Systems biology of bacterial nitrogen fixation: High-throughput technology and its integrative description with constraint-based modeling

<p>Abstract</p> <p>Background</p> <p>Bacterial nitrogen fixation is the biological process by which atmospheric nitrogen is uptaken by bacteroids located in plant root nodules and converted into ammonium through the enzymatic activity of nitrogenase. In practice, this biological process serves as a natural form of fertilization and its optimization has significant implications in sustainable agricultural programs. Currently, the advent of high-throughput technology supplies with valuable data that contribute to understanding the metabolic activity during bacterial nitrogen fixation. This undertaking is not trivial, and the development of computational methods useful in accomplishing an integrative, descriptive and predictive framework is a crucial issue to decoding the principles that regulated the metabolic activity of this biological process.</p> <p>Results</p> <p>In this work we present a systems biology description of the metabolic activity in bacterial nitrogen fixation. This was accomplished by an integrative analysis involving high-throughput data and constraint-based modeling to characterize the metabolic activity in <it>Rhizobium etli </it>bacteroids located at the root nodules of <it>Phaseolus vulgaris (</it>bean plant). Proteome and transcriptome technologies led us to identify 415 proteins and 689 up-regulated genes that orchestrate this biological process. Taking into account these data, we: 1) extended the metabolic reconstruction reported for <it>R. etli</it>; 2) simulated the metabolic activity during symbiotic nitrogen fixation; and 3) evaluated the <it>in silico </it>results in terms of bacteria phenotype. Notably, constraint-based modeling simulated nitrogen fixation activity in such a way that 76.83% of the enzymes and 69.48% of the genes were experimentally justified. Finally, to further assess the predictive scope of the computational model, gene deletion analysis was carried out on nine metabolic enzymes. Our model concluded that an altered metabolic activity on these enzymes induced different effects in nitrogen fixation, all of these in qualitative agreement with observations made in <it>R. etli </it>and other <it>Rhizobiaceas</it>.</p> <p>Conclusions</p> <p>In this work we present a genome scale study of the metabolic activity in bacterial nitrogen fixation. This approach leads us to construct a computational model that serves as a guide for 1) integrating high-throughput data, 2) describing and predicting metabolic activity, and 3) designing experiments to explore the genotype-phenotype relationship in bacterial nitrogen fixation.</p

Metabolic Reconstruction and Modeling of Nitrogen Fixation in Rhizobium etli

Rhizobiaceas are bacteria that fix nitrogen during symbiosis with plants. This symbiotic relationship is crucial for the nitrogen cycle, and understanding symbiotic mechanisms is a scientific challenge with direct applications in agronomy and plant development. Rhizobium etli is a bacteria which provides legumes with ammonia (among other chemical compounds), thereby stimulating plant growth. A genome-scale approach, integrating the biochemical information available for R. etli, constitutes an important step toward understanding the symbiotic relationship and its possible improvement. In this work we present a genome-scale metabolic reconstruction (iOR363) for R. etli CFN42, which includes 387 metabolic and transport reactions across 26 metabolic pathways. This model was used to analyze the physiological capabilities of R. etli during stages of nitrogen fixation. To study the physiological capacities in silico, an objective function was formulated to simulate symbiotic nitrogen fixation. Flux balance analysis (FBA) was performed, and the predicted active metabolic pathways agreed qualitatively with experimental observations. In addition, predictions for the effects of gene deletions during nitrogen fixation in Rhizobia in silico also agreed with reported experimental data. Overall, we present some evidence supporting that FBA of the reconstructed metabolic network for R. etli provides results that are in agreement with physiological observations. Thus, as for other organisms, the reconstructed genome-scale metabolic network provides an important framework which allows us to compare model predictions with experimental measurements and eventually generate hypotheses on ways to improve nitrogen fixation

In Silico Insights into the Symbiotic Nitrogen Fixation in Sinorhizobium meliloti via Metabolic Reconstruction

