Identifying functional multi-host shuttle plasmids to advance synthetic biology applications in Mesorhizobium and Bradyrhizobium.

Ammonia availability has a crucial role in agriculture as it ensures healthy plant growth and increased crop yields. Since diazotrophs are the only organisms capable of reducing dinitrogen to ammonia, they have great ecological importance and potential to mitigate the environmental and economic costs of synthetic fertilizer use. Rhizobia are especially valuable being that they can engage in nitrogen-fixing symbiotic relationships with legumes, and they demonstrate great diversity and plasticity in genomic and phenotypic traits. However, few rhizobial species have sufficient genetic tractability for synthetic biology applications. This study established a basic genetic toolbox with antibiotic resistance markers, multi-host shuttle plasmids and a streamlined protocol for biparental conjugation with Mesorhizobium and Bradyrhizobium species. We identified two repABC origins of replication from Sinorhizobium meliloti (pSymB) and Rhizobium etli (p42d) that were stable across all three strains of interest. Furthermore, the NZP2235 genome was sequenced and phylogenetic analysis determined its reclassification to Mesorhizobium huakuii. These tools will enable the use of plasmid-based strategies for more advanced genetic engineering projects and ultimately contribute towards the development of more sustainable agriculture practices by means of novel nitrogen-fixing organelles, elite bioinoculants, or symbiotic association with nonlegumes.

Refining the taxonomy of the order Hyphomicrobiales (Rhizobiales) based on whole genome comparisons of over 130 type strains.

The alphaproteobacterial order Hyphomicrobiales consists of 38 families comprising at least 152 validly published genera as of January 2024. The order Hyphomicrobiales was first described in 1957 and underwent important revisions in 2020. However, we show that several inconsistencies in the taxonomy of this order remain and we argue that there is a need for a consistent framework for defining families within the order. We propose a common genome-based framework for defining families within the order Hyphomicrobiales, suggesting that families represent monophyletic groups in core-genome phylogenies that share pairwise average amino acid identity values above ~75 % when calculated from a core set of 59 proteins. Applying this framework, we propose the formation of four new families and to reassign the genera Salaquimonas, Rhodoblastus, and Rhodoligotrophos into Salaquimonadaceae fam. nov., Rhodoblastaceae fam. nov., and Rhodoligotrophaceae fam. nov., respectively, and the genera Albibacter, Chenggangzhangella, Hansschlegelia, and Methylopila into Methylopilaceae fam. nov. We further propose to unify the families Bartonellaceae, Brucellaceae, Phyllobacteriaceae, and Notoacmeibacteraceae as Bartonellaceae; the families Segnochrobactraceae and Pseudoxanthobacteraceae as Segnochrobactraceae; the families Lichenihabitantaceae and Lichenibacteriaceae as Lichenihabitantaceae; and the families Breoghaniaceae and Stappiaceae as Stappiaceae. Lastly, we propose to reassign several genera to existing families. Specifically, we propose to reassign the genus Pseudohoeflea to the family Rhizobiaceae; the genera Oricola, Roseitalea, and Oceaniradius to the family Ahrensiaceae; the genus Limoniibacter to the emended family Bartonellaceae; the genus Faunimonas to the family Afifellaceae; and the genus Pseudochelatococcus to the family Chelatococcaceae. Our data also support the recent proposal to reassign the genus Prosthecomicrobium to the family Kaistiaceae.

A genome-scale metabolic reconstruction of soybean and Bradyrhizobium diazoefficiens reveals the cost-benefit of nitrogen fixation.

Nitrogen-fixing symbioses allow legumes to thrive in nitrogen-poor soils at the cost of diverting some photoassimilate to their microsymbionts. Effort is being made to bioengineer nitrogen fixation into nonleguminous crops. This requires a quantitative understanding of its energetic costs and the links between metabolic variations and symbiotic efficiency. A whole-plant metabolic model for soybean (Glycine max) with its associated microsymbiont Bradyrhizobium diazoefficiens was developed and applied to predict the cost-benefit of nitrogen fixation with varying soil nitrogen availability. The model predicted a nitrogen-fixation cost of c. 4.13 g C g-1 N, which when implemented into a crop scale model, translated to a grain yield reduction of 27% compared with a non-nodulating plant receiving its nitrogen from the soil. Considering the lower nitrogen content of cereals, the yield cost to a hypothetical N-fixing cereal is predicted to be less than half that of soybean. Soybean growth was predicted to be c. 5% greater when the nodule nitrogen export products were amides versus ureides. This is the first metabolic reconstruction in a tropical crop species that simulates the entire plant and nodule metabolism. Going forward, this model will serve as a tool to investigate carbon use efficiency and key mechanisms within N-fixing symbiosis in a tropical species forming determinate nodules.

