Next Article in Journal
Effect of Ridge–Furrow with Plastic Film Mulching System and Different Nitrogen Fertilization Rates on Lodging Resistance of Spring Maize in Loess Plateau China
Previous Article in Journal
Are Heat Shock Proteins Important in Low-Temperature-Stressed Plants? A Minireview
Previous Article in Special Issue
Effects of Film Mulching on Soil Microbial Diversity and Community Structure in the Maize Root Zone under Drip Irrigation in Northwest China
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Agronomy
Volume 14
Issue 6
10.3390/agronomy14061295
agronomy-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Nodulating Aeschynomene indica without Nod Factor Synthesis Genes: In Silico Analysis of Evolutionary Relationship

by
Mengguang Zhao
1,†,
Jingyi Dong
1,†,
Zhenpeng Zhang
2,
Entao Wang
3,
Dandan Wang
1,
Huijie Xie
1,
Chao Wang
1 and
Zhihong Xie
1,*
1
National Engineering Laboratory for Efficient Utilization of Soil and Fertilizer Resources, Shandong Engineering Research Center of Plant-Microbia Restoration for Saline-Alkali Land, College of Resources and Environment, Shandong Agricultural University, Taian 271000, China
2
Key Laboratory of Coastal Environmental Processes and Ecological Remediation, Yantai Institute of Coastal Zone Research, Chinese Academy of Sciences, Yantai 264003, China
3
Departamento de Microbiología, Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional, Mexico City 11350, Mexico
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Agronomy 2024, 14(6), 1295; https://doi.org/10.3390/agronomy14061295
Submission received: 29 April 2024 / Revised: 11 June 2024 / Accepted: 12 June 2024 / Published: 15 June 2024
(This article belongs to the Special Issue Rhizosphere Microorganisms—2nd Edition)
Browse Figures
Versions Notes

Abstract

:
Aeschynomene indica rhizobia (AIRs) are special classes of bacteria capable of nodulating without nodulation factors and have photosynthetic capacity. With an aim to characterize the structural variations in Bradyrhizobium genomes during its evolution, the genomes of AIRs and the reference Bradyrhizobium strains were compared utilizing molecular biology, bioinformatics, and biochemistry techniques. The presence of symbiotic nitrogen fixation (nif) genes and photosynthetic genes, as well as components of the T3SS (Type III secretion system) and T3CP (Type III chaperone) in the genome of AIRs, was also assessed. Additionally, the origin, evolutionary history, and genes associated with the NF-independent nodulation pattern in AIRs were explored. The results indicate that horizontal gene transfer events have occurred in AIRs, and three distinct origins of AIRs were estimated: early differentiated AIRs, non-symbiotic Bradyrhizobium, and non-AIRs. In contrast to the significant genetic transformations observed in the second and third groups, the first group of AIRs displays a rich evolutionary history, exhibits high species diversity, and primarily relies on vertical transmission of nitrogen fixation and photosynthetic genes. Overall, the findings provide a fundamental theoretical foundation for gaining a deeper understanding of the phylogeny and genealogy of AIRs.

1. Introduction

Aeschynomene indica L. is an annual herb or subshrub within the Leguminosae family that can form both stem and root nodules with rhizobia [1,2]. Based on their differences in nodulation patterns with Bradyrhizobium, Aeschynomene species can be classified into three cross-inoculation (CI) groups [3]: Group CI-I species represented by A. elaphroxylon can only form root nodules with non-photosynthetic and weakly specific Bradyrhizobium strains (like ORS301) [4]. This nodulation process relies on signal exchange between the rhizobia and the hosts. Species in CI-II, like A. afraspera, are capable of nodulating with non-photosynthetic strains (such as ORS354) on roots only and with photosynthetic strains (such as ORS285) on both roots and stems. And CI-III species, like A. indica and A. sensitiva, can nodulate on both roots and stems with photosynthetic strains (such as ORS278). Notably, some CI-II rhizobia have Nod genes and can produce tumorigenic factors, while no strain containing Nod genes has been found in CI-III rhizobia [1]. In association with Aeschynomene in these three CI groups, the symbiotic Bradyrhizobium strains present differences in three aspects: nodulating on roots only or on both roots and stems, photosynthesis or non-photosynthesis, and with/without nod genes. Therefore, the Aeschynomene-nodulating bradyrhizobia should present great variations in chromosome structure or gene composition during their co-evolution with the host plants. A thorough comparative study of genomes of different types of Aeschynomene-nodulating bradyrhizobia might give information about how the rhizobia evolved to nodulate the diverse CI groups of Aeschynomene species.
The photosynthetic ability of Aeschynomene-nodulating bradyrhizobia was believed to be similar to that in cyanobacteria and a number of other bacterial groups [5], while all of the photosynthesis species in Bradyrhizobium have been classified into the photosynthetic supergroup [5,6], corresponding to the dominant symbionts (B. oligotrophicum, B. denitrificans, and B. aeschynomenes) of Aeschynomene species [5,7,8]. Previously, a complex evolutionary history of photosynthesis has been found in Bradyrhizobium strains [9], and Bradyrhizobium sp. BTAi1 was the first strain found to nodulate with A. indica and capable of photosynthesis [10,11,12], which has been studied as a model of photosynthetic bradyrhizobia. This photosynthetic pigment-dependent property of AIRs is similar to that of photoheterotrophs [13,14,15], such as in the purple bacteria of Rhodopseudomonas. In these bacteria, bch and crt genes encode bacterial chlorophylls and carotenoids, respectively, while puf genes encode components of light capture complexes (pufA and pufB genes) and reaction centers (pufL and pufM genes).
Researchers have identified five types of puf operons, differing mainly in the number and arrangement of genes. However, it is noteworthy to mention that pufL and pufM genes are consistently present in all five puf operons. Consequently, the pufLM gene is commonly used as a target gene for photosynthetic centers [16,17]. Subsequently, it was discovered that some rhizobia isolated from stem nodules of A. indica also possessed photosynthetic pigments. Notably, all AIR strains containing photosynthetic pigments were capable of forming stem nodules with A. indica [4]. Furthermore, mutations in the photosynthetic genes reduced the number of nodules by 50% and had a corresponding impact on nif activity and plant growth [16]. So, the photosynthetic ability is also linked to the procedure of symbiosis, but its mechanism is still not clear [9]. It could be expected that a comparison of the genomes between the photosynthetic and non-photosynthetic strains would offer some information about their evolutionary relationships, while the genes mentioned above might be valuable markers for this kind of study.
It is widely accepted that the initiation of symbiosis between legumes and rhizobia relies on nodulation factor (NF) signaling [18]; however, the absence of nod genes in the genomes of two photosynthetic Bradyrhizobium strains (BTAi1 and ORS278) has been demonstrated [19,20,21]. So, the photosynthetic Bradyrhizobium strains presented an NF-independent nodulation mode in which the type III secretion system (T3SS) played a crucial role in symbiosis [22,23,24]. Similar to that of the nodulation genes, activation of T3SS in rhizobia is triggered by flavonoids secreted by the plant root system [25,26]: when rhizobia sense signals such as flavonoids, the T3SS transcriptional regulator TtsI binds to the tts box locus within the T3SS promoter, initiating T3SS activation [27,28]. It has been reported that T3SS-encoding genes are also located in the symbiosis island, similar to the nod genes, which is able to disperse between species via horizontal gene transfer (HGT) [29]. Based on the information mentioned above, it could be estimated that nod gene loss and T3SS gene adaptation might have happened in the photosynthetic Bradyrhizobium since both the gene loss events and HGT are substantial for genome alterations in bacteria [30,31]. Moreover, it is indispensable to gain symbiotic genes through HGT [32].
AIRs are a unique group of rhizobia that can form nodules with their host plants without the need for nodulation factors. Additionally, they also possess the ability to photosynthesize. This research study has a research hypothesis that during the long and complex evolutionary process, the functional genomes of Bradyrhizobium, such as nitrogen fixation genes, photosynthesis genes, and T3SS genes, must have undergone great changes, which makes AIRs form a separate branch that differs significantly from other Bradyrhizobium traits in the phylogenetic tree [9]. The purpose of this research is to figure out the changes that occurred in the genome of AIRs, investigate the diverse origins and evolutionary history of AIRs, and establish a solid theoretical basis for further understanding the phylogeny and genealogy of AIRs; the genomes of AIRs and the reference strains of Bradyrhizobium were compared in the present study.