BACKGROUND: Sinorhizobium meliloti is a soil bacterium, known for its capability to establish symbiotic nitrogen fixation (SNF) with leguminous plants such as alfalfa. S. meliloti 1021 is the most extensively studied strain to understand the mechanism of SNF and further to study the legume-microbe interaction. In order to provide insight into the metabolic characteristics underlying the SNF mechanism of S. meliloti 1021, there is an increasing demand to reconstruct a metabolic network for the stage of SNF in S. meliloti 1021. RESULTS: Through an iterative reconstruction process, a metabolic network during the stage of SNF in S. meliloti 1021 was presented, named as iHZ565, which accounts for 565 genes, 503 internal reactions, and 522 metabolites. Subjected to a novelly defined objective function, the in silico predicted flux distribution was highly consistent with the in vivo evidences reported previously, which proves the robustness of the model. Based on the model, refinement of genome annotation of S. meliloti 1021 was performed and 15 genes were re-annotated properly. There were 19.8% (112) of the 565 metabolic genes included in iHZ565 predicted to be essential for efficient SNF in bacteroids under the in silico microaerobic and nutrient sharing condition. CONCLUSIONS: As the first metabolic network during the stage of SNF in S. meliloti 1021, the manually curated model iHZ565 provides an overview of the major metabolic properties of the SNF bioprocess in S. meliloti 1021. The predicted SNF-required essential genes will facilitate understanding of the key functions in SNF and help identify key genes and design experiments for further validation. The model iHZ565 can be used as a knowledge-based framework for better understanding the symbiotic relationship between rhizobia and legumes, ultimately, uncovering the mechanism of nitrogen fixation in bacteroids and providing new strategies to efficiently improve biological nitrogen fixation

Interaction and Regulation of Carbon, Nitrogen, and Phosphorus Metabolisms in Root Nodules of Legumes

Members of the plant family Leguminosae (Fabaceae) are unique in that they have evolved a symbiotic relationship with rhizobia (a group of soil bacteria that can fix atmospheric nitrogen). Rhizobia infect and form root nodules on their specific host plants before differentiating into bacteroids, the symbiotic form of rhizobia. This complex relationship involves the supply of C4-dicarboxylate and phosphate by the host plants to the microsymbionts that utilize them in the energy-intensive process of fixing atmospheric nitrogen into ammonium, which is in turn made available to the host plants as a source of nitrogen, a macronutrient for growth. Although nitrogen-fixing bacteroids are no longer growing, they are metabolically active. The symbiotic process is complex and tightly regulated by both the host plants and the bacteroids. The metabolic pathways of carbon, nitrogen, and phosphate are heavily regulated in the host plants, as they need to strike a fine balance between satisfying their own needs as well as those of the microsymbionts. A network of transporters for the various metabolites are responsible for the trafficking of these essential molecules between the two partners through the symbiosome membrane (plant-derived membrane surrounding the bacteroid), and these are in turn regulated by various transcription factors that control their expressions under different environmental conditions. Understanding this complex process of symbiotic nitrogen fixation is vital in promoting sustainable agriculture and enhancing soil fertility

Genetically diverse lentil- and faba bean-nodulating rhizobia are present in soils across Central and Southern Ethiopia

In total 196 bacterial isolates were obtained from root nodules of lentil (Lens culinaris) and faba bean (Vicia faba) grown on soil samples collected from 10 different sites in central and southern parts of Ethiopia. All isolates were identified as members of the genus Rhizobium by using recA gene sequence analysis. In the recA phylogenetic tree 195 rhizobial strains were classified into nine genospecies. The phylogeny of symbiotic genes nodC and nifH revealed five and six distinct groups respectively, largely dominated by symbiovar viciae. A multivariate analysis showed that environmental variables of the sampling sites considered in this study had more effect on the distribution and composition of the genospecies than the host legumes of the strains. Twenty representative strains, selected based on their isolation site, host plant and nodC group, were able to nodulate all lentil, faba bean, field pea (Pisum abyssinicum) and grass pea (Lathyrus sativus) plants in a greenhouse test in axenic conditions. The majority of the rhizobial strains were effective nitrogen-fixing symbionts for all tested legumes, indicating their potential to serve as broad host-range inoculants in agriculture. The present work suggests the presence of taxonomically and symbiotically diverse rhizobial species for legumes in the Viciae tribe in Ethiopia.Peer reviewe