Diversity and Evolutionary History of Ti Plasmids of "tumorigenes" Clade of Rhizobium spp. and Their Differentiation from Other Ti and Ri Plasmids.

Agrobacteria are important plant pathogens responsible for crown/cane gall and hairy root diseases. Crown/cane gall disease is associated with strains carrying tumor-inducing (Ti) plasmids, while hairy root disease is caused by strains harboring root-inducing (Ri) plasmids. In this study, we analyzed the sequences of Ti plasmids of the novel "tumorigenes" clade of the family Rhizobiaceae ("tumorigenes" Ti plasmids), which includes two species, Rhizobium tumorigenes and Rhizobium rhododendri. The sequences of reference Ti/Ri plasmids were also included, which was followed by a comparative analysis of their backbone and accessory regions. The "tumorigenes" Ti plasmids have novel opine signatures compared with other Ti/Ri plasmids characterized so far. The first group exemplified by pTi1078 is associated with production of agrocinopine, nopaline, and ridéopine in plant tumors, while the second group comprising pTi6.2 is responsible for synthesis of leucinopine. Bioinformatic and chemical analyses, including opine utilization assays, indicated that leucinopine associated with pTi6.2 most likely has D,L stereochemistry, unlike the L,L-leucinopine produced in tumors induced by reference strains Chry5 and Bo542. Most of the "tumorigenes" Ti plasmids have conjugative transfer system genes that are unusual for Ti plasmids, composed of avhD4/avhB and traA/mobC/parA regions. Next, our results suggested that "tumorigenes" Ti plasmids have a common origin, but they diverged through large-scale recombination events, through recombination with single or multiple distinct Ti/Ri plasmids. Lastly, we showed that Ti/Ri plasmids could be differentiated based on pairwise Mash or average amino-acid identity distance clustering, and we supply a script to facilitate application of the former approach by other researchers.

Complete Genome Sequences of Rhizobium leguminosarum bv. phaseoli BT01, Rhizobium sp. Strain BT03, and Rhizobium sp. Strain BT04, Isolated from Black Turtle Bean Nodules in Ontario, Canada.

We report the complete genome sequences of three Rhizobium strains isolated from nodules of heritage black turtle bean (Phaseolus vulgaris) plants grown in a community garden in Ontario, Canada. The genomes are between 6.91 Mb and 7.98 Mb long and consist of five to seven DNA replicons.

Complete Genome Sequences of the Species Type Strains Sinorhizobium garamanticum LMG 24692 and Sinorhizobium numidicum LMG 27395 and CIP 109850.

The genus Sinorhizobium comprises rhizobia that fix nitrogen in symbiosis with legumes. To support taxonomic studies of this genus and of rhizobia more broadly, we report complete genome sequences and annotations for the species type strains Sinorhizobium garamanticum LMG 24692 and Sinorhizobium numidicum LMG 27395 and CIP 109850.

Independent origins and evolution of the secondary replicons of the class Gammaproteobacteria.

Multipartite genomes, consisting of more than one replicon, have been found in approximately 10 % of bacteria, many of which belong to the phylum Proteobacteria. Many aspects of their origin and evolution, and the possible advantages related to this type of genome structure, remain to be elucidated. Here, we performed a systematic analysis of the presence and distribution of multipartite genomes in the class Gammaproteobacteria, which includes several genera with diverse lifestyles. Within this class, multipartite genomes are mainly found in the order Alteromonadales (mostly in the genus Pseudoalteromonas) and in the family Vibrionaceae. Our data suggest that the emergence of secondary replicons in Gammaproteobacteria is rare and that they derive from plasmids. Despite their multiple origins, we highlighted the presence of evolutionary trends such as the inverse proportionality of the genome to chromosome size ratio, which appears to be a general feature of bacteria with multipartite genomes irrespective of taxonomic group. We also highlighted some functional trends. The core gene set of the secondary replicons is extremely small, probably limited to essential genes or genes that favour their maintenance in the genome, while the other genes are less conserved. This hypothesis agrees with the idea that the primary advantage of secondary replicons could be to facilitate gene acquisition through horizontal gene transfer, resulting in replicons enriched in genes associated with adaptation to different ecological niches. Indeed, secondary replicons are enriched both in genes that could promote adaptation to harsh environments, such as those involved in antibiotic, biocide and metal resistance, and in functional categories related to the exploitation of environmental resources (e.g. carbohydrates), which can complement chromosomal functions.

