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The production of ammonia (NH3) from nitrogen sources involves competitive adsorption of different intermediates and multiple electron and proton transfers, presenting grand challenges in catalyst design. In nature nitrogenases reduce dinitrogen to NH3 using two component proteins, in which electrons and protons are delivered from Fe protein to the active site in MoFe protein for transfer to the bound N2. We draw inspiration from this structural enzymology, and design a two-component metal-sulfur-carbon (M-S-C) catalyst composed of sulfur-doped carbon-supported ruthenium (Ru) single atoms (SAs) and nanoparticles (NPs) for the electrochemical reduction of nitrate (NO3-) to NH3. The catalyst demonstrates a remarkable NH3 yield rate of ~37 mg L-1 h-1 and a Faradaic efficiency of ~97% for over 200 hours, outperforming those consisting solely of SAs or NPs, and even surpassing most reported electrocatalysts. Our experimental and theoretical investigations reveal the critical role of Ru SAs with the coordination of S in promoting the formation of the HONO intermediate and the subsequent reduction reaction over the NP-surface nearby. This study proves a better understanding of how M-S-Cs act as a synthetic nitrogenase mimic during ammonia synthesis, and contributes to the future mechanism-based catalyst design.

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Prokaryotes contribute to the global sulfur cycle by using diverse sulfur compounds as sulfur sources or electron acceptors. In this study, we report that a nitrogenase-like enzyme (NFL) and a radical SAM enzyme (RSE) are involved in the novel anaerobic assimilation pathway of a sulfonate, isethionate, in the photosynthetic bacterium Rhodobacter capsulatus. The nflHDK genes for NFL are localized at a locus containing genes for known sulfonate metabolism in the genome. A gene nflB encoding an RSE is present just upstream of nflH, forming a small gene cluster nflBHDK. Mutants lacking any nflBHDK genes are incapable of growing with isethionate as the sole sulfur source under anaerobic photosynthetic conditions, indicating that all four NflBHDK proteins are essential for the isethionate assimilation pathway. Heterologous expression of the islAB genes encoding a known isethionate lyase that degrades isethionate to sulfite and acetaldehyde restored the isethionate-dependent growth of a mutant lacking nflDK, indicating that the enzyme encoding nflBHDK is involved in an isethionate assimilation reaction to release sulfite. Furthermore, the heterologous expression of nflBHDK and ssuCAB encoding an isethionate transporter in the closely related species R. sphaeroides, which does not have nflBHDK and cannot grow with isethionate as the sole sulfur source, conferred isethionate-dependent growth ability to this species. We propose to rename nflBHDK as isrBHDK (isethionate reductase). The isrBHDK genes are widely distributed among various prokaryote phyla. Discovery of the isethionate assimilation pathway by IsrBHDK provides a missing piece for the anaerobic sulfur cycle and for understanding the evolution of ancient sulfur metabolism.IMPORTANCENitrogenase is an important enzyme found in prokaryotes that reduces atmospheric nitrogen to ammonia and plays a fundamental role in the global nitrogen cycle. It has been noted that nitrogenase-like enzymes (NFLs), which share an evolutionary origin with nitrogenase, have evolved to catalyze diverse reactions such as chlorophyll biosynthesis (photosynthesis), coenzyme F430 biosynthesis (methanogenesis), and methionine biosynthesis. In this study, we discovered that an NFL with unknown function in the photosynthetic bacterium Rhodobacter capsulatus is a novel isethionate reductase (Isr), which catalyzes the assimilatory degradation of isethionate, a sulfonate, releasing sulfite used as the sulfur source under anaerobic conditions. Isr is widely distributed among various bacterial phyla, including intestinal bacteria, and is presumed to play an important role in sulfur metabolism in anaerobic environments such as animal guts and microbial mats. This finding provides a clue for understanding ancient metabolism that evolved under anaerobic environments at the dawn of life.

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Nitrogenases are the only enzymes that activate the strong triple bond in N2. The mechanism for the activation has been very difficult to determine in spite of decades of work. In previous modeling studies it has been suggested that the mechanism for nitrogen activation starts out by four pre-activation steps (A0-A4) before catalysis. That suggestion led to excellent agreement with experimental Elecrtron Paramagnetic Resonance (EPR) observations in the step where N2 becomes protonated (E4). An important part of the pre-activation is that a sulfide is released. In the present paper, the details of the pre-activation are modeled, including the release of the sulfide. Several possible transition states for the release have been obtained. An A4(E0) state is reached which is very similar to the E4 state. For completeness, the steps going back from A4(E0) to A0 after catalysis are also modeled, including the insertion of a sulfide.

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Microscale thermophoresis (MST) is a technique used to measure the strength of molecular interactions. MST is a thermophoretic-based technique that monitors the change in fluorescence associated with the movement of fluorescent-labeled molecules in response to a temperature gradient triggered by an IR LASER. MST has advantages over other approaches for examining molecular interactions, such as isothermal titration calorimetry, nuclear magnetic resonance, biolayer interferometry, and surface plasmon resonance, requiring a small sample size that does not need to be immobilized and a high-sensitivity fluorescence detection. In addition, since the approach involves the loading of samples into capillaries that can be easily sealed, it can be adapted to analyze oxygen-sensitive samples. In this Bio-protocol, we describe the troubleshooting and optimization we have done to enable the use of MST to examine protein-protein interactions, protein-ligand interactions, and protein-nanocrystal interactions. The salient elements in the developed procedures include 1) loading and sealing capabilities in an anaerobic chamber for analysis using a NanoTemper MST located on the benchtop in air, 2) identification of the optimal reducing agents compatible with data acquisition with effective protection against trace oxygen, and 3) the optimization of data acquisition and analysis procedures. The procedures lay the groundwork to define the determinants of molecular interactions in these technically demanding systems. Key features • Established procedures for loading and sealing tubes in an anaerobic chamber for subsequent analysis. • Sodium dithionite (NaDT) could easily be substituted with one electron-reduced 1,1'-bis(3-sulfonatopropyl)-4,4'-bipyridinium [(SPr)2V•] to perform sensitive biophysical assays on oxygen-sensitive proteins like the MoFe protein. • Established MST as an experimental tool to quantify binding affinities in novel enzyme-quantum dot biohybrid complexes that are extremely oxygen-sensitive.

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The transfer of nitrogen fixation (nif) genes from diazotrophs to non-diazotrophic hosts is of increasing interest for engineering biological nitrogen fixation. A recombinant Escherichia coli strain expressing Azotobacter vinelandii 18 nif genes (nifHDKBUSVQENXYWZMF, nifiscA, and nafU) were previously constructed and showed nitrogenase activity. In the present study, we constructed several E. coli strain derivatives in which all or some of the 18 nif genes were additionally integrated into the fliK locus of the chromosome in various combinations. E. coli derivatives with the chromosomal integration of nifiscA, nifU, and nifS, which are involved in the biosynthesis of the [4Fe-4S] cluster of dinitrogenase reductase, exhibited enhanced nitrogenase activity. We also revealed that overexpression of E. coli fldA and ydbK, which encode flavodoxin and flavodoxin-reducing enzyme, respectively, enhanced nitrogenase activity, likely by facilitating electron transfer to dinitrogenase reductase. The additional expression of nifM, putatively involved in maturation of dinitrogenase reductase, further enhanced nitrogenase activity and the amount of soluble NifH. By combining these factors, we successfully improved nitrogenase activity 10-fold.

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Current views of O2 accumulation in Earth history depict three phases: The onset of O2 production by ∼2.4 billion years ago; 2 billion years of stasis at ∼1 % of modern atmospheric levels; and a rising phase, starting about 500 million years ago, in which oxygen eventually reached modern values. Purely geochemical mechanisms have been proposed to account for this tripartite time course of Earth oxygenation. In particular the second phase, the long period of stasis between the advent of O2 and the late rise to modern levels, has posed a puzzle. Proposed solutions involve Earth processes (geochemical, ecosystem, day length). Here we suggest that Earth oxygenation was not determined by geochemical processes. Rather it resulted from emergent biological innovations associated with photosynthesis and the activity of only three enzymes: 1) The oxygen evolving complex of cyanobacteria that makes O2; 2) Nitrogenase, with its inhibition by O2 causing two billion years of oxygen level stasis; 3) Cellulose synthase of land plants, which caused mass deposition and burial of carbon, thus removing an oxygen sink and therefore increasing atmospheric O2. These three enzymes are endogenously produced by, and contained within, cells that have the capacity for exponential growth. The catalytic properties of these three enzymes paved the path of Earth's atmospheric oxygenation, requiring no help from Earth other than the provision of water, CO2, salts, colonizable habitats, and sunlight.

