| Nitrogen fixation by Rhizobia-legume symbiosis is one of the important forms of biological nitrogen fixation and is an important component of sustainable agriculture.In-depth research on the diversity,evolutionary dynamics,genomic characteristics,and gene regulation and metabolic strategies of rhizobia can deepen our understanding of the origin and mechanisms of symbiotic nitrogen fixation（SNF）.Furthermore,it has the potential to enhance rhizobia’s symbiotic nitrogen fixation capabilities through selective breeding and genetic modification,presenting crucial theoretical and practical value.Although researchers have explored these aspects in depth,the conclusions have mainly focused on a few type rhizobia strains,lacking large-scale comparative analyses based on known rhizobia genomes,limiting the understanding on diversity on rhizobia genome and metabolic characteristics.Moreover,the understanding on metabolic strategies during SNF and oxygenic stress is limited to key metabolic pathways in type strains,thus overlooking the contribution of overall metabolic network to SNF and defense against oxygen stress.To dissolve these issues,the thesis uses6,378 genomes of rhizobia and close-related non-rhizobia to study the taxonomy distribution,genomic characteristics,diversity of symbiotic gene clusters and its correlation to host specificity,and evolutionary dynamics of symbiotic nitrogen fixation phenotypes.Using Azorhizobium caulinodans which has nitrogen fixation ability in free-living,stem nodulation and nodule nodulation as material,the thesis analyzed transcriptional and metabolic responses of A.caulinodans under H2O2 stress and in symbiosis.Moreover,the thesis established a bioinformatics pipeline named‘micro GEM’to generate high-quality microbial metabolic models,and use this pipeline to construct 282 rhizobia metabolic models,analyzed the metabolic diversity and core metabolic network of rhizobia.The detailed results are as following:1.Large-scale Genomic Study of Rhizobia and Establishment of a Rhizobia Genomic and Evolution Database:（1）Through statistical analysis of genomic data,showing the distribution of Rhizobium species is uneven,which indicates significant differences in the emerge and spread of symbiotic nitrogen fixation phenotypes within different bacterial branches;（2）Rhizobia in the genera Rhizobium,Microvirga,Bradyrhizobium,Paraburkholderia,Mesorhizobium,and Cupriavidus have significantly larger genome sizes compared to non-symbiotic relatives within the same genera.This suggests that the successful evolution of symbiotic nitrogen fixation in these genera likely requires additional gene sets;（3）Comparative and classification analyses of symbiotic gene clusters based on genomic differences,similarity of nodulation and nitrogen fixation genes,and co-linearity revealed the diversity of rhizobial symbiotic gene clusters.However,the types of symbiotic gene clusters only partially reflect their host specificity;（4）Frequency statistics of changes in symbiotic nitrogen fixation phenotypes indicates that within the Bradyrhizobium and Rhizobium,rhizobia tend to lose their symbiotic nitrogen fixation ability.Conversely,the frequency of non-symbiotic rhizobia（Nod-Nif-）evolving into symbiotic rhizobia（Nod+Nif+）is very low within these genera.In contrast,within the Paraburkholderia,the frequency of non-symbiotic rhizobia evolving into symbiotic rhizobia is significantly higher than the frequency of symbiotic rhizobia losing their nitrogen fixation ability;（5）The Rhizobium Genomics and Evolution Database named Rhizobia Wiki（https://rhizobiawiki.com/）were established,providing statistical analysis and high-quality datasets for genomic and evolutionary research on rhizobia.2.Transcriptional Regulation Characteristics and Metabolic Mechanisms of Efficient Symbiotic Nitrogen Fixation in Azorhizobium caulinodans.（1）Metabolic flux simulations using a genome-scale metabolic network model revealed that A.caulinodans can engage in co-metabolism of different carbon sources to enhance the maximum theoretical flux of symbiotic nitrogen fixation.Additionally,it can stabilize and regulate the metabolic balance between carbon sources and oxygen during symbiotic nitrogen fixation;（2）Transcriptomic analysis showed that the differences in nitrogen-fixing abilities of A.caulinodans during stem nodule and root nodule symbiosis are not significantly associated with variations in gene transcription levels under these two conditions;（3）Through metabolic flux predictions,the metabolic strategies of A.caulinodans during symbiotic nitrogen fixation were depicted.It was revealed that pyruvate oxidase serves as the primary metabolic contribution for providing energy to nitrogenase under low oxygen conditions during symbiotic nitrogen fixation.3.Omic Integrated Metabolic Modeling Reveals the Oxygen Tolerance Mechanisms of A.caulinodans（1）A systematic description of A.caulinodans’s transcriptional regulatory changes under hydrogen peroxide stress was explored,including the transcriptional suppression of genes involved in energy production and conversion,inorganic ion transport,and amino acid transport;（2）Multiple-copy genes in A.caulinodans can be categorized into"symbiosis-specific copies"and"non-symbiosis-specific copies."These categories displayed significant differences in transcriptional regulation features,evolutionary origins,sequence characteristics,and three-dimensional structures,indicating possible subfunctionalization;（3）Metabolic flux simulations revealed the metabolic strategies employed by A.caulinodans in response to hydrogen peroxide stress.These strategies included inhibition of ATP synthesis,amino acid synthesis,cofactor synthesis,lipopolysaccharide synthesis,heme synthesis,peptidoglycan synthesis and de novo nucleotide synthesis reactions.Additionally,enzyme-catalyzed reactions utilizing cytochrome c and thioredoxin as electron transfer agents were enhanced to cope with oxidative stress.4.Development of an Automated Microbial Metabolic Network Modeling Workflow and Its Application in Rhizobium Metabolic Network（1）A workflow named micro GEM was established,enabling the automated construction of microbial metabolic network models based on the association between genome annotations and biochemical reactions.The accuracy of the constructed metabolic network models was validated through simulations of carbon and nitrogen source utilization and model breakpoint analysis;（2）The micro GEM workflow was utilized to construct and analyze whole-genome-scale metabolic network models for 282 rhizobia strains with complete genomic data.Based on these models,the commonalities and diversities within the metabolic networks of rhizobia were further summarized.The development of this workflow and the construction of these models have significantly enriched our understanding of rhizobia metabolic capabilities and characteristics.Furthermore,this advancement provides effective tools to support large-scale studies in microbial metabolism. |