Genomics of the "tumorigenes" clade of the family Rhizobiaceae and description of Rhizobium rhododendri sp. nov.

Tumorigenic members of the family Rhizobiaceae, known as agrobacteria, are responsible for crown and cane gall diseases of various crops worldwide. Tumorigenic agrobacteria are commonly found in the genera Agrobacterium, Allorhizobium, and Rhizobium. In this study, we analyzed a distinct "tumorigenes" clade of the genus Rhizobium, which includes the tumorigenic species Rhizobium tumorigenes, as well as strains causing crown gall disease on rhododendron. Here, high-quality, closed genomes of representatives of the "tumorigenes" clade were generated, followed by comparative genomic and phylogenomic analyses. Additionally, the phenotypic characteristics of representatives of the "tumorigenes" clade were analyzed. Our results showed that the tumorigenic strains isolated from rhododendron represent a novel species of the genus Rhizobium for which the name Rhizobium rhododendri sp. nov. is proposed. This species also includes additional strains originating from blueberry and Himalayan blackberry in the United States, whose genome sequences were retrieved from GenBank. Both R. tumorigenes and R. rhododendri contain multipartite genomes, including a chromosome, putative chromids, and megaplasmids. Synteny and phylogenetic analyses indicated that a large putative chromid of R. rhododendri resulted from the cointegration of an ancestral megaplasmid and two putative chromids, following its divergence from R. tumorigenes. Moreover, gene clusters specific for both species of the "tumorigenes" clade were identified, and their biological functions and roles in the ecological diversification of R. rhododendri and R. tumorigenes were predicted and discussed.

Whole genome assemblies of Zophobas morio and Tenebrio molitor.

Zophobas morio (=Zophobas atratus) and Tenebrio molitor are darkling beetles with industrial importance due to their use as feeder insects and their apparent ability to biodegrade plastics. High quality genome assemblies were recently reported for both species. Here, we report additional independent Z. morio and T. molitor genome assemblies generated from Nanopore and Illumina data. Following scaffolding against the published genomes, haploid assemblies of 462 Mb (scaffold N90 of 16.8 Mb) and 258 Mb (scaffold N90 of 5.9 Mb) were produced for Z. morio and T. molitor, respectively. Gene prediction led to the prediction of 28,544 and 19,830 genes for Z. morio and T. molitor, respectively. Benchmarking Universal Single Copy Orthologs (BUSCO) analyses suggested that both assemblies have a high level of completeness; 91.5 and 89.0% of the BUSCO endopterygota marker genes were complete in the Z. morio assembly and proteome, respectively, while 99.1 and 92.8% were complete in the T. molitor assembly and proteome, respectively. Phylogenomic analyses of four genera from the family Tenebrionidae yielded phylogenies consistent with those previously constructed based on mitochondrial genomes. Synteny analyses revealed large stretches of macrosynteny across the family Tenebrionidae, as well as numerous within-chromosome rearrangements. Finally, orthogroup analysis identified ∼28,000 gene families across the family Tenebrionidae, of which 8,185 were identified in all five of the analyzed species, and 10,837 were conserved between Z. morio and T. molitor. We expect that the availability of multiple whole genome sequences for Z. morio and T. molitor will facilitate population genetics studies to identify genetic variation associated with industrially relevant phenotypes.

The evolutionary ecology of rhizobia: multiple facets of competition before, during, and after symbiosis with legumes.

Rhizobial bacteria have complex lifestyles that involve growth and survival in bulk soil, plant rhizospheres and rhizoplanes, legume infection threads, and mature and senescing legume nodules. In nature, rhizobia coexist and compete with many other rhizobial strains and species to form host associations. We review recent work defining competitive interactions across these environments. We highlight the use of sophisticated measurement tools and sequencing technologies to examine competition mechanisms in planta, and highlight environments (e.g. soil and senescing nodules) where we still know exceedingly little. We argue that moving toward an explicitly ecological framework (types of competition, resources, and genetic differentiation) will clarify the evolutionary ecology of these foundational organisms and open doors for engineering sustainable, beneficial associations with hosts.

Cold-inducible promoter-driven knockdown of Brachypodium antifreeze proteins confers freezing and phytopathogen susceptibility.