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Biological nitrogen fixation catalyzed by nitrogenase contributes greatly to the global nitrogen cycle. Nitrogenase expression is subject to regulation in response to nitrogen availability. However, the mechanism through which the transcriptional activator NifA regulates nitrogenase expression by interacting with PII nitrogen regulatory proteins remains unclear in diazotrophic proteobacteria lacking NifL. Here, we demonstrate that in Rhodopseudomonas palustris grown with ammonium, NifA bound deuridylylated PII proteins to form an inactive NifA-PII complex, thereby inhibiting the expression of nitrogenase. Upon nitrogen limitation, the dissociation of uridylylated PII proteins from NifA resulted in the full restoration of NifA activity, and, simultaneously, uridylylation of the significantly up-regulated PII protein GlnK2 led to the increased expression of NifA in R. palustris. This insight into how NifA interacts with PII proteins and controls nitrogenase expression sets the stage for creating highly efficient diazotrophs, reducing the need for energy-intensive chemical fertilizers and helping to diminish carbon emissions.

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The nitrogenase reductase NifH catalyses ATP-dependent electron delivery to the Mo-nitrogenase, a reaction central to biological dinitrogen (N2) fixation. While NifHs have been extensively studied in bacteria, structural information about their archaeal counterparts is limited. Archaeal NifHs are considered more ancient, particularly those from Methanococcales, a group of marine hydrogenotrophic methanogens, which includes diazotrophs growing at temperatures near 92 °C. Here, we structurally and biochemically analyse NifHs from three Methanococcales, offering the X-ray crystal structures from meso-, thermo-, and hyperthermophilic methanogens. While NifH from Methanococcus maripaludis (37 °C) was obtained through heterologous recombinant expression, the proteins from Methanothermococcus thermolithotrophicus (65 °C) and Methanocaldococcus infernus (85 °C) were natively purified from the diazotrophic archaea. The structures from M. thermolithotrophicus crystallised as isolated exhibit high flexibility. In contrast, the complexes of NifH with MgADP obtained from the three methanogens are superposable, more rigid, and present remarkable structural conservation with their homologues. They retain key structural features of P-loop NTPases and share similar electrostatic profiles with the counterpart from the bacterial model organism Azotobacter vinelandii. In comparison to the NifH from the phylogenetically distant Methanosarcina acetivorans, these reductases do not cross-react significantly with Mo-nitrogenase from A. vinelandii. However, they associate with bacterial nitrogenase when ADP· AlF 4 - is added to mimic a transient reactive state. Accordingly, detailed surface analyses suggest that subtle substitutions would affect optimal binding during the catalytic cycle between the NifH from Methanococcales and the bacterial nitrogenase, implying differences in the N2-machinery from these ancient archaea.

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Microbial nitrogen fixation presents a viable alternative to chemical fertilizers, yet the limited colonization and specificity of naturally occurring nitrogen-fixing microorganisms present significant challenges to their widespread application. In this study, we identified a nitrogen fixation gene cluster (VNnif) in Vibrio natriegens (VN) and tested its nitrogenase activity through the acetylene reduction assay. We investigated the potential utilization of nitrogenase by incorporating the nitrogenase gene cluster from VN into plant growth-promoting rhizosphere bacteria Pseudomonas protegens CHA0 and enhancing its activity to 48.16 nmol C2H2/mg/h through promoter replacement and cluster rearrangement. The engineered strain CHA0-PVNnif was found to positively impact the growth of Arabidopsis thaliana col-0 and Triticum aestivum L. (wheat). This study expanded the role of plant growth-promoting rhizobacteria (PGPR) and provided a research foundation for enhancing nitrogenase activity.

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Azotobacter vinelandii is a genetically tractable Gram-negative proteobacterium able to fix nitrogen (N2) under aerobic growth conditions. This narrative describes how biochemical-genetic approaches using A. vinelandii to study nitrogen fixation led to the formulation of the "scaffold hypothesis" for the assembly of both simple and complex [Fe-S] clusters associated with biological nitrogen fixation. These studies also led to the discovery of a parallel, but genetically distinct, pathway for maturation of [Fe-S] proteins that support central metabolic processes.

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Nitrogen fixation is a key process that sustains life on Earth. Nitrogenase is the sole enzyme capable of fixing nitrogen under ambient conditions. Extensive research efforts have been dedicated to elucidating the enzyme mechanism and its artificial activation through high applied voltage, photochemistry, or strong reducing agents. Harnessing light irradiation to minimize the required external bias can lower the process's high energy investment. Herein, we present the development of photo-bioelectrochemical cells (PBECs) utilizing BiVO4/CoP or CdS/NiO photoanodes for nitrogenase activation toward N2 fixation. The constructed PBEC based on BiVO4/CoP photoanode requires minimal external bias (200 mV) and suppresses O2 generation that allows efficient activation of the nitrogenase enzyme, using glucose as an electron donor. In a second developed PBEC configuration, CdS/NiO photoanode was used, enabling bias-free activation of the nitrogenase-based cathode to produce 100 µM of ammonia at a faradaic efficiency (FE) of 12%. The ammonia production was determined by a commonly used fluorescence probe and further validated using 1H-NMR spectroscopy. The presented PBECs lay the foundation for biotic-abiotic systems to directly activate enzymes toward value-added chemicals by light-driven reactions.

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Coenzyme F430 is a nickel-containing tetrapyrrole, serving as the prosthetic group of methyl-coenzyme M reductase in methanogenic and methanotrophic archaea. During coenzyme F430 biosynthesis, the tetrapyrrole macrocycle is reduced by the nitrogenase-like CfbC/D system consisting of the reductase component CfbC and the catalytic component CfbD. Both components are homodimeric proteins, each carrying a [4Fe-4S] cluster. Here, the ligands of the [4Fe-4S] clusters of CfbC2 and CfbD2 were identified revealing an all cysteine ligation of both clusters. Moreover, the midpoint potentials of the [4Fe-4S] clusters were determined to be -256 mV for CfbC2 and -407 mV for CfbD2. These midpoint potentials indicate that the consecutive thermodynamically unfavorable 6 individual "up-hill" electron transfers to the organic moiety of the Ni2+-sirohydrochlorin a,c-diamide substrate require an intricate interplay of ATP-binding, hydrolysis, protein complex formation and release to drive product formation, which is a common theme in nitrogenase-like systems.

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Increasing the soybean-planting area and increasing the soybean yield per unit area are two effective solutions to improve the overall soybean yield. Northeast China has a large saline soil area, and if soybeans could be grown there with the help of isolated saline-tolerant rhizobia, the soybean cultivation area in China could be effectively expanded. In this study, soybeans were planted in soils at different latitudes in China, and four strains of rhizobia were isolated and identified from the soybean nodules. According to the latitudes of the soil-sampling sites from high to low, the four isolated strains were identified as HLNEAU1, HLNEAU2, HLNEAU3, and HLNEAU4. In this study, the isolated strains were identified for their resistances, and their acid and saline tolerances and nitrogen fixation capacities were preliminarily identified. Ten representative soybean germplasm resources in Northeast China were inoculated with these four strains, and the compatibilities of these four rhizobium strains with the soybean germplasm resources were analyzed. All four isolates were able to establish different extents of compatibility with 10 soybean resources. Hefeng 50 had good compatibility with the four isolated strains, while Suinong 14 showed the best compatibility with HLNEAU2. The isolated rhizobacteria could successfully establish symbiosis with the soybeans, but host specificity was also present. This study was a preliminary exploration of the use of salinity-tolerant rhizobacteria to help the soybean nitrogen fixation in saline soils in order to increase the soybean acreage, and it provides a valuable theoretical basis for the application of saline-tolerant rhizobia.

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Nitrogenase reduces N2 to NH3 at its active-site cofactor. Previous studies of an N2-bound Mo-nitrogenase from Azotobacter vinelandii suggest binding of three N2 species via asymmetric belt-sulfur displacements in the two cofactors of its catalytic component (designated Av1*), leading to the proposal of stepwise N2 reduction involving all cofactor belt-sulfur sites; yet, the evidence for the existence of multiple N2 species on Av1* remains elusive. Here we report a study of ATP-independent, EuII/SO3 2--driven turnover of Av1* using GC-MS and frequency-selective pulse NMR techniques. Our data demonstrate incorporation of D2-derived D by Av1* into the products of C2H2- and H+-reduction, and decreased formation of NH3 by Av1* concomitant with the release of N2 under H2; moreover, they reveal a strict dependence of these activities on SO3 2-. These observations point to the presence of distinct N2 species on Av1*, thereby providing strong support for our proposed mechanism of stepwise reduction of N2 via belt-sulfur mobilization.

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Molecular innovations within key metabolisms can have profound impacts on element cycling and ecological distribution. Yet, much of the molecular foundations of early evolved enzymes and metabolisms are unknown. Here, we bring one such mystery to relief by probing the birth and evolution of the G-subunit protein, an integral component of certain members of the nitrogenase family, the only enzymes capable of biological nitrogen fixation. The G-subunit is a Paleoproterozoic-age orphan protein that appears more than 1 billion years after the origin of nitrogenases. We show that the G-subunit arose with novel nitrogenase metal dependence and the ecological expansion of nitrogen-fixing microbes following the transition in environmental metal availabilities and atmospheric oxygenation that began ∼2.5 billion years ago. We identify molecular features that suggest early G-subunit proteins mediated cofactor or protein interactions required for novel metal dependency, priming ancient nitrogenases and their hosts to exploit these newly diversified geochemical environments. We further examined the degree of functional specialization in G-subunit evolution with extant and ancestral homologs using laboratory reconstruction experiments. Our results indicate that permanent recruitment of the orphan protein depended on the prior establishment of conserved molecular features and showcase how contingent evolutionary novelties might shape ecologically important microbial innovations.

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Photosynthetic diazotrophs expressing iron-only (Fe-only) nitrogenase can be developed into a promising biofertilizer, as it is independent on the molybdenum availability in the soil. However, the expression of Fe-only nitrogenase in diazotrophs is repressed by the fixed nitrogen of the soil, limiting the efficiency of nitrogen fixation in farmland with low ammonium concentrations that are inadequate for sustainable crop growth. Here, we succeeded in constitutively expressing the Fe-only nitrogenase even in the presence of ammonium by controlling the transcription of Fe-only nitrogenase gene cluster (anfHDGK) with the transcriptional activator of Mo nitrogenase (NifA*) in several different ways, indicating that the engineered NifA* strains can be used as promising chassis cells for efficient expression of different types of nitrogenases. When applied as a biofertilizer, the engineered Rhodopseudomonas palustris effectively stimulated rice growth, contributing to the reduced use of chemical fertilizer and the development of sustainable agriculture.

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Biological nitrogen fixation by microbial diazotrophs can contribute significantly to nitrogen availability in non-nodulating plant species. In this study of molecular mechanisms and gene expression relating to biological nitrogen fixation, the aerobic nitrogen-fixing endophyte Burkholderia vietnamiensis, strain WPB, isolated from Populus trichocarpa served as a model for endophyte-poplar interactions. Nitrogen-fixing activity was observed to be dynamic on nitrogen-free medium with a subset of colonies growing to form robust, raised globular like structures. Secondary ion mass spectrometry (NanoSIMS) confirmed that N-fixation was uneven within the population. A fluorescent transcriptional reporter (GFP) revealed that the nitrogenase subunit nifH is not uniformly expressed across genetically identical colonies of WPB and that only ~11% of the population was actively expressing the nifH gene. Higher nifH gene expression was observed in clustered cells through monitoring individual bacterial cells using single-molecule fluorescence in situ hybridization. Through 15N2 enrichment, we identified key nitrogenous metabolites and proteins synthesized by WPB and employed targeted metabolomics in active and inactive populations. We cocultivated WPB Pnif-GFP with poplar within a RhizoChip, a synthetic soil habitat, which enabled direct imaging of microbial nifH expression within root epidermal cells. We observed that nifH expression is localized to the root elongation zone where the strain forms a unique physical interaction with the root cells. This work employed comprehensive experimentation to identify novel mechanisms regulating both biological nitrogen fixation and beneficial plant-endophyte interactions.

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Nitrogen is essential for all organisms, but biological nitrogen fixation (BNF) occurs only in a small fraction of prokaryotes. Previous studies divided nitrogenase-gene-carrying prokaryotes into Groups I to IV and provided evidence that BNF first evolved in bacteria. This study constructed a timetree of the evolution of nitrogen-fixation genes and estimated that archaea evolved BNF much later than bacteria and that nitrogen-fixing cyanobacteria evolved later than 1,900 MYA, considerably younger than the previous estimate of 2,200 MYA. Moreover, Groups III and II/I diverged ∼2,280 MYA, after the Kenorland supercontinent breakup (∼2,500-2,100 MYA) and the Great Oxidation Event (∼2,400-2,100 MYA); Groups III and Vnf/Anf diverged ∼2,086 MYA, after the Yarrabubba impact (∼2,229 MYA); and Groups II and I diverged ∼1,920 MYA, after the Vredefort impact (∼2,023 MYA). In summary, this study provided a timescale of BNF events and discussed the possible effects of geological events on BNF evolution.

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A grand challenge for the next century is in facing a changing climate through bioengineering solutions. Biological nitrogen fixation, the globally consequential, nitrogenase-catalyzed reduction of atmospheric nitrogen to bioavailable ammonia, is a vital area of focus. Nitrogen fixation engineering relies upon extensive understanding of underlying genetics in microbial models, including the broadly utilized gammaproteobacterium, Azotobacter vinelandii (A. vinelandii). Here, we report the first CRISPR interference (CRISPRi) system for targeted gene silencing in A. vinelandii that integrates genomically via site-specific transposon insertion. We demonstrate that CRISPRi can repress transcription of an essential nitrogen fixation gene by ~60%. Further, we show that nitrogenase genes are suitably expressed from the transposon insertion site, indicating that CRISPRi and engineered nitrogen fixation genes can be co-integrated for combinatorial studies of gene expression and engineering. Our established CRISPRi system fills an important gap for engineering microbial nitrogen fixation for desired purposes.IMPORTANCEAll life on Earth requires nitrogen to survive. About 78% of the atmosphere alone is nitrogen, yet humans cannot use it directly. Instead, we obtain the nitrogen we need for our survival through the food we eat. For more than 100 years, a substantial portion of agricultural productivity has relied on industrial methods for nitrogen fertilizer synthesis, which consumes significant amounts of nonrenewable energy resources and exacerbates environmental degradation and human-induced climate change. Promising alternatives to these industrial methods rely on engineering the only biological pathway for generating bioaccessible nitrogen: microbial nitrogen fixation. Bioengineering strategies require an extensive understanding of underlying genetics in nitrogen-fixing microbes, but genetic tools for this critical goal remain lacking. The CRISPRi gene silencing system that we report, developed in the broadly utilized nitrogen-fixing bacterial model, Azotobacter vinelandii, is an important step toward elucidating the complexity of nitrogen fixation genetics and enabling their manipulation.

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The novel genus Aquibium that lacks nitrogenase was recently reclassified from the Mesorhizobium genus. The genomes of Aquibium species isolated from water were smaller and had higher GC contents than those of Mesorhizobium species. Six Mesorhizobium species lacking nitrogenase were found to exhibit low similarity in the average nucleotide identity values to the other 24 Mesorhizobium species. Therefore, they were classified as the non-N2-fixing Mesorhizobium lineage (N-ML), an evolutionary intermediate species. The results of our phylogenomic analyses and the loss of Rhizobiales-specific fur/mur indicated that Mesorhizobium species may have evolved from Aquibium species through an ecological transition. Halotolerant and alkali-resistant Aquibium and Mesorhizobium microcysteis belonging to N-ML possessed many tripartite ATP-independent periplasmic transporter and sodium/proton antiporter subunits composed of seven genes (mrpABCDEFG). These genes were not present in the N2-fixing Mesorhizobium lineage (ML), suggesting that genes acquired for adaptation to highly saline and alkaline environments were lost during the evolution of ML as the habitat changed to soil. Land-to-water habitat changes in Aquibium species, close relatives of Mesorhizobium species, could have influenced their genomic evolution by the gain and loss of genes. Our study indicated that lineage-specific evolution could have played a significant role in shaping their genome architecture and conferring their ability to thrive in different habitats.IMPORTANCEPhylogenetic analyses revealed that the Aquibium lineage (AL) and non-N2-fixing Mesorhizobium lineage (N-ML) were monophyletically grouped into distinct clusters separate from the N2-fixing Mesorhizobium lineage (ML). The N-ML, an evolutionary intermediate species having characteristics of both ancestral and descendant species, could provide a genomic snapshot of the genetic changes that occur during adaptation. Genomic analyses of AL, N-ML, and ML revealed that changes in the levels of genes related to transporters, chemotaxis, and nitrogen fixation likely reflect adaptations to different environmental conditions. Our study sheds light on the complex and dynamic nature of the evolution of rhizobia in response to changes in their environment and highlights the crucial role of genomic analysis in understanding these processes.

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