparison of the S-matrix logos showed a greater frequency and/or re-definition of specific-position nucleotides found in the O-matrices. Moreover, S-matrices were better at detecting genes in the genome, and there was a more significant number of transcription factors (TFs) in the vicinity than O-matrices, corresponding to a more significant genomic coverage for S-matrices. O-matrices of 3187 TFs and S-matrices of 2754 TFs from 9 species were deposited in RhizoBindingSites and RhizoBindingSites v2.0, respectively. The homology between the matrices of TFs from a genome showed inter-regulation between the clustered TFs. In addition, matrices of AraC, ArsR, GntR, and LysR ortholog TFs showed different motifs, suggesting distinct regulation. Benchmarking showed 72%, 68%, and 81% of common genes per regulon for O-matrices and approximately 14% less common genes with S-matrices of Rhizobium etli CFN42, Rhizobium leguminosarum bv. viciae 3841, and Sinorhizobium meliloti 1021. These data were deposited in RhizoBindingSites and the RhizoBindingSites v2.0 database (http://rhizobindingsites.ccg.unam.mx/).
ABSTRACT
The phosphate (P)-solubilizing potential of rhizobia isolated from active root nodules of Brazilian native Mimosa and Desmodium was assessed. Out of the 15 strains selected, five Paraburkholderia isolated from Mimosa spp. grown in rocky outcrops stood out. The Ca3(PO4)2-solubilizing efficiency of these strains ranged from 110.67 to 356.3 mgL-1, with less expressive results for FePO4 and Al(H2PO4)3, that might be attributed to the low solubility of these two P compounds. Paraburkholderia strains CNPSo 3281 and CNPSo 3076 were the most efficient siderophore producers (44.17 and 41.87 µMol EDTA) and two of the top FePO4 solubilizers. Acidification of the culture media was observed for all the strains and P sources. Regarding Ca3(PO4)2 solubilization, the main organic acids detected were glucuronic (an important component of rhizobia exopolysaccharides) and gluconic acids. Genomic analysis of P. nodosa CNPSo 3281 and CNPSo 3076 along with other phosphate-solubilizing Paraburkholderia species of the genus pointed out a conserved gene organization of phoUBR, pstSCAB, ppk and ppx. Greenhouse experiment revealed that P. nodosa CNPSo 3281 and CNPSo 3076 promoted maize growth under low P. Our results indicate the relevance of native rhizobia as multifunctional plant-associated bacteria and the rocky outcrops ecosystems as hotspots for bioprospection.
ABSTRACT
This study characterizes seedling exudates of peas, tomatoes, and cucumbers at the level of chemical composition and functionality. A plant experiment confirmed that Rhizobium leguminosarum bv. viciae 3841 enhanced growth of pea shoots, while Azospirillum brasilense Sp7 supported growth of pea, tomato, and cucumber roots. Chemical analysis of exudates after 1 day of seedling incubation in water yielded differences between the exudates of the three plants. Most remarkably, cucumber seedling exudate did not contain detectable sugars. All exudates contained amino acids, nucleobases/nucleosides, and organic acids, among other compounds. Cucumber seedling exudate contained reduced glutathione. Migration on semi solid agar plates containing individual exudate compounds as putative chemoattractants revealed that R. leguminosarum bv. viciae was more selective than A. brasilense, which migrated towards any of the compounds tested. Migration on semi solid agar plates containing 1:1 dilutions of seedling exudate was observed for each of the combinations of bacteria and exudates tested. Likewise, R. leguminosarum bv. viciae and A. brasilense grew on each of the three seedling exudates, though at varying growth rates. We conclude that the seedling exudates of peas, tomatoes, and cucumbers contain everything that is needed for their symbiotic bacteria to migrate and grow on.