The model forage crop, Brachypodium distachyon, has a cluster of ice recrystallization inhibition (BdIRI) genes, which encode antifreeze proteins that function by adsorbing to ice crystals and inhibiting their growth. The genes were targeted for knockdown using a cold-induced promoter from rice (prOsMYB1R35) to drive miRNA. The transgenic lines showed no apparent pleiotropic developmental defects but had reduced antifreeze activity as assessed by assays for ice-recrystallization inhibition, thermal hysteresis, electrolyte leakage, and leaf infrared thermography. Strikingly, the number of cold-acclimated transgenic plants that survived freezing at -8°C was reduced by half or killed entirely, depending on the line, compared with cold-acclimated wild type plants. In addition, more leaf damage was apparent at subzero temperatures in knockdowns after infection with an ice nucleating pathogen, Pseudomonas syringae. Although antifreeze proteins have been studied for almost 60 years, this is the first unequivocal demonstration of their function by knockdown in any organism, and their dual contribution to freeze protection as well as pathogen susceptibility, independent of obvious developmental defects. These proteins are thus of potential interest in a wide range of biotechnological applications from cryopreservation, to frozen product additives, to the engineering of transgenic crops with enhanced pathogen and freezing tolerance.

Whole genome sequencing of mesorhizobia isolated from northern Canada.

Rhizobia are soil-dwelling bacteria that can form N2-fixing symbioses with legume plant species (Fabaceae). These bacteria are globally distributed; however, few studies have examined the genomics of rhizobia that live in cold environments. Here, we isolated and characterized three rhizobial strains from legume nodules collected at a pair of distant low Arctic tundra and boreal forest sites in northern Canada. Phylogenetic and average nucleotide identity measurements suggested that the three strains are members of the genus Mesorhizobium, and that each strain represents a novel genospecies. Intriguingly, whereas most mesorhizobia contain the classical determinants of nodulation and nitrogen fixation on their chromosome, whole genome sequencing revealed that all three strains carry these genes on large symbiotic megaplasmids of ∼750 to ∼1000 kb. Phylogenetic and sequence analyses of the common nodulation genes revealed highly conserved alleles amongst these northern mesorhizobia, leading us to propose that they belong to a novel symbiovar that we termed symbiovar oxytropis. Interestingly, these nod gene alleles are uncommon in mesorhizobia isolated from similar plant hosts in other climatic regions, suggesting potential functional adaptive differences.

Reference nodule transcriptomes for Melilotus officinalis and Medicago sativa cv. Algonquin.

Host/symbiont compatibility is a hallmark of the symbiotic nitrogen-fixing interaction between rhizobia and legumes, mediated in part by plant-produced nodule-specific cysteine-rich (NCR) peptides and the bacterial BacA membrane protein that can act as a NCR peptide transporter. In addition, the genetic and metabolic properties supporting symbiotic nitrogen fixation often differ between compatible partners, including those sharing a common partner, highlighting the need for multiple study systems. Here, we report high-quality nodule transcriptome assemblies for Medicago sativa cv. Algonquin and Melilotus officinalis, two legumes able to form compatible symbioses with Sinorhizobium meliloti. The compressed M. sativa and M. officinalis assemblies consisted of 79,978 and 64,593 contigs, respectively, of which 33,341 and 28,278 were assigned putative annotations, respectively. As expected, the two transcriptomes showed broad similarity at a global level. We were particularly interested in the NCR peptide profiles of these plants, as these peptides drive bacterial differentiation during the symbiosis. A total of 412 and 308 NCR peptides were predicted from the M. sativa and M. officinalis transcriptomes, respectively, with approximately 9% of the transcriptome of both species consisting of NCR transcripts. Notably, transcripts encoding highly cationic NCR peptides (isoelectric point > 9.5), which are known to have antimicrobial properties, were ∼2-fold more abundant in M. sativa than in M. officinalis, and ∼27-fold more abundant when considering only NCR peptides in the six-cysteine class. We hypothesize that the difference in abundance of highly cationic NCR peptides explains our previous observation that some rhizobial bacA alleles which can support symbiosis with M. officinalis are unable to support symbiosis with M. sativa.

Brachypodium Antifreeze Protein Gene Products Inhibit Ice Recrystallisation, Attenuate Ice Nucleation, and Reduce Immune Response.