Characterization of nitrogen-fixing bacteria from Phaseolus vulgaris L. in Kenya

Phaseolus vulgaris (common bean) is an important food crop in Sub-Saharan Africa. Low soil nitrogen limits the productivity of P. vulgaris in Kenya and a greater exploitation of symbiotic nitrogen fixation, resulting from interactions with rhizobia, has the potential to improve yields. To enable the increased use of the symbiosis in Kenyan agriculture in the future, studies in this thesis examined the genetic diversity of rhizobia that nodulate P. vulgaris in the central and western parts of Kenya, their nitrogen-fixing capabilities, and their competitiveness against Rhizobium tropici CIAT 899, a leading commercial inoculant strain for P. vulgaris. Lastly, studies investigated the relative importance of the genotypes of resident soil rhizobia, soil rhizobial population densities, inocula densities, and levels of soil nitrogen, in determining nodule occupancy by R. tropici CIAT 899 inoculated onto P. vulgaris. Phylogenetic studies using 16S rRNA and recA genes indicated at least five species of Rhizobium viz., R. sophoriradicis, R. phaseoli, R. leucaenae, R. paranaense and R. etli nodulate P. vulgaris in Kenya. In addition to the five species, strains that likely belong to new species in the genus Rhizobium also widely nodulate P. vulgaris in Kenyan soils. In glasshouse studies, recovered strains were variably effective on Kenyan cultivars of P. vulgaris and 11 fixed as much nitrogen as R. tropici CIAT 899. From the 11, strains such as NAK 227, NAK 288, NAK 214 and NAK 157 were also highly competitive in liquid co-inoculation assays, carried out with the aid of gusA and celB marker genes, and are potential future inoculants for P. vulgaris in Kenya. The genotype of the rhizobia in the soil was found to be the primary determinant of the nodule occupancy achieved by the inoculant strain, a finding that conflicts previous reports that indicated nodule occupancy was mainly determined by soil rhizobial densities. The rhizobial genotypes varied in their rhizosphere competence and in their ability to preferentially nodulate the host, suggesting these two traits are important for the successful colonization of P. vulgaris nodules by rhizobia. It is anticipated that future studies will leverage on the results in this thesis, to develop locally-targeted inoculation solutions that optimize nitrogen fixation in P. vulgaris in Kenya, and to elucidate the molecular basis for preferential nodulation in P. vulgaris

Advances in Host Plant and Rhizobium Genomics to Enhance Symbiotic Nitrogen Fixation in Grain Legumes

Legumes form symbiotic relationship with root-nodule, rhizobia. The nitrogen (N2) fixed by legumes is a renewable source and of great importance to agriculture. Symbiotic nitrogen fixation (SNF) is constrained by multiple stresses and alleviating them would improve SNF contribution to agroecosystems. Genetic differences in adaptation tolerance to various stresses are known in both host plant and rhizobium. The discovery and use of promiscuous germplasm in soybean led to the release of high-yielding cultivars in Africa. High N2-fixing soybean cultivars are commercially grown in Australia and some countries in Africa and South America and those of pea in Russia. SNF is a complex trait, governed by multigenes with varying effects. Few major quantitative trait loci (QTL) and candidate genes underlying QTL are reported in grain and model legumes. Nodulating genes in model legumes are cloned and orthologs determined in grain legumes. Single nucleotide polymorphism (SNP) markers from nodulation genes are available in common bean and soybean. Genomes of chickpea, pigeonpea, and soybean; and genomes of several rhizobium species are decoded. Expression studies revealed few genes associated with SNF in model and grain legumes. Advances in host plant and rhizobium genomics are helping identify DNA markers to aid breeding of legume cultivars with high symbiotic efficiency. A paradigm shift is needed by breeding programs to simultaneously improve host plant and rhizobium to harness the strength of positive symbiotic interactions in cultivar development. Computation models based on metabolic reconstruction pathways are providing greater insights to explore genotype–phenotype relationships in SNF. Models to simulate the response of N2 fixation to a range of environmental variables and crop growth are assisting researchers to quantify SNF for efficient and sustainable agricultural production systems. Such knowledge helps identifying bottlenecks in specific legume–rhizobia systems that could be overcome by legume breeding to enhance SNF. This review discusses the recent developments to improve SNF and productivity of grain legumes