Subject(s)
Azospirillum brasilense , Cucumis sativus , Pisum sativum , Rhizobium leguminosarum , Seedlings , Solanum lycopersicum , Solanum lycopersicum/microbiology , Solanum lycopersicum/growth & development , Cucumis sativus/microbiology , Cucumis sativus/growth & development , Seedlings/growth & development , Seedlings/microbiology , Rhizobium leguminosarum/growth & development , Rhizobium leguminosarum/metabolism , Azospirillum brasilense/growth & development , Azospirillum brasilense/metabolism , Pisum sativum/microbiology , Pisum sativum/growth & development , Plant Roots/microbiology , Plant Roots/growth & development , Chemotaxis , Plant Exudates/chemistry , Plant Exudates/metabolismABSTRACT
Recent advancements in plant biotechnology have highlighted the potential of hairy roots as a biotechnological platform, primarily due to their rapid growth and ability to produce specialized metabolites. This study aimed to delve deeper into hairy root development in C. asiatica and explore the optimization of genetic transformation for enhanced bioactive compound production. Previously established hairy root lines of C. asiatica were categorized based on their centelloside production capacity into HIGH, MID, or LOW groups. These lines were then subjected to a meticulous label-free proteomic analysis to identify and quantify proteins. Subsequent multivariate and protein network analyses were conducted to discern proteome differences and commonalities. Additionally, the quantification of rol gene copy numbers was undertaken using qPCR, followed by gene expression measurements. From the proteomic analysis, 213 proteins were identified. Distinct proteome differences, especially between the LOW line and other lines, were observed. Key proteins related to essential processes like photosynthesis and specialized metabolism were identified. Notably, potential biomarkers, such as the Tr-type G domain-containing protein and alcohol dehydrogenase, were found in the HIGH group. The presence of ornithine cyclodeaminase in the hairy roots emerged as a significant biomarker linked with centelloside production capacity lines, indicating successful Rhizobium-mediated genetic transformation. However, qPCR results showed an inconsistency with rol gene expression levels, with the HIGH line displaying notably higher expression, particularly of the rolD gene. The study unveiled the importance of ornithine cyclodeaminase as a traceable biomarker for centelloside production capacity. The strong correlation between this biomarker and the rolD gene emphasizes its potential role in optimizing genetic transformation processes in C. asiatica.
ABSTRACT
The rhizobia-Desmodium (Leguminosae, Papilionoideae) symbiosis is generally described by its specificity with alpha-rhizobia, especially with Bradyrhizobium. Our study aimed to isolate rhizobia from root nodules of native D. barbatum, D. incanum, and D. discolor, collected in remnants of the biomes of Atlantic Forest and Cerrado in protected areas of the Paraná State, southern Brazil. Based on the 16S rRNA phylogeny, 18 out of 29 isolates were classified as Alphaproteobacteria (Bradyrhizobium and Allorhizobium/Rhizobium) and 11 as Betaproteobacteria (Paraburkholderia). Phylogeny of the recA gene of the alpha-rhizobia resulted in ten main clades, of which two did not group with any described rhizobial species. In the 16S rRNA phylogeny of the beta-rhizobia, Paraburkholderia strains from the same host and conservation unity occupied the same clade. Phenotypic characterization of representative strains revealed the ability of Desmodium rhizobia to grow under stressful conditions such as high temperature, salinity, low pH conditions, and tolerance of heavy metals and xenobiotic compounds. Contrasting with previous reports, our results revealed that Brazilian native Desmodium can exploit symbiotic interactions with stress-tolerant strains of alpha- and beta-rhizobia. Stress tolerance can highly contribute to the ecological success of Desmodium in this phytogeographic region, possibly relating to its pioneering ability in Brazil. We propose Desmodium as a promising model for studies of plant-rhizobia interactions.