Antifreeze proteins (AFPs) from the model crop, Brachypodium distachyon, allow freeze survival and attenuate pathogen-mediated ice nucleation. Intriguingly, Brachypodium AFP genes encode two proteins, an autonomous AFP and a leucine-rich repeat (LRR). We present structural models which indicate that ice-binding motifs on the ~13 kDa AFPs can "spoil" nucleating arrays on the ~120 kDa bacterial ice nucleating proteins used to form ice at high sub-zero temperatures. These models are consistent with the experimentally demonstrated decreases in ice nucleating activity by lysates from wildtype compared to transgenic Brachypodium lines. Additionally, the expression of Brachypodium LRRs in transgenic Arabidopsis inhibited an immune response to pathogen flagella peptides (flg22). Structural models suggested that this was due to the affinity of the LRR domains to flg22. Overall, it is remarkable that the Brachypodium genes play multiple distinctive roles in connecting freeze survival and anti-pathogenic systems via their encoded proteins' ability to adsorb to ice as well as to attenuate bacterial ice nucleation and the host immune response.

DNA Methylation in Ensifer Species during Free-Living Growth and during Nitrogen-Fixing Symbiosis with Medicago spp.

Methylation of specific DNA sequences is ubiquitous in bacteria and has known roles in immunity and regulation of cellular processes, such as the cell cycle. Here, we explored DNA methylation in bacteria of the genus Ensifer, including its potential role in regulating terminal differentiation during nitrogen-fixing symbiosis with legumes. Using single-molecule real-time sequencing, six genome-wide methylated motifs were identified across four Ensifer strains, five of which were strain-specific. Only the GANTC motif, recognized by the cell cycle-regulated CcrM methyltransferase, was methylated in all strains. In actively dividing cell cultures, methylation of GANTC motifs increased progressively from the ori to ter regions in each replicon, in agreement with a cell cycle-dependent regulation of CcrM. In contrast, there was near full genome-wide GANTC methylation in the early stage of symbiotic differentiation. This was followed by a moderate decrease in the overall extent of methylation and a progressive decrease in chromosomal GANTC methylation from the ori to ter regions in later stages of differentiation. Based on these observations, we suggest that CcrM activity is dysregulated and constitutive during terminal differentiation, which we hypothesize is a driving factor for endoreduplication of terminally differentiated bacteroids. IMPORTANCE Nitrogen fixation by rhizobia in symbiosis with legumes is economically and ecologically important. The symbiosis can involve a complex bacterial transformation-terminal differentiation-that includes major shifts in the transcriptome and cell cycle. Epigenetic regulation is an important regulatory mechanism in diverse bacteria; however, the roles of DNA methylation in rhizobia and symbiotic nitrogen fixation have been poorly investigated. We show that aside from cell cycle regulation, DNA methyltransferases are unlikely to have conserved roles in the biology of bacteria of the genus Ensifer. However, we present evidence consistent with an interpretation that the cell cycle methyltransferase CcrM is dysregulated during symbiosis, which we hypothesize may be a key factor driving the cell cycle switch in terminal differentiation required for effective symbioses.

Cold Acclimation in Brachypodium Is Accompanied by Changes in Above-Ground Bacterial and Fungal Communities.

Shifts in microbiota undoubtedly support host plants faced with abiotic stress, including low temperatures. Cold-resistant perennials prepare for freeze stress during a period of cold acclimation that can be mimicked by transfer from growing conditions to a reduced photoperiod and a temperature of 4 °C for 2-6 days. After cold acclimation, the model cereal, Brachypodium distachyon, was characterized using metagenomics supplemented with amplicon sequencing (16S ribosomal RNA gene fragments and an internal transcribed spacer region). The bacterial and fungal rhizosphere remained largely unchanged from that of non-acclimated plants. However, leaf samples representing bacterial and fungal communities of the endo- and phyllospheres significantly changed. For example, a plant-beneficial bacterium, Streptomyces sp. M2, increased more than 200-fold in relative abundance in cold-acclimated leaves, and this increase correlated with a striking decrease in the abundance of Pseudomonas syringae (from 8% to zero). This change is of consequence to the host, since P. syringae is a ubiquitous ice-nucleating phytopathogen responsible for devastating frost events in crops. We posit that a responsive above-ground bacterial and fungal community interacts with Brachypodium's low temperature and anti-pathogen signalling networks to help ensure survival in subsequent freeze events, underscoring the importance of inter-kingdom partnerships in the response to cold stress.

The Brachypodium distachyon cold-acclimated plasma membrane proteome is primed for stress resistance.