2. Materials and Methods

2.1. Selection of Test Strains

The complete genome map of strain B. oligotrophicum S58T was readily accessible in the GenBank database. In this study, we conducted second-generation sequencing on 19 representative strains of AIRs and on B. denitrificans LMG8443T. The genome sequences were obtained from GenBank, serving as a reference for our comparative genomic analysis with the genome of AIRs. Based on their symbiotic characteristics, Bradyrhizobium strains were categorized into three groups: AIRs nodulating with A. indica (including CI-I, CI-II, and CI-III types); non-AIRs associating with legumes other than A. indica; and non-symbiotic Bradyrhizobium lacking the ability to nodulate. The genomes utilized in this study are detailed in Table S1.

2.2. Genome Sequencing, Splicing, Optimization

To harvest the biomass, each of the bacterial solutions was centrifuged in a cryogenic centrifuge at 4 °C and 8000 rpm. The collected pellet was frozen and dispatched to Shanghai Meiji Biomedical Technology Co company (Shanghai, China) for comprehensive sequencing. The gene sketches were sequenced on the Illumina (San Diego, CA, USA) second-generation sequencing platform at a depth of 100×. Subsequently, the genome sketches were assembled using SPAdes software (v3.15.5) [33]. Pilon software (v1.23) [34] was employed to refine the genome based on the second-generation sequencing data. Through a comparison of Illumina sequencing data with the genome, Pilon software (v1.23) was employed to generate a BAM file and a genomic FASTA file as input. To assess the genetic relatedness, Average Nucleotide Identity (ANI) values were computed using OrthoANI software (http://www.ezbiocloud.net/sw/oat) [35]. This involved pairwise comparisons of genomes, and the results were aggregated to form an ANI matrix. For constructing species trees, OrthoFinder2 software was utilized [36]. The tree file was opened using FigTree software (v1.4.3), and the results were subsequently optimized with iTOL software (v5) [37].

2.3. Genome Prediction, Annotation

For predicting genomic functional genes, we employed Prodigal software (v2.6.3) [38]. To annotate the functionality of these genes, we utilized DIAMOND software (v4.6.3) [39]. This annotation process involved referencing the Nr database and the Swiss-Prot database [40,41]. For the analysis of homologous gene families, we turned to OrthoFinder software (v2.0.0).

2.4. Analysis of Functional Gene Components

A comprehensive analysis of the functional genes related to the symbiotic process was conducted in AIRs. The genes implicated in symbiosis encompass symbiotic genes, nif genes, T3SS genes, T3SS effectors, and photosynthetic genes. Utilizing the BlastGUI software (https://github.com/byemaxx/BlastGUI) [35], gene comparisons were performed to construct a library. Subsequently, the results were extracted and organized using a custom Python (v3.10.8) script, allowing us to create a comparative heat map. To conduct covariance analysis of the nif gene clusters, genome annotation was carried out for B. oligotrophicum S58. Subsequently, the locations of nif gene clusters in the annotation file were identified by extracting these clusters from the genomic species of B. oligotrophicum S58 for constructing a database. All strains were screened with BlastGUI [35] to identify the existence of nif gene clusters. Following the results of the BLAST comparison, appropriate strains harboring the nif gene clusters were selected for creating a circos map to visualize and analyze the differences in nif gene clusters among the selected strains, taking B. oligotrophicum S58 as reference. Using a similar methodology, covariance analysis of the photosynthetic gene clusters was also conducted. This allowed us to generate a comparative genomic analysis of representative strains of AIRs, with the genome of B. oligotrophicum S58T serving as the reference. The findings were then presented using Galactic Circos (usegalaxy.eu, accessed on 26 April 2024) for clarity [42].

2.5. Evolutionary Analysis of AIRs

The evolutionary history of AIRs was explored through an analysis involving the species tree and statistical assessments of homologous gene families within the reference strains of AIRs and Bradyrhizobium. This analysis was executed using the Count software (http://www.iro.umontreal.ca/~csuros/gene_content/count.html). Subsequently, we utilized Count to analyze the differentiation of AIRs into three specific nodes. Specifically, we identified gene families in which acquisition and loss events occurred at these nodes. Subsequently, these gene families were subjected to KEGG (Kyoto Encyclopedia of Genes and Genomes) analysis to determine if they were enriched in specific metabolic pathways [43].

3. Results and Discussion

3.1. Genome Profile

The sketch of the genome of AIRs was sequenced, and the genome profile is shown in Table 1. The genome size of AIRs ranged from 6.63 M in the case of Bradyrhizobium sp. VII 81001 to 8.84 M in the case of B. denitrificans 80005. The GC content of the genomes ranged from 64.55% for Bradyrhizobium sp. I 82044 to 65.78% Bradyrhizobium sp. IV 81033.
The large variation in genome size of AIRs, which is consistent with previous studies [44,45,46,47,48,49,50,51,52], suggests a long evolutionary history, leading to divergence in genome size. Variations in mean genome size were observed among different classes of Bradyrhizobium. The average genome size is 7.6 M for AIRs, 8.7 M for non-AIRs, and the average genome size is 8.0 M for non-symbiotic Bradyrhizobium, which might be related to distinct selection pressures from their hosts and environments on these three classes of rhizobia [53].