Subject(s)
Bradyrhizobium , Burkholderiaceae , Fabaceae , Rhizobium , Rhizobium/genetics , RNA, Ribosomal, 16S/genetics , Fabaceae/microbiology , Forests , Burkholderiaceae/genetics , Phylogeny , Symbiosis , Root Nodules, Plant/microbiology , DNA, Bacterial/genetics , Sequence Analysis, DNAABSTRACT
Common bean is considered a legume of great socioeconomic importance, capable of establishing symbioses with a wide variety of rhizobial species. However, the legume has also been recognized for its low efficiency in fixing atmospheric nitrogen. Brazil is a hotspot of biodiversity, and in a previous study, we identified 13 strains isolated from common bean (Phaseolus vulgaris) nodules in three biomes of Mato Grosso do Sul state, central-western Brazil, that might represent new phylogenetic groups, deserving further polyphasic characterization. The phylogenetic tree of the 16S rRNA gene split the 13 strains into two large clades, seven in the R. etli and six in the R. tropici clade. The MLSA with four housekeeping genes (glnII, gyrB, recA, and rpoA) confirmed the phylogenetic allocation. Genomic comparisons indicated eight strains in five putative new species and the remaining five as R. phaseoli. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) comparing the putative new species and the closest neighbors ranged from 81.84 to 92.50% and 24.0 to 50.7%, respectively. Other phenotypic, genotypic, and symbiotic features were evaluated. Interestingly, some strains of both R. etli and R. tropici clades lost their nodulation capacity. The data support the description of the new species Rhizobium cerradonense sp. nov. (CNPSo 3464T), Rhizobium atlanticum sp. nov. (CNPSo 3490T), Rhizobium aureum sp. nov. (CNPSo 3968T), Rhizobium pantanalense sp. nov. (CNPSo 4039T), and Rhizobium centroccidentale sp. nov. (CNPSo 4062T).
Subject(s)
Phaseolus , Rhizobium , Brazil , Rhizobium/genetics , Phylogeny , RNA, Ribosomal, 16S/genetics , Vegetables , DNAABSTRACT
The genome sequences of two nitrogen-fixing type strains of the Rhizobium tropici group were obtained: Rhizobium calliandrae CCGE524T and R. mayense CCGE526T. Genomic analyses confirmed their taxonomic position and identified three complete sequences of the repABC genes, indicative of three plasmids, one of them carrying symbiotic genes.
ABSTRACT
Lentil, which is an important grain legume, can be co-inoculated with plant growth-promoting rhizobia and rhizobacteria to boost nitrogen fixation, increase biomass, and a possibility for early nodulation. The goal of the ongoing study was to identify plant growth-promoting rhizobacteria (PGPR) in the rhizosphere of lentil growing soils in eastern India. Sixteen rhizosphere bacteria were isolated from two different soil orders, and their capacity to solubilize phosphate and generate hydrogen cyanide (HCN), siderophore, and indole acetic acid (IAA) was assessed. The three best strains were selected for compatibility study with twenty Rhizobium isolated from lentil root nodules. The isolated rhizobacteria were able to produce ammonia and different mycolytic enzymes. Isolate B3 produced the highest amount of IAA and siderophore; the highest amount of phosphate solubilized by PSB1 strain; and isolates AB1, AB2, B3, PS2, and PSB2 produced considerable amount of HCN gas. Among all the isolates, B3, PSB1, and PS2 performed better based on different plant growth-promoting abilities. These three bacterial isolates showed compatible reaction with most of the Rhizobium strains. Isolates B3, PS2, and PSB1 were identified as Bacillus subtilis (MT729775), Pseudomonas palmensis (MT729782), and Paraburkholderia caribenis (MZ956803), respectively. Lentil shoot weight, root length, nodule number, N uptake, and P uptake were increased in the pot culture experiment when inoculated with these strains. PGPR strain B3 performed best among the three strains in the pot culture experiment. Strain B3 can be used as potential biofertilizer along with compatible Rhizobium species for better production of lentil.