In order to survive subzero temperatures, some plants undergo cold acclimation (CA) where low, nonfreezing temperatures, and/or shortened day lengths allow cold-hardening and survival during subsequent freeze events. Central to this response is the plasma membrane (PM), where low temperature is perceived and cellular homeostasis must be preserved by maintaining membrane integrity. Here, we present the first PM proteome of cold-acclimated Brachypodium distachyon, a model species for the study of monocot crops. A time-course experiment investigated CA-induced changes in the proteome following two-phase partitioning PM enrichment and label-free quantification by nano-liquid chromatography-mass spectrophotometry. Two days of CA were sufficient for membrane protection as well as an initial increase in sugar levels and coincided with a significant change in the abundance of 154 proteins. Prolonged CA resulted in further increases in soluble sugars and abundance changes in more than 680 proteins, suggesting both a necessary early response to low-temperature treatment, as well as a sustained CA response elicited over several days. A meta-analysis revealed that the identified PM proteins have known roles in low-temperature tolerance, metabolism, transport, and pathogen defense as well as drought, osmotic stress, and salt resistance suggesting crosstalk between stress responses, such that CA may prime plants for other abiotic and biotic stresses. The PM proteins identified here present keys to an understanding of cold tolerance in monocot crops and the hope of addressing economic losses associated with modern climate-mediated increases in frost events.

Proteobacteria Contain Diverse flg22 Epitopes That Elicit Varying Immune Responses in Arabidopsis thaliana.

Bacterial flagellin protein is a potent microbe-associated molecular pattern. Immune responses are triggered by a 22-amino-acid epitope derived from flagellin, known as flg22, upon detection by the pattern recognition receptor FLAGELLIN-SENSING2 (FLS2) in multiple plant species. However, increasing evidence suggests that flg22 epitopes of several bacterial species are not universally immunogenic to plants. We investigated whether flg22 immunogenicity systematically differs between classes of the phylum Proteobacteria, using a dataset of 2,470 flg22 sequences. To predict which species encode highly immunogenic flg22 epitopes, we queried a custom motif (11[ST]xx[DN][DN]xAGxxI21) in the flg22 sequences, followed by sequence conservation analysis and protein structural modeling. These data led us to hypothesize that most flg22 epitopes of the γ- and ß-Proteobacteria are highly immunogenic, whereas most flg22 epitopes of the α-, δ-, and ε-Proteobacteria are weakly to moderately immunogenic. To test this hypothesis, we generated synthetic peptides representative of the flg22 epitopes of each proteobacterial class, and we monitored their ability to elicit an immune response in Arabidopsis thaliana. The flg22 peptides of γ- and ß-Proteobacteria triggered strong oxidative bursts, whereas peptides from the ε-, δ-, and α-Proteobacteria triggered moderate, weak, or no response, respectively. These data suggest flg22 immunogenicity is not highly conserved across the phylum Proteobacteria. We postulate that sequence divergence of each taxonomic class was present prior to the evolution of FLS2, and that the ligand specificity of A. thaliana FLS2 was driven by the flg22 epitopes of the γ- and ß-Proteobacteria, a monophyletic group containing many common phytopathogens.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Minimal gene set from Sinorhizobium (Ensifer) meliloti pSymA required for efficient symbiosis with Medicago.

Reduction of N2 gas to ammonia in legume root nodules is a key component of sustainable agricultural systems. Root nodules are the result of a symbiosis between leguminous plants and bacteria called rhizobia. Both symbiotic partners play active roles in establishing successful symbiosis and nitrogen fixation: while root nodule development is mostly controlled by the plant, the rhizobia induce nodule formation, invade, and perform N2 fixation once inside the plant cells. Many bacterial genes involved in the rhizobia-legume symbiosis are known, and there is much interest in engineering the symbiosis to include major nonlegume crops such as corn, wheat, and rice. We sought to identify and combine a minimal bacterial gene complement necessary and sufficient for symbiosis. We analyzed a model rhizobium, Sinorhizobium (Ensifer) meliloti, using a background strain in which the 1.35-Mb symbiotic megaplasmid pSymA was removed. Three regions representing 162 kb of pSymA were sufficient to recover a complete N2-fixing symbiosis with alfalfa, and a targeted assembly of this gene complement achieved high levels of symbiotic N2 fixation. The resulting gene set contained just 58 of 1,290 pSymA protein-coding genes. To generate a platform for future synthetic manipulation, the minimal symbiotic genes were reorganized into three discrete nod, nif, and fix modules. These constructs will facilitate directed studies toward expanding the symbiosis to other plant partners. They also enable forward-type approaches to identifying genetic components that may not be essential for symbiosis, but which modulate the rhizobium's competitiveness for nodulation and the effectiveness of particular rhizobia-plant symbioses.