3.2. Analysis of the Origin of AIRs

Orthologous gene family (orthogroup) analysis revealed that a total of 421,466 genes were identified in the genomes of 56 tested Bradyrhizobium strains, which were organized into 17,858 gene families. Out of all the genes, 408,370 (96.9%) were assigned to these gene families, while 13,096 genes (3.1%) were found in only one strain. Additionally, there were 36 specific gene families encompassing 95 genes (less than 0.1%). Furthermore, 2074 orthogroups presented in all the 56 Bradyrhizobium strains, and 1098 of these were single-copy gene families.
A species tree was constructed based on 56 homologous gene families of AIRs (Figure 1). In Figure 1, the tested strains in Bradyrhizobium were clearly divided into three branches, with AIRs located in two of them. The 20 strains in Cluster I were all able to nodulate with Indian AIRs. In this cluster, Bradyrhizobium sp. ORS285 is a CI-II type photosynthetic strain, which could form root and stem nodules with Aeschynomene species via NF-dependent and NF-independent mechanisms [54]. No AIR strain was included in Cluster II, which consisted mainly of non-symbiotic Bradyrhizobium strains and Bradyrhizobium strains that nodulate with other legumes. Four strains of AIRs were grouped in Cluster III, in which Bradyrhizobium sp. I 82044, 82054, and Bradyrhizobium sp. II 80013 were isolated for this study, while Bradyrhizobium sp. DOA9 was a CI-I type strain nodulating A. indica but originally isolated from America and could fix nitrogen in free living conditions [54]. In Cluster III, Bradyrhizobium sp. I 82044, 82054, and Bradyrhizobium sp. II 80013 are clustered in a small branch together with B. liaoningense and B. yuanmingense. The hosts of B. liaoningense include peanut, a legume species infected by bradyrhizobia via root cracks. Furthermore, B. liaoningense also shared the same geographic distribution with the three isolates of AIRs in this study. The close kinship between the hosts, the consistent infestation pattern in both rhizobia nodulation processes, and the similar geographic locations suggest that Bradyrhizobium sp. I and B. liaoningense share a common ancestor, with the two Bradyrhizobium strains differentiating later throughout their subsequent long-term and complex evolutionary history, as reported in other studies [55,56,57]. The original host of Bradyrhizobium sp. DOA9 and B. yuanmingense were close relatives in Figure 1, and their symbiotic nodulation relied on the exchange of nodulation factors. In cluster III, Bradyrhizobium sp.II 80013 presented a close relationship with non-symbiotic Bradyrhizobium strain Bradyrhizobium sp. S23321 isolated from the rhizosphere of rice. Moreover, B. betae PL7HG1 (isolated from the beetroot sphere) [58], Bradyrhizobium sp. YR681, and Bradyrhizobium sp. OK095 (isolated from the poplar rhizosphere) [59] also have very similar phylogenetic positions to 80013 (Figure 1), indicating a possible origin of Bradyrhizobium sp. II 80013 from non-symbiotic Bradyrhizobium.
Based on the phylogenetic relationships in Figure 1, it is speculated that AIRs can be categorized into three origin modes. The first mode involves AIRs originating from the early differentiation of Bradyrhizobium species (cluster I), which has a long evolutionary history and has accumulated great genetic variations, resulting in a high level of diversity. This is evidenced by the significant differences in genome size and GC content, as well as the formation of cluster I as a separate and highly differentiated branch in the species tree (Figure 1) of housekeeping genes. The second group covers the strains Bradyrhizobium sp. DOA9 and Bradyrhizobium sp. I 82044 and 82054, which originated relatively later from species of non-AIRs and might share an ancestor with the peanut rhizobia since the nodulation mode of all rhizobia with peanut is fissure infection [60], which is conducive to the mutual transformation of their symbiotic bacteria. The third group, represented by Bradyrhizobium sp. 80013, might have originated from non-symbiotic Bradyrhizobium in the soil and cannot synthesize nodulation factors, but some strains have photosynthetic ability. The above traits overlap with AIRs and can be easily selected and differentiated into AIRs by nodulating A. indica. The low diversity of the second and third groups suggests that they have evolved recently.

3.3. Comparative Genome Analysis of AIRs

The different origins of AIRs have necessarily undergone distinct evolutionary histories, potentially resulting in significant differences in their genome components. To investigate these differences, a comparative genomic analysis was conducted on three different origins of AIRs.
Bradyrhizobium sp. VIII 83002 belongs to the branch of Cluster I, which exhibits early formation and high diversity. AIRs within this branch appear to share similarities due to the selection pressure of the host and, as a result, correspond closely to the genomic components of 83002, characterized by the presence of a large number of contiguous fragments. Bradyrhizobium sp. III 80001 has the smallest genome, but it has more gaps than 83002. Both 80001 and Bradyrhizobium sp. ORS285 are clustered in the same branch on the species tree, and the original host of ORS285 was the CI-II type of A. indica, which contains the Nod gene. Therefore, 80001 likely originated from the CI-II type of A. indica and underwent a large number of gene loss events to adapt to an NF-independent nodulation mode. Bradyrhizobium sp. VIII 83002 has differentiated significantly from non-A. indica rhizobia (B. yuanmingense CCBAU10071), non-symbiotic Bradyrhizobium (B. betae PL7HG1, Bradyrhizobium sp. S23321), and Cluster III-branched AIRs (Bradyrhizobium sp. I 82044, Bradyrhizobium sp. II 80013), with substantial differences in genomic components and poor genomic covariance. Genome 83002 has several regions with low GC content (indicated by black arrows in the figure), and gaps exist in other strains, suggesting that HGT has occurred in these regions (Figure 2A). Bradyrhizobium sp. II 80013 is a strain of AIRs in the Cluster III branch, forming a small sub-branch alongside non-symbiotic Bradyrhizobium in the species tree. The 80013 genome is characterized by the presence of numerous significant endemic segments (indicated by black arrows in the figure), and the GC content of these regions suggests that horizontal gene transfer has occurred, along with significant changes in the genome structure during the transformation of 80013 into AIRs (Figure 2B). Bradyrhizobium sp. I 82044 is also identified as an AIR within Cluster III. According to the Bradyrhizobium species tree, it is assumed that this strain originated from the transformation of a non-symbiotic Bradyrhizobium. The genome of Bradyrhizobium sp. I 82044 also contains regions with a low GC content (indicated by black arrows in the figure). However, these regions are not unique to 82044, suggesting that they may have originated from AIRs. The average genome size of non-AIRs is 8.7 M, while that of 82044 is only 6.9 M. This size difference suggests that the transformation of 82044 into A. indica was accompanied by the loss of a significant number of genomic fragments. These fragments may include genes associated with the host symbiosis of 82044’s ancestors, aligning with the possible origin of 82044 from non-AIRs (Figure 2C).
HGT occurs in several regions of the genome of AIRs. There are three main forms of HGT [61]. First is transformation, in which AIRs take up naked DNA fragments directly from the environment, integrate them into their own genome, and perform their functions. However, the low concentration of DNA fragments in the environment makes the transformation process difficult to observe, and it can only be deduced backwards from phylogenetic trees or genomic information. Second, conjugative transfer occurs [62,63,64], which is the process of DNA transfer between the donor and the recipient bacterium through contact. The DNA fragments are recombined at the att site (usually one of the loci is adjacent to phe-tRNA) by the action of integrase and transferred via T4SS [63]. This process requires a high structural basis for the genome, and the analysis of the genome of A. indica in this study did not reveal any relevant structures at either end of the low GC fragment. Furthermore, the absence of the T4SS-encoding gene in AIRs does not support DNA translocation. Third, phage-mediated horizontal gene transfer [65,66,67] occurs, in which phages take the genomic DNA of the former host and integrate it into the next host when they infest between different bacteria. The presence of prophage integrase in the genome of AIRs suggests that phages play a role in the horizontal gene transfer of AIRs.