Subject(s)
Lens Plant , Rhizobium , Soil , Rhizosphere , Siderophores , Bacillus subtilis , Phosphates , Soil MicrobiologyABSTRACT
Introduction: The extensive use of chemical fertilizers has served as a response to the increasing need for crop production in recent decades. While it addresses the demand for food, it has resulted in a decline in crop productivity and a heightened negative environmental impact. In contrast, plant probiotic bacteria (PPB) offer a promising alternative to mitigate the negative consequences of chemical fertilizers. PPB can enhance nutrient availability, promote plant growth, and improve nutrient uptake efficiency, thereby reducing the reliance on chemical fertilizers. Methods: This study aimed to evaluate the impact of native Rhizobium strains, specifically Rhizobium calliandrae LBP2-1, Rhizobium mayense NSJP1-1, and Rhizobium jaguaris SJP1- 2, on the growth, quality, and rhizobacterial community of tomato crops. Various mechanisms promoting plant growth were investigated, including phosphate solubilization, siderophore production, indole acetic acid synthesis, and cellulose and cellulase production. Additionally, the study involved the assessment of biofilm formation and root colonization by GFP-tagged strains, conducted a microcosm experiment, and analyzed the microbial community using metagenomics of rhizospheric soil. Results: The results showed that the rhizobial strains LBP2-1, NSJP1-1 and SJP1-2 had the ability to solubilize dicalcium phosphate, produce siderophores, synthesize indole acetic acid, cellulose production, biofilm production, and root colonization. Inoculation of tomato plants with native Rhizobium strains influenced growth, fruit quality, and plant microbiome composition. Metagenomic analysis showed increased Proteobacteria abundance and altered alpha diversity indices, indicating changes in rhizospheric bacterial community. Discussion: Our findings demonstrate the potential that native Rhizobium strains have to be used as a plant probiotic in agricultural crops for the generation of safe food and high nutritional value.
ABSTRACT
Cysteine-rich receptor-like kinases (CRKs) are a type of receptor-like kinases (RLKs) that are important for pathogen resistance, extracellular reactive oxygen species (ROS) signaling, and programmed cell death in plants. In a previous study, we identified 46 CRK family members in the Phaseolus vulgaris genome and found that CRK12 was highly upregulated under root nodule symbiotic conditions. To better understand the role of CRK12 in the Phaseolus-Rhizobia symbiotic interaction, we functionally characterized this gene by overexpressing (CRK12-OE) and silencing (CRK12-RNAi) it in a P. vulgaris hairy root system. We found that the constitutive expression of CRK12 led to an increase in root hair length and the expression of root hair regulatory genes, while silencing the gene had the opposite effect. During symbiosis, CRK12-RNAi resulted in a significant reduction in nodule numbers, while CRK12-OE roots showed a dramatic increase in rhizobial infection threads and the number of nodules. Nodule cross sections revealed that silenced nodules had very few infected cells, while CRK12-OE nodules had enlarged infected cells, whose numbers had increased compared to controls. As expected, CRK12-RNAi negatively affected nitrogen fixation, while CRK12-OE nodules fixed 1.5 times more nitrogen than controls. Expression levels of genes involved in symbiosis and ROS signaling, as well as nitrogen export genes, supported the nodule phenotypes. Moreover, nodule senescence was prolonged in CRK12-overexpressing roots. Subcellular localization assays showed that the PvCRK12 protein localized to the plasma membrane, and the spatiotemporal expression patterns of the CRK12-promoter::GUS-GFP analysis revealed a symbiosis-specific expression of CRK12 during the early stages of rhizobial infection and in the development of nodules. Our findings suggest that CRK12, a membrane RLK, is a novel regulator of Phaseolus vulgaris-Rhizobium tropici symbiosis.
Subject(s)
Phaseolus , Rhizobium tropici , Rhizobium , Symbiosis/genetics , Rhizobium tropici/genetics , Rhizobium tropici/metabolism , Phaseolus/metabolism , Reactive Oxygen Species/metabolism , Plant Roots/genetics , Plant Roots/metabolism , Rhizobium/metabolism , Nitrogen Fixation/genetics , Root Nodules, Plant/metabolismABSTRACT
BACKGROUND: Hairy roots are a plant-tissue culture raised by Rhizobium rhizogenes infection (formerly known as Agrobacterium rhizogenes). Nowadays, these roots have been gaining more space in biotechnology due to their benefits for the recombinant expression of valuables proteins; it includes simplified downstream processing, protein rhizosecretion, and scalability in bioreactors. However, due to methodological inconsistency among reports, the tissue platform is still a promising technology. METHODS AND RESULTS: In the current paper, we propose the first step to overcome this issue through a systematic review of studies that employ Nicotiana hairy roots for recombinant expression. We conducted a qualitative synthesis of 36 out of 387 publications initially selected. Following the PRISMA procedure, all papers were assessed for exclusion and inclusion criteria. Multiple points of root culture were explored, including transformation methods, root growth curve, external additives, and scale-up with bioreactors to determine which approaches performed best and what is still required to achieve a robust protocol. CONCLUSION: The information presented here may help researchers who want to work with hairy roots in their laboratories trace a successful path to appraisal the literature status.
Subject(s)
Biotechnology , Nicotiana , Nicotiana/genetics , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Biotechnology/methods , Bioreactors , Plants, Genetically Modified/genetics , Plant Roots/metabolism , Transformation, GeneticABSTRACT
Black wattle (Acacia mearnsii) is a forest species of significant economic importance in southern Brazil; as a legume, it forms symbiotic associations with rhizobia, fixing atmospheric nitrogen. Nonetheless, little is known about native rhizobia in soils where the species is cultivated. Therefore, this study aimed to evaluate the diversity and symbiotic efficiency of rhizobia nodulating A. mearnsii in commercial planting areas and validate the efficiency of a potential strain in promoting seedling development. To this end, nodules were collected from four A. mearnsii commercial plantations located in Rio Grande do Sul State, southern Brazil. A total of 80 rhizobia isolates were obtained from black wattle nodules, and thirteen clusters were obtained by rep-PCR. Higher genetic diversity was found within the rhizobial populations from the Duas Figueiras (H' = 2.224) and Seival (H' = 2.112) plantations. Twelve isolates were evaluated belonging to the genus Bradyrhizobium, especially to the species Bradyrhizobium guangdongense. The principal component analysis indicated an association between rhizobia diversity and the content of clay, Ca, Mg, and K. Isolates and reference strains (SEMIA 6163 and 6164) induced nodulation and fixed N via symbiosis with black wattle plants after 60 days of germination. The isolates DF2.4, DF2.3, DF3.3, SEMIA 6164, SEMIA 6163, CA4.3, OV3.4, and OV1.4 showed shoot nitrogen accumulation values similar to the N + control treatment. In the second experiment (under nursery conditions), inoculation with the reference strain SEMIA 6164 generally improved the growth of A. mearnsii seedlings, reinforcing its efficiency even under production conditions.
Subject(s)
Acacia , Bradyrhizobium , Rhizobium , Rhizobium/genetics , Seedlings , Nitrogen Fixation , Symbiosis/genetics , Phylogeny , Root Nodules, Plant/microbiology , Bradyrhizobium/geneticsABSTRACT
Sucrose non-fermentation-related protein kinase 1 (SnRK1) a Ser/Thr protein kinase, is known to play a crucial role in plants during biotic and abiotic stress responses by activating protein phosphorylation pathways. SnRK1 and some members of the plant-specific SnRK2 and SnRK3 sub-families have been studied in different plant species. However, a comprehensive study of the SnRK gene family in Phaseolus vulgaris is not available. Symbiotic associations of P. vulgaris with Rhizobium and/or mycorrhizae are crucial for the growth and productivity of the crop. In the present study, we identified PvSnRK genes and analysed their expression in response to the presence of the symbiont. A total of 42 PvSnRK genes were identified in P. vulgaris and annotated by comparing their sequence homology to Arabidopsis SnRK genes. Phylogenetic analysis classified the three sub-families into individual clades, and PvSnRK3 was subdivided into two groups. Chromosome localization analysis showed an uneven distribution of PvSnRK genes on 10 of the 11 chromosomes. Gene structural analysis revealed great variation in intron number in the PvSnRK3 sub-family, and motif composition is specific and highly conserved in each sub-family of PvSnRKs. Analysis of cis-acting elements suggested that PvSnRK genes respond to hormones, symbiosis and other abiotic stresses. Furthermore, expression data from databases and transcriptomic analyses revealed differential expression patterns for PvSnRK genes under symbiotic conditions. Finally, an in situ gene interaction network of the PvSnRK gene family with symbiosis-related genes showed direct and indirect interactions. Taken together, the present study contributes fundamental information for a better understanding of the role of the PvSnRK gene family not only in symbiosis but also in other biotic and abiotic interactions in P. vulgaris.