Metabolic modelling reveals the specialization of secondary replicons for niche adaptation in Sinorhizobium meliloti

The genome of about 10% of bacterial species is divided among two or more large chromosome-sized replicons. The contribution of each replicon to the microbial life cycle (for example, environmental adaptations and/or niche switching) remains unclear. Here we report a genome-scale metabolic model of the legume symbiont Sinorhizobium meliloti that is integrated with carbon utilization data for 1,500 genes with 192 carbon substrates. Growth of S. meliloti is modelled in three ecological niches (bulk soil, rhizosphere and nodule) with a focus on the role of each of its three replicons. We observe clear metabolic differences during growth in the tested ecological niches and an overall reprogramming following niche switching. In silico examination of the inferred fitness of gene deletion mutants suggests that secondary replicons evolved to fulfil a specialized function, particularly host-associated niche adaptation. Thus, genes on secondary replicons might potentially be manipulated to promote or suppress host interactions for biotechnological purposes

The complete genome of Burkholderia phenoliruptrix strain BR3459a, a symbiont of Mimosa flocculosa: highlighting the coexistence of symbiotic and pathogenic genes.

Burkholderia species play an important ecological role related to xenobiosis, the promotion of plant growth, the biocontrol of agricultural diseases, and symbiotic and non-symbiotic biological nitrogen fixation. Here, we highlight our study as providing the first complete genome of a symbiotic strain of B. phenoliruptrix, BR3459a (=CLA1), which was originally isolated in Brazil from nodules of Mimosa flocculosa and is effective in fixing nitrogen in association with this leguminous species. Genomic comparisons with other pathogenic and non-pathogenic Burkholderia strains grouped B. phenoliruptrix BR3459a with plant-associated beneficial and environmental species, although it shares a high percentage of its gene repertoire with species of the B. cepacia complex (Bcc) and "pseudomallei" group. The genomic analyses showed that the bce genes involved in exopolysaccharide production are clustered together in the same genomic region, constituting part of the Group III cluster of non-pathogenic bacteria. Regarding environmental stresses, we highlight genes that might be relevant in responses to osmotic, heat, cold and general stresses. Furthermore, a number of particularly interesting genes involved in the machinery of the T1SS, T2SS, T3SS, T4ASS and T6SS secretion systems were identified. The xenobiotic properties of strain BR3459a were also investigated, and some enzymes involved in the degradation of styrene, nitrotoluene, dioxin, chlorocyclohexane, chlorobenzene and caprolactam were identified. The genomic analyses also revealed a large number of antibiotic-related genes, the most important of which were correlated with streptomycin and novobiocin. The symbiotic plasmid showed high sequence identity with the symbiotic plasmid of B. phymatum. Additionally, comparative analysis of 545 housekeeping genes among pathogenic and non-pathogenic Burkholderia species strongly supports the definition of a new genus for the second branch, which would include BR3459a. The analyses of B. phenoliruptrix BR3459a showed key property of fixing nitrogen that together with genes for high tolerance to environmental stresses might explain a successful strategy of symbiosis in the tropics. The strain also harbours interesting sets of genes with biotechnological potential. However, the resemblance of certain genes to those of pathogenic Burkholderia raise concerns about large-scale applications in agriculture or for bioremediation

Metabolic modelling reveals the specialization of secondary replicons for niche adaptation in Sinorhizobium meliloti

The genome of about 10% of bacterial species is divided among two or more large chromosome-sized replicons. The contribution of each replicon to the microbial life cycle (for example, environmental adaptations and/or niche switching) remains unclear. Here we report a genome-scale metabolic model of the legume symbiont Sinorhizobium meliloti that is integrated with carbon utilization data for 1,500 genes with 192 carbon substrates. Growth of S. meliloti is modelled in three ecological niches (bulk soil, rhizosphere and nodule) with a focus on the role of each of its three replicons. We observe clear metabolic differences during growth in the tested ecological niches and an overall reprogramming following niche switching. In silico examination of the inferred fitness of gene deletion mutants suggests that secondary replicons evolved to fulfil a specialized function, particularly host-associated niche adaptation. Thus, genes on secondary replicons might potentially be manipulated to promote or suppress host interactions for biotechnological purposes