3.4. Analysis of Symbiotic Nitrogen Fixation Gene Components

Nod genes encode enzymes for nodulation factor (NF) synthesis, and rhizobia with nodulation capacity generally have the nodABC genes for synthesis of the main structure of NF, as well as a number of genes for modifying the NF. Most of the AIR strains in clusters I and II, except Bradyrhizobium sp. DOA9 and Bradyrhizobium sp. ORS 285, only harbored nodN and nodQ, while several of them also harbored nodM and nodP1/nodP2 but were absent for the regulation and constructure nod genes (Figure 3). A similar situation was observed in the 12 non-symbiotic Bradyrhizobium strains. Among the AIR strains, Bradyrhizobium sp. DOA9 and Bradyrhizobium sp. ORS 285, which nodulate with CI-I and CI-II types of Aeschynomene species, respectively, contain all the essential conserved genes for nodulation factor synthesis in their genomes, demonstrating they are symbionts with an NF-dependent strategy, which is similar to the Bradyrhizobium strains nodulating with other legume species (Figure 3). It was interesting that all the 56 tested Bradyrhizobium strains in this study contained nodN and nodQ genes, demonstrating that they may also play some essential role in a pathway(s) without relation to symbiosis. For example, nodM and nodN genes present common functions as glucosamine synthase, playing a crucial role in the biosynthesis of hexosamine [68], while nodO gene encodes a hemolysin homolog [69].
The nif (Nif) genes were more widely distributed than Nod genes in A. indica-nodulating bradyrhizobia. As shown in Figure 4, nifHDKNEX genes were harbored in the genomes of all the tested symbiotic Bradyrhizobium strains except Bradyrhizobium sp. VII 81001, suggesting that all of these gene strains possessed the potential of nif. Our previous study has indicated that mutations contribute more to evolution than recombination [1]. Strain 81001 has a genome size of only 6.1 M, which is the smallest among A. indica-nodulating strains, suggesting that this strain has undergone a large number of gene loss events during its evolutionary history, and nif genes were lost at some point (Figure 4). Nitrogen fixation is a very energy-intensive biological process [70,71], and the ability of rhizobia to fix nitrogen does not contribute significantly to the environmental adaptability and competitiveness of Bradyrhizobium when they are saprophytic in soil; as a result, most non-symbiotic strains do not have Nif genes in their genomes. Bradyrhizobium sp. S23321 and Bradyrhizobium sp. AT1 were found to have a more complete cluster of nitrogen fixation genes [72], suggesting that the ancestors of these two strains may have had the ability to have symbiosis with legumes.
In most cases, Nif genes presented a co-evolutionary tendency with nod genes, and host specificity was observed since Nif genes from the same host can often transcend phylogenetic boundaries and cluster in the same branch. Among the nif gene clusters of AIRs, nifDKNEX is a conserved nif gene operon and is consistently arranged in the same order in different bacteria. In this study, a phylogenetic tree was constructed for NifDKNEX gene clusters to analyze their evolution (Figure 5). B. oligotrophicum S58T has been sequenced, and the nif gene Cluster I is located in the region spanning 2584552′ to 2632812′, with a total length of 48,270 bp. This strain was used as a reference for a comparative analysis of the nif genomic score of Bradyrhizobium. The phylogenetic tree depicts the nifDKNEX gene clustering into three distinct branches, consistent with that defined in Figure 1. AIRs within Branch I covered all strains in Cluster I of Figure 1 and separated from the other Bradyrhizobium species, suggesting a common origin for the Nif genes in this branch, with an evolutionary history distinguished from the other Bradyrhizobium species. It further supports the idea that Bradyrhizobium within Branch I has a unique origin. Additionally, nif capacity is a crucial component of the mutually beneficial symbiotic system. The nif gene experiences more significant host selection pressure than even the nodulation gene. Within the Branch I, the nifDKNEX branch appears to be more diverse and abundant, indicating a longer evolutionary history for this particular branch. Bradyrhizobium sp. I 82044, 82054, Bradyrhizobium sp. II 80013, and Bradyrhizobium sp. DOA9 harbor the nifDKNEX genes distinct from that of AIRs in the branch of Cluster I but more similar with that of B. guangxiense, suggesting that their nifDKNEX genes have origins different from those in Cluster I. The nifDKNEX genes on the chromosome of B. guangxiense presented closer phylogenetic relationships to those of Bradyrhizobium sp. I 82044, 82054, and Bradyrhizobium sp. DOA9, suggesting that they might have the same origin. A previous study evidenced that B. guangxiense can cross-nodulate with Indian A. indica, further demonstrating that non-AIRs could be transformed into AIRs. The phylogenetic tree based on the NifDKNEX genes shows that B. liangningense is distantly related to Bradyrhizobium sp. I 82044, 82054, and Bradyrhizobium sp. DOA9. Instead, they were clustered together with other Bradyrhizobium strains that share the same host, suggesting the existence of HGT of nif genes in B. liangningense. In this study, the nif gene cluster was only found in Bradyrhizobium sp. S23321 and Bradyrhizobium sp. AT1, both of which clustered in the same branch as Bradyrhizobium sp. II 80013 in the phylogenetic tree. This finding further demonstrates that Bradyrhizobium sp. II 80013 is more closely related to non-symbiotic Bradyrhizobium. The position of AIRs in the nifDKNEX phylogenetic tree closely corresponds to the species tree (Figure 5). AIR strains of the same genetic species cluster in the same branch, indicating that the nif genes of AIRs are primarily transmitted vertically, which is highly in accordance with previous research [6].
The B. oligotrophicum S58T nif gene cluster is 48,270 bp in length, with an average GC content of 64.11%, ranging from 47% to 80% GC per 100 bp [12]. The gene cluster contains 64 genes encoding nif molybdenum iron proteins, nif-related transporter proteins, and regulatory proteins. The S58 nif gene Cluster I is almost identical to the component of AIRs of Cluster I. The strains in the branch of Cluster III differ from S58 in their nif gene cluster components. The nif gene cluster components of Bradyrhizobium sp. I 80013 (transformation origin of non-AIRs) and Bradyrhizobium sp. II 82044 and 82054 (origin of non-symbiotic Bradyrhizobium) are consistent, indicating that both types of AIRs have the same origin of nif genes. Compared to strain S58, only 17.5% of the nif gene clusters in B. japonicum USDA 6 exhibit consistency, implying greater divergence in nif gene clusters among Bradyrhizobium in association with different hosts. B. betae PL7HG1, isolated from sugar beet roots [58], contains a minimal number of nitrogen-fixing genes and lacks nitrogen-fixing capacity, with just 4.7% of its genetic sequence aligning with the nif gene cluster of strain S58. The high collinearity observed in the nif gene clusters among closely related strains suggests that these nif genes in AIRs primarily undergo vertical transmission (Figure 6).
It is evident that nitrogen fixation genes (nif genes) are highly conserved in AIRs and show high covariance among different strains, which suggests that these genes are mainly transmitted through vertical rather than horizontal gene transfer (HGT). However, HGT still plays a role in AIRs. This is important for understanding the evolution of AIRs, as it indicates the strong pressure of host selection on these genes during symbiosis. In addition, our study showed that the nif gene clusters of different strains exhibited some differences in composition and arrangement, which may be related to the ecological environment and biological characteristics of different host plants. Although some strains lost some of their nif genes during evolution, the majority of these gene clusters retained and maintained their functions.