Subject(s)
Mycorrhizae , Phaseolus , Protein Serine-Threonine Kinases , Rhizobium , Mycorrhizae/physiology , Phaseolus/genetics , Phaseolus/microbiology , Phylogeny , Protein Serine-Threonine Kinases/genetics , Rhizobium/physiology , Symbiosis/geneticsABSTRACT
Biological nitrogen fixation (BNF) is a key process for the N input in agriculture, with outstanding economic and environmental benefits from the replacement of chemical fertilizers. However, not all symbioses are equally effective in fixing N2, and a major example relies on the high contribution associated with the soybean (Glycine max), contrasting with the low rates reported with the common bean (Phaseolus vulgaris) crop worldwide. Understanding these differences represents a major challenge that can help to design strategies to increase the contribution of BNF, and next-generation sequencing (NGS) analyses of the nodule and root microbiomes may bring new insights to explain differential symbiotic performances. In this study, three treatments evaluated in non-sterile soil conditions were investigated in both legumes: (i) non-inoculated control; (ii) inoculated with host-compatible rhizobia; and (iii) co-inoculated with host-compatible rhizobia and Azospirillum brasilense. In the more efficient and specific symbiosis with soybean, Bradyrhizobium presented a high abundance in nodules, with further increases with inoculation. Contrarily, the abundance of the main Rhizobium symbiont was lower in common bean nodules and did not increase with inoculation, which may explain the often-reported lack of response of this legume to inoculation with elite strains. Co-inoculation with Azospirillum decreased the abundance of the host-compatible rhizobia in nodules, probably because of competitiveness among the species at the rhizosphere, but increased in root microbiomes. The results showed that several other bacteria compose the nodule microbiomes of both legumes, including nitrogen-fixing, growth-promoters, and biocontrol agents, whose contribution to plant growth deserves further investigation. Several genera of bacteria were detected in root microbiomes, and this microbial community might contribute to plant growth through a variety of microbial processes. However, massive inoculation with elite strains should be better investigated, as it may affect the root microbiome, verified by both relative abundance and diversity indices, that might impact the contribution of microbial processes to plant growth.
Subject(s)
Microbiota , Phaseolus , Rhizobium , Fertilizers , Nitrogen , Nitrogen Fixation , Phaseolus/microbiology , Plant Roots/microbiology , Rhizobium/physiology , Root Nodules, Plant/microbiology , Soil , Glycine max/microbiology , SymbiosisABSTRACT
A comparative proteomic study at 6 h of growth in minimal medium (MM) and bacteroids at 18 days of symbiosis of Rhizobium etli CFN42 with the Phaseolus vulgaris leguminous plant was performed. A gene ontology classification of proteins in MM and bacteroid, showed 31 and 10 pathways with higher or equal than 30 and 20% of proteins with respect to genome content per pathway, respectively. These pathways were for energy and environmental compound metabolism, contributing to understand how Rhizobium is adapted to the different conditions. Metabolic maps based on orthology of the protein profiles, showed 101 and 74 functional homologous proteins in the MM and bacteroid profiles, respectively, which were grouped in 34 different isoenzymes showing a great impact in metabolism by covering 60 metabolic pathways in MM and symbiosis. Taking advantage of co-expression of transcriptional regulators (TF's) in the profiles, by selection of genes whose matrices were clustered with matrices of TF's, Transcriptional Regulatory networks (TRN´s) were deduced by the first time for these metabolic stages. In these clustered TF-MM and clustered TF-bacteroid networks, containing 654 and 246 proteins, including 93 and 46 TFs, respectively, showing valuable information of the TF's and their regulated genes with high stringency. Isoenzymes were specific for adaptation to the different conditions and a different transcriptional regulation for MM and bacteroid was deduced. The parameters of the TRNs of these expected biological networks and biological networks of E. coli and B. subtilis segregate from the random theoretical networks. These are useful data to design experiments on TF gene-target relationships for bases to construct a TRN.
ABSTRACT
Inoculants with beneficial microorganisms comprise both selected strains and carriers that ensure a favorable microenvironment for cell survival and stability. Formulations of inoculants using synthetic polymers as carriers are common. However, only a few studies are available in the literature regarding the formulation of inoculants using natural biomolecules as carriers. Exopolysaccharides (EPS) are biomolecules produced by a vast array of microbial species, including symbiotic nitrogen-fixing bacteria, commonly known as rhizobia. EPS perform several functions, such as the protection against the deleterious effects of diverse environmental soil stresses. Two Rhizobium tropici strains and one Paraburkholderia strain were selected after semiquantitative analysis by scanning electron microscopy (SEM) of their EPS production in liquid YMA medium. Their EPS were characterized through a series of analytical techniques, aiming at their use in the formulation of plant inoculants. In addition, the effect of the carbon source on EPS yield was evaluated. Multi-stage fragmentation analysis showed the presence of xylose, glucose, galactose, galacturonic acid, and glucuronic acid in EPS chemical composition, which was confirmed by FT-IR spectra and 13C NMR spectroscopy. Thermal stability (thermogravimetric) was close to 270 °C and viscosity ranged from 120 to 1053.3 mPa.s. Surface morphology (SEM) was rough and irregular, with a cross-linked spongy matrix, which, together with the hydrophilic functional groups, confers water holding capacity. The present study showed that the three EPS have potential as microorganism carriers for formulation of microbial inoculants to be applied in plants.
Subject(s)
Rhizobium tropici , Rhizobium , Spectroscopy, Fourier Transform Infrared , Rhizobium tropici/metabolism , Symbiosis , Biopolymers/metabolism , Polysaccharides, Bacterial/metabolismABSTRACT
Rhizobia are Gram-negative bacteria that are able to establish a nitrogen-fixing symbiotic interaction with leguminous plants. Rhizobia genomes usually harbor several plasmids which can be transferred to other organisms by conjugation. Two main mechanisms of the regulation of rhizobial plasmid transfer have been described: quorum sensing (QS) and the rctA/rctB system. Nevertheless, new genes and molecules that modulate conjugative transfer have recently been described, demonstrating that new actors can tightly regulate the process. In this work, by means of bioinformatics tools and molecular biology approaches, two hypothetical genes are identified as playing key roles in conjugative transfer. These genes are located between conjugative genes of plasmid pRfaLPU83a from Rhizobium favelukesii LPU83, a plasmid that shows a conjugative transfer behavior depending on the genomic background. One of the two mentioned genes, rcgA, is essential for conjugation, while the other, rcgR, acts as an inhibitor of the process. In addition to introducing this new regulatory system, we show evidence of the functions of these genes in different genomic backgrounds and confirm that homologous proteins from non-closely related organisms have the same functions. These findings set up the basis for a new regulatory circuit of the conjugative transfer of plasmids. IMPORTANCE Extrachromosomal DNA elements, such as plasmids, allow for the adaptation of bacteria to new environments by conferring new determinants. Via conjugation, plasmids can be transferred between members of the same bacterial species, different species, or even to organisms belonging to a different kingdom. Knowledge about the regulatory systems of plasmid conjugative transfer is key in understanding the dynamics of their dissemination in the environment. As the increasing availability of genomes raises the number of predicted proteins with unknown functions, deeper experimental procedures help to elucidate the roles of these determinants. In this work, two uncharacterized proteins that constitute a new regulatory circuit with a key role in the conjugative transfer of rhizobial plasmids were discovered.
Subject(s)
Conjugation, Genetic , Quorum Sensing , Plasmids/genetics , Bacteria/genetics , Nitrogen , DNA , Gene Transfer, HorizontalABSTRACT
This study describes novel single-stranded DNA phages isolated from common bean agriculture soils by infection of the nitrogen-fixing symbiotic bacteria Rhizobium etli and R. phaseoli. A total of 29 phages analyzed have 4.3-6 kb genomes in size and GC 59-60%. They belong to different clades unrelated to other Microviridae subfamilies. Three-dimensional models of the major capsid protein (MCP) showed a conserved ß-barrel structural "jelly-roll" fold. A variable-length loop in the MCPs distinguished three Rhizobium microvirus groups. Microviridae subfamilies were consistent with viral clusters determined by the protein-sharing network. All viral clusters, except for Bullavirinae, included mostly microviruses identified in metagenomes from distinct ecosystems. Two Rhizobium microvirus clusters, chaparroviruses, and chicoviruses, were included within large viral unknown clusters with microvirus genomes identified in diverse metagenomes. A third Rhizobium microvirus cluster belonged to the subfamily Amoyvirinae. Phylogenetic analysis of the MCP confirms the divergence of the Rhizobium microviruses into separate clades. The phylogeny of the bacterial hosts matches the microvirus MCP phylogeny, suggesting a coevolutionary history between the phages and their bacterial host. This study provided essential biological information on cultivated microvirus for understanding the evolution and ecological diversification of the Microviridae family in diverse microbial ecosystems.
ABSTRACT
Rhizobium leguminosarum synthesizes an acidic polysaccharide mostly secreted to the extracellular medium, known as exopolysaccharide (EPS) and partially retained on the bacterial surface as a capsular polysaccharide (CPS). Rap proteins, extracellular protein substrates of the PrsDE type I secretion system (TISS), share at least one Ra/CHDL (cadherin-like) domain and are involved in biofilm matrix development either through cleaving the polysaccharide by Ply glycanases or by altering the bacterial adhesive properties. It was shown that the absence or excess of extracellular RapA2 (a monomeric CPS calcium-binding lectin) alters the biofilm matrix's properties. Here, we show evidence of the role of a new Rap protein, RapD, which comprises an N-terminal Ra/CHDL domain and a C-terminal region of unknown function. RapD was completely released to the extracellular medium and co-secreted with the other Rap proteins in a PrsDE-dependent manner. Furthermore, high levels of RapD secretion were found in biofilms under conditions that favor EPS production. Interestingly, size exclusion chromatography of the EPS produced by the ΔrapA2ΔrapD double mutant showed a profile of EPS molecules of smaller sizes than those of the single mutants and the wild type strain, suggesting that both RapA2 and RapD proteins influence EPS processing on the cell surface. Biophysical studies showed that calcium triggers proper folding and multimerization of recombinant RapD. Besides, further conformational changes were observed in the presence of EPS. Enzyme-Linked ImmunoSorbent Assay (ELISA) and Binding Inhibition Assays (BIA) indicated that RapD specifically binds the EPS and that galactose residues would be involved in this interaction. Taken together, these observations indicate that RapD is a biofilm matrix-associated multimeric protein that influences the properties of the EPS, the main structural component of the rhizobial biofilm.
ABSTRACT
The acidity of soils significantly reduces the productivity of legumes mainly because of the detrimental effects of hydrogen ions on the legume plants, leading to the establishment of an inefficient symbiosis and poor biological nitrogen fixation. We recently reported the analysis of the fully sequenced genome of Rhizobium favelukesii LPU83, an alfalfa-nodulating rhizobium with a remarkable ability to grow, nodulate and compete in acidic conditions. To gain more insight into the genetic mechanisms leading to acid tolerance in R. favelukesii LPU83, we constructed a transposon mutant library and screened for mutants displaying a more acid-sensitive phenotype than the parental strain. We identified mutant Tn833 carrying a single-transposon insertion within LPU83_2531, an uncharacterized short ORF located immediately upstream from ubiF homolog. This gene encodes a protein with an enzymatic activity involved in the biosynthesis of ubiquinone. As the transposon was inserted near the 3' end of LPU83_2531 and these genes are cotranscribed as a part of the same operon, we hypothesized that the phenotype in Tn833 is most likely due to a polar effect on ubiF transcription.We found that a mutant in ubiF was impaired to grow at low pH and other abiotic stresses including 5 mM ascorbate and 0.500 mM Zn2+. Although the ubiF mutant retained the ability to nodulate alfalfa and Phaseolus vulgaris, it was unable to compete with the R. favelukesii LPU83 wild-type strain for nodulation in Medicago sativa and P. vulgaris, suggesting that ubiF is important for competitiveness. Here, we report for the first time an ubiF homolog being essential for nodulation competitiveness and tolerance to specific stresses in rhizobia.