3.5. Analysis of Photosynthetic Gene Components

Bacterial chlorophylls, carotenoids, and photoreaction centers are the structural basis for photosynthesis in Bradyrhizobium [73]. In this study, we analyzed the components of thirteen bacterial chlorophyll-encoding genes, six carotenoid-encoding genes, and nine photoreaction center-encoding genes in Bradyrhizobium (Figure 6), which were extracted from the genomes. Among the 16 strains of AIRs in Cluster I, only Bradyrhizobium sp. VI 80004 lacked a complete set of photosynthetic genes in its genome. In Cluster III, the genomes of Bradyrhizobium sp. I 82044 and 82054 contained partial bacterial chlorophyll (bch)- and carotenoid (crt)-coding genes but lacked photoreaction center (puf)-coding genes. Conversely, the genome of Bradyrhizobium sp. II 80013 included the complete photosynthetic gene set, consistent with the PCR amplification results of the pufLM gene. The photosynthetic genome component of non-AIRs exhibited high similarity to strains 82044 and 82054, indicating a close phylogenetic relationship between these groups of Bradyrhizobium. This similarity further supports the notion that non-AIRs may represent one of the origins of AIRs. Notably, as demonstrated in a previous study [3], similar results were obtained for the two strains (B. betae PL7HG1 and Bradyrhizobium sp. S23321) that are most closely related to 80013 in the species tree and contain photosynthetic genes in their genomes [58,72]. This suggests that although the photosynthetic genes of 80013 originated from non-symbiotic Bradyrhizobium, they have undergone more substantial modifications in subsequent evolutionary history (Figure 7).
In the genome of AIRs, the composition and arrangement of genomes within the photosynthetic gene cluster vary significantly among strains. To depict the phylogenetic relationships of photosynthetic genes in different strains, chlorophyll-coding genes (bchF, bchG, bchN), carotenoid-coding genes (crt I, crtB, crtC), and photoreaction center-coding genes (pufA, pufB, pufL) were extracted from the genomes of AIRs and reference strains. The sequences of these genes were then concatenated to construct a phylogenetic tree (Figure 8). In Figure 8, Rhodopseudomonas, the oldest photosynthetic bacterium on the Earth, is positioned on the outermost branch, suggesting that the photosynthetic capacity of AIRs may have a common origin with Rhodopseudomonas. B. betae PL7HG1 and Bradyrhizobium sp. S23321 are located between Rhodopseudomonas and AIRs but closer to Rhodopseudomonas, suggesting that non-symbiotic Bradyrhizobium strains might be the first within the Bradyrhizobium genus to acquire photosynthetic capabilities. Previous studies have demonstrated that the photosynthetic genes of Bradyrhizobium were obtained through multiple HGTs [6,9]. Therefore, the initial photosynthetic capacity of AIRs likely originated from the common ancestor with Rhodopseudomonas and was subsequently acquired by non-symbiotic Bradyrhizobium strains. In Figure 8, strain 80013 is positioned at the outermost part of AIRs and exhibits significant differences from non-symbiotic Bradyrhizobium strains, suggesting that strain 80013 acquired photosynthetic genes earlier and accumulated a substantial amount of genetic variation throughout its evolutionary history. With the exception of Bradyrhizobium sp. VI 80004, all genomes of AIRs within the Cluster branch exhibit the presence of photosynthetic genes. These genes also display a high level of diversity, indicating their existence during the formation of the common ancestral species of AIRs within this group and their subsequent preservation throughout evolutionary history. It is possible that Bradyrhizobium sp. VI 80004 experienced deletions of photosynthetic genes during its own evolutionary trajectory. Interestingly, the phylogenetic tree of photosynthetic genes in Bradyrhizobium sp. VI 80004 closely aligns with the species tree, suggesting that photosynthetic genes are primarily transmitted vertically within AIRs, and no HGT events have been detected (Figure 8).
The photosynthetic gene cluster of B. oligotrophicum S58T is mainly concentrated in a 59,571 bp fragment with an average GC content of 66.77% and GC content between 45% and 80% per 100 bp [12]. The gene cluster contains 53 genes, most of which encode for the synthesis of bacterial chlorophylls, carotenoids, and photoreaction centers. There are also some genes of unknown function in the gene cluster, and whether these genes are related to photosynthesis still needs further study. The results of comparative analysis of symbiotic and non-symbiotic photosynthetic rhizobial strains, as well as the phototrophic gene cluster components of the genus Bradyrhizobium and Rhodopseudomonas, indicate the phototrophic gene cluster of strain S58 is identical to that of its counterpart strain 80025, which shows a higher degree of collinearity with the components of Cluster I of symbiotic Bradyrhizobium, suggesting a shared origin of phototrophic genes in these strains. However, among the genomes studied, only Bradyrhizobium sp. II 80013 contains photosynthetic gene clusters in Cluster II. These components differ more significantly from that of S58, displaying more gaps between them. The proportion of congruent components between the two clusters is approximately 76.29%. The nif gene cluster in the non-symbiotic strains B. betae PL7HG1 and Bradyrhizobium sp. S23321, as well as Rhodopseudomonas palustris BisA53 and BisB18, exhibit more and larger gaps with that of S58. These gaps correspond to consistency components of 48.99%, 41.53%, 47.76%, and 48.99%, respectively. The higher collinearity observed in the photosynthetic gene clusters of strains with closer phylogenetic relationships suggests that the transmission of photosynthetic genes in AIRs is predominantly vertical. Additionally, the presence of a significant number of gaps in the photosynthetic gene cluster of Bradyrhizobium sp. VI 80004 indicates substantial gene losses in this particular strain (Figure 9).
The photosynthetic gene cluster in AIRs contains genes encoding bacterial chlorophylls, carotenoids, and photoreaction centers. There were also significant differences in the distribution and arrangement of these genes in different strains, indicating events of acquisition and loss of these genes in different evolutionary pathways. In particular, strains such as Bradyrhizobium sp. II 80013 may have acquired photosynthetic genes earlier in their evolutionary history and accumulated a large amount of genetic variation during subsequent evolution. In addition, the transmission of photosynthetic genes in AIRs is mainly vertical, which is similar to the mode of transmission of nif genes.

3.6. Analysis of T3SS Components

A widespread feature in rhizobia is the presence of T3SS, which consists of a core component (rhizobium conserved, rch) and effector proteins (Nops). These effector proteins, secreted by the T3SS, play a crucial role in modulating the extent of rhizobial nodulation. Analysis of the composition of 11 genes encoding T3SS in Bradyrhizobium (Figure 10) revealed that the genes responsible for encoding the T3SS structure were commonly found across the Bradyrhizobium strains. Among the non-AIR strains, all 11 components were almost completely present. In contrast, among the 24 AIRs, only 9 strains had the complete set of genes encoding the T3SS structure, while the remaining strains primarily lacked the rhcC1, rhcL, and rhcS genes. In addition to the genomes of non-symbiotic Bradyrhizobium, the rhcC1, rhcL, and rhcS genes were also commonly absent. Furthermore, the rhcT gene was frequently missing as well. The similarity between the T3SS structure-encoding genes of AIRs and the reference strains displayed substantial variation. Sequence similarity ranged between 70% and 100% when comparing the T3SS structure-encoding genes of the reference strains with those in the T3SS database. Among the AIRs, Bradyrhizobium sp. DOA9 (CI-I type) and Bradyrhizobium ORS285 (CI-II type) exhibited high sequence similarity in the T3SS genes, which is consistent with previous research [74]. However, the similarities among the other strains were generally below 30%. The observed differences between the T3SS of AIRs and the reference strains suggest that there are structural variations in T3SS among them (Figure 10).
T3SS plays a crucial role in modulating the physiological state of plants through the secretion of effector proteins, which subsequently impact the extent of nodulation. This study also performed an analysis of 23 effector proteins in Bradyrhizobium (Figure 11), and the findings imply that in contrast to the T3SS structural genes, the effector proteins displayed substantial variation across different Bradyrhizobium taxa. The genome of non-AIRs contains numerous genes encoding effector proteins, and the repertoire of these proteins differs between strains. These variations in effector proteins are an important contributing factor to the host-specific differences observed among the strains. The lower levels of effector proteins in non-symbiotic Bradyrhizobium are related to its lifestyle. The genomes of Bradyrhizobium sp. DOA9 and Bradyrhizobium sp. ORS285 had a high number of effector proteins, while the rest of the genomes of Bradyrhizobium species had a low number of genes encoding effector proteins, and nopM, nopU, nopV, and nopY genes were present in the genomes of some strains. Albin experimented with the ernA gene mutant of Bradyrhizobium sp. ORS3257 (whose original host is cowpea) on nodulation with AIRs, and the mutant lost its ability to nodulate, indicating that the effector protein encoded by the ernA gene is important for the nodulation of AIRs [22]. We discovered that only the genome of Bradyrhizobium sp. ORS285 contained the ernA gene in AIRs. Additionally, a homolog of ernA was also found in the DOA9 genome, which is known to have a high number of effector proteins. However, no homolog of ernA was identified in the Indian AIRs. These findings suggest that the ernA gene plays a crucial role in the nodulation process of CI-I and CI-II types of AIRs but has little influence on the nodulation of CI-III type (Figure 11).
Recent studies have shown that the role of T3SS in symbiosis goes beyond the regulation of plant immune responses [75] and plays an important role in the NF-independent nodulation mode [74]. Bradyrhizobium elkanii USDA61 can nodulate symbiotically with soybean nfr mutants (which do not have the sensing ability of nodulation factor), whereas T3SS mutants cannot nodulate with them. Transcriptional analysis revealed increased expression of the soybean nodulation-specific genes ENOD40 and NIN in roots of soybean plants inoculated with B. elkanii but not with the T3SS mutant, suggesting that T3SS activates the host nodulation signaling pathway by bypassing NF recognition [76]. T3SS structure does not appear to be a major influence on NF-independent nodal patterns, with Bradyrhizobium sp. ORS278 and BTAi1 able to nodulate with Aeschynomene indica in the absence of T3SS [19].

3.7. Analysis of the Genetic Evolution of Bradyrhizobium

Prokaryotic genomes are highly flexible, accompanied by the acquisition and loss of a large number of gene families in their evolutionary history [77]. To gain insight into the changes in the composition and content of gene families in the evolutionary history of AIRs, this study analyzed AIRs and reference strains of Bradyrhizobium and counted the gains and losses of gene families at different nodes in the species tree.
The results (Figure 12, Table S2) indicate that the evolutionary history of Bradyrhizobium was characterized by significant gene acquisition events. Analysis of the species tree and gene gains and losses revealed that the common ancestor genome of Bradyrhizobium initially possessed 3692 gene families. Subsequently, one lineage acquired 1072 gene families, leading to the evolution of the ancestor of AIRs. Another lineage acquired 310 gene families, resulting in the diversification of Bradyrhizobium and the emergence of non-symbiotic Bradyrhizobium strains. These findings highlight the importance of gene acquisition events in shaping the adaptive evolution of Bradyrhizobium. In Cluster I of the species tree, Bradyrhizobium sp. VII 81001 experienced a substantial loss of gene families during the species formation phase. A total of 1190 gene families were lost, which is the highest number observed among the 56 Bradyrhizobium strains. This loss of gene families also resulted in a significantly smaller genome size of 6.1 M compared to other strains. On the other hand, all Bradyrhizobium strains in Cluster II were found to be symbiotic, and no significant gene family losses were observed in this cluster. This suggests that Bradyrhizobium strains in Cluster II have been actively acquiring new gene families throughout their evolutionary history to adapt to different host selections. Cluster III of the species tree branch consists of a diverse group of Bradyrhizobium strains, including non-AIRs (such as B. gunagxiense, B. ottawaense, etc.), non-symbiotic Bradyrhizobium (e.g., B. betae, Bradyrhizobium sp. S23321, etc.), and AIRs (e.g., Bradyrhizobium sp. I, Bradyrhizobium sp. II, etc.). Similar to Cluster II, non-AIR strains have a history characterized by the acquisition of new gene families. Additionally, the presence of gene family losses in the evolutionary history of AIRs suggests that these host plants have selective preferences for certain rhizobial genomes. Consequently, Bradyrhizobium strains must undergo the loss of some functional genes to establish symbiosis with A. indica. This observation aligns with the absence of genes encoding nodulation factors in AIRs (Figure 12).
Gene loss can enable bacteria to adapt to specific niches [78]. For example, the loss of unnecessary metabolic genes can reduce energy consumption [79], thereby improving environmental adaptability and competitiveness. The newly obtained genes may give bacteria new functions [80], such as improving the ability to adapt to host plants. Throughout the whole evolution process, the loss of the gene family of AIRs was the most obvious, which indicated that Bradyrhizobium continuously lost genes during the symbiosis with AIRs, thus adapting and colonizing the host rhizosphere. In addition, the proportion of acquired and lost gene families during the evolution of Bradyrhizobium varies at different nodes, which may be related to the ecological environment and biological characteristics of different species.

3.8. Analysis of Differential Genes in Bradyrhizobium

The considerable phylogenetic distance observed between AIRs and the reference Bradyrhizobium strains (non-AIRs and non-symbiotic Bradyrhizobium), along with the patterns of gene acquisition and loss, strongly suggest that the genomes of both types of rhizobia have undergone extensive changes throughout their evolutionary history (Figure 3, Figure 4, Figure 6, Figure 7, Figure 9, Figure 10 and Figure 11). While rhizobia can live in the soil as saprophytes and engage in HGT with one another, host selection exerts a directed influence, imposing stringent requirements on the genomic background of rhizobia. Consequently, over long periods of selection, the genome of AIRs is expected to retain specific genes that facilitate symbiosis with A. indica due to the specific demands of the host and the symbiotic relationship between them.
Based on these hypotheses, we extracted genes from different gene families of AIRs and the reference Bradyrhizobium strains and analyzed the KEGG pathway for these genes to investigate the genomic background that may contribute to the formation of AIR ancestry and the NF-independent nodulation pattern. In total, we identified 1877 gene families unique to the 24 strains of AIRs and 5211 gene families unique to the 32 reference strains of Bradyrhizobium. The results revealed that the gene families specific to AIRs were enriched in 159 metabolic pathways, while the gene families specific to the reference strains of Bradyrhizobium were enriched in 221 metabolic pathways. Meanwhile, 143 metabolic pathways that overlapped between the two taxa were identified (Table S3). The specific pathways enriched in AIRs were associated with bacterial invasion of epithelial cells, plant-pathogen interactions, Nod-like receptor signaling pathways, protein transport, photosynthesis, and carotenoid synthesis. These findings align with the trait of AIRs being photosynthetic [1,3,4,7,81] and adopting fissure infection [7]. The presence of the Nod-like receptor signaling pathway in AIRs suggests the existence of a system for sensing plant secretions, similar to NF-dependent types of rhizobia. On the other hand, the enrichment of metabolic pathways in the reference strains of Bradyrhizobium indicated the presence of pathways related to bacterial secretion systems, biofilm synthesis, and group-sensing responses. Additionally, this group of strains exhibited a high enrichment of disease-associated pathways, which could potentially be attributed to HGT events between non-symbiotic Bradyrhizobium and pathogenic bacteria present in the environment. This transfer might have resulted in the acquisition of accessory genes associated with disease pathways. Alternatively, it is possible that certain genes within Bradyrhizobia have functional similarities to proteins or enzymes involved in these disease pathways (Table 2).
Specifically, genes involved in carotenoid synthesis encode phytoene synthase, phytoene desaturase, lycopene-β-cyclase, and ε-ring hydroxylase [82]. Phytoene synthase catalyzes the first committed step in the carotenoid biosynthesis pathway and is a major rate-limiting enzyme of carotenogenesis [83]. Phytoene desaturase and ε-ring hydroxylase are essential plant carotenoid biosynthetic enzymes [84,85]. Lycopene-β-cyclases are key enzymes located at the branch point of the carotenoid biosynthesis pathway [86]. The functions of these proteins may be related to the photosynthetic ability of AIRs.

4. Conclusions

The NF-independent nodulation mechanism of A. indica currently lacks a universally accepted explanation; in this study, a comprehensive analysis of the acquisition and loss of gene families of AIRs and reference strains, followed by KEGG annotation of the gene families specific to AIRs, was performed. The findings revealed that AIRs have three distinct origins: early AIRs, non-symbiotic Bradyrhizobium, and non-AIRs. The phylogenetic tree of the nif gene cluster in AIRs exhibited a high degree of concordance with the housekeeping genes, suggesting that significant HGT events have not occurred. Furthermore, the GC content of several regions in the AIR genome was significantly lower than the average GC content of the genome, indicating the occurrence of HGT. A comparative analysis of the genomes of AIRs and the reference strains of Bradyrhizobium revealed that AIRs generally lack nodulation genes, while the abundance of type III secretion system effectors in their genome is lower. However, a great conservation level was observed in nif genes and photosynthesis genes. Most of the genes specific to the genome of AIRs are associated with pathogen infestation. These genes may play a role in the NF-independent nodulation mode observed in AIRs. The KEGG analysis showed that genes specific to AIRs are enriched in epithelial cell invasion, carotenoid synthesis, nod-like receptor signaling pathway, O-glycan biosynthesis, photosynthesis, plant-pathogen interactions, protein transport, and other pathways. However, the elucidation of the NF-independent nodulation mechanism requires further investigation.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/agronomy14061295/s1, Table S1: The genome used in comparative genomic analysis of A. indica Bradyrhizobium; Table S2: The gains and losses of gene families of different nodes; Table S3: Enrichment and analysis of homologous gene family difference gene KEGG in A. indica Bradyrhizobium and reference strain.

Author Contributions

M.Z.: Data Curation, Writing—original draft. J.D.: Data Curation, Formal Analysis. Z.Z.: Data Curation, Formal Analysis, Methodology, Visualization. E.W.: Writing—Review and Editing. H.X.: Visualization. C.W.: Writing—Review and Editing. D.W.: Writing—Review and Editing. Z.X.: Data curation, Supervision, Project Administration, Funding Acquisition, Writing—Review and Editing. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the Shandong Key Research and Development Program (2021CXGC010804) and the National Natural Science Foundation of China (32270065).

Data Availability Statement

The original contributions presented in the study are included in the article, further inquiries can be directed to the corresponding author.

Acknowledgments

We thank Yan Li for his valuable suggestions.

Conflicts of Interest

The authors declare no conflicts of interest.

References

  1. Zhang, Z.; Li, Y.; Pan, X.; Shao, S.; Liu, W.; Wang, E.T.; Xie, Z. Aeschynomene indica-Nodulating Rhizobia Lacking Nod Factor Synthesis Genes: Diversity and Evolution in Shandong Peninsula, China. Appl. Environ. Microbiol. 2019, 85, e00782-19. [Google Scholar] [CrossRef]
  2. Boivin, C.; Ndoye, I.; Molouba, F.; de Lajudie, P.; Dupuy, N.; Dreyfus, B.; de Bruijn, F.J. Stem Nodulation in Legumes: Diversity, Mechanisms, and Unusual Characteristics. Crit. Rev. Plant Sci. 1997, 16, 1–30. [Google Scholar] [CrossRef]
  3. Miche, L.; Moulin, L.; Chaintreuil, C.; Contreras-Jimenez, J.L.; Munive-Hernandez, J.A.; Del Carmen Villegas-Hernandez, M.; Crozier, F.; Bena, G. Diversity analyses of Aeschynomene symbionts in Tropical Africa and Central America reveal that nod-independent stem nodulation is not restricted to photosynthetic bradyrhizobia. Environ. Microbiol. 2010, 12, 2152–2164. [Google Scholar] [CrossRef]
  4. Molouba, F.; Lorquin, J.; Willems, A.; Hoste, B.; Giraud, E.; Dreyfus, B.; Gillis, M.; de Lajudie, P.; Masson-Boivin, C. Photosynthetic bradyrhizobia from Aeschynomene spp. are specific to stem-nodulated species and form a separate 16S ribosomal DNA restriction fragment length polymorphism group. Appl. Environ. Microbiol. 1999, 65, 3084–3094. [Google Scholar] [CrossRef] [PubMed]
  5. Mornico, D.; Miché, L.; Béna, G.; Nouwen, N.; Verméglio, A.; Vallenet, D.; Smith, A.A.; Giraud, E.; Médigue, C.; Moulin, L. Comparative genomics of Aeschynomene symbionts: Insights into the ecological lifestyle of nod-independent photosynthetic bradyrhizobia. Genes 2011, 3, 35–61. [Google Scholar] [CrossRef] [PubMed]
  6. Avontuur, J.R.; Palmer, M.; Beukes, C.W.; Chan, W.Y.; Coetzee, M.P.A.; Blom, J.; Stępkowski, T.; Kyrpides, N.C.; Woyke, T.; Shapiro, N.; et al. Genome-informed Bradyrhizobium taxonomy: Where to from here? Syst. Appl. Microbiol. 2019, 42, 427–439. [Google Scholar] [CrossRef]
  7. Bonaldi, K.; Gargani, D.; Prin, Y.; Fardoux, J.; Gully, D.; Nouwen, N.; Goormachtig, S.; Giraud, E. Nodulation of Aeschynomene afraspera and A. indica by photosynthetic Bradyrhizobium Sp. strain ORS285: The nod-dependent versus the nod-independent symbiotic interaction. Mol. Plant Microbe Interact. 2011, 24, 1359–1371. [Google Scholar] [CrossRef]
  8. Chaintreuil, C.; Gully, D.; Hervouet, C.; Tittabutr, P.; Randriambanona, H.; Brown, S.C.; Lewis, G.P.; Bourge, M.; Cartieaux, F.; Boursot, M.; et al. The evolutionary dynamics of ancient and recent polyploidy in the African semiaquatic species of the legume genus Aeschynomene. New Phytol. 2016, 211, 1077–1091. [Google Scholar] [CrossRef] [PubMed]
  9. Avontuur, J.R.; Wilken, P.M.; Palmer, M.; Coetzee, M.P.A.; Stępkowski, T.; Venter, S.N.; Steenkamp, E.T. Complex evolutionary history of photosynthesis in Bradyrhizobium. Microb. Genom. 2023, 9, 001105. [Google Scholar] [CrossRef]
  10. Wettlaufer, S.H.; Hardy, R.W. Effect of Light and Organic Acids on Oxygen Uptake by BTAi 1, a Photosynthetic Rhizobium. Appl. Environ. Microbiol. 1992, 58, 3830–3833. [Google Scholar] [CrossRef]
  11. Evans, W.R.; Fleischman, D.E.; Calvert, H.E.; Pyati, P.V.; Alter, G.M.; Rao, N.S. Bacteriochlorophyll and Photosynthetic Reaction Centers in Rhizobium Strain BTAi 1. Appl. Environ. Microbiol. 1990, 56, 3445–3449. [Google Scholar] [CrossRef] [PubMed]
  12. Okubo, T.; Fukushima, S.; Itakura, M.; Oshima, K.; Longtonglang, A.; Teaumroong, N.; Mitsui, H.; Hattori, M.; Hattori, R.; Hattori, T.; et al. Genome analysis suggests that the soil oligotrophic bacterium Agromonas oligotrophica (Bradyrhizobium oligotrophicum) is a nitrogen-fixing symbiont of Aeschynomene indica. Appl. Environ. Microbiol. 2013, 79, 2542–2551. [Google Scholar] [CrossRef] [PubMed]
  13. Yurkov, V.; Beatty, J.T. Isolation of aerobic anoxygenic photosynthetic bacteria from black smoker plume waters of the juan de fuca ridge in the pacific ocean. Appl. Environ. Microbiol. 1998, 64, 337–341. [Google Scholar] [CrossRef] [PubMed]
  14. Knaff, D.B. Anoxygenic photosynthetic bacteria. Photosynth. Res. 1996, 47, 199–200. [Google Scholar] [CrossRef]
  15. Jaubert, M.; Vuillet, L.; Hannibal, L.; Adriano, J.M.; Fardoux, J.; Bouyer, P.; Bonaldi, K.; Fleischman, D.; Giraud, E.; Verméglio, A. Control of peripheral light-harvesting complex synthesis by a bacteriophytochrome in the aerobic photosynthetic bacterium Bradyrhizobium strain BTAi1. J. Bacteriol. 2008, 190, 5824–5831. [Google Scholar] [CrossRef] [PubMed]
  16. Giraud, E.; Hannibal, L.; Fardoux, J.; Vermeglio, A.; Dreyfus, B. Effect of Bradyrhizobium photosynthesis on stem nodulation of Aeschynomene sensitiva. Proc. Natl. Acad. Sci. USA 2000, 97, 14795–14800. [Google Scholar] [CrossRef]
  17. Tank, M.; Thiel, V.; Imhoff, J.F. Phylogenetic relationship of phototrophic purple sulfur bacteria according to pufL and pufM genes. Int. Microbiol. 2009, 12, 175–185. [Google Scholar]
  18. Chen, W.F.; Wang, E.T.; Ji, Z.J.; Zhang, J.J. Recent development and new insight of diversification and symbiosis specificity of legume rhizobia: Mechanism and application. J. Appl. Microbiol. 2021, 131, 553–563. [Google Scholar] [CrossRef]
  19. Giraud, E.; Moulin, L.; Vallenet, D.; Barbe, V.; Cytryn, E.; Avarre, J.C.; Jaubert, M.; Simon, D.; Cartieaux, F.; Prin, Y.; et al. Legumes symbioses: Absence of Nod genes in photosynthetic bradyrhizobia. Science 2007, 316, 1307–1312. [Google Scholar] [CrossRef]
  20. Guha, S.; Molla, F.; Sarkar, M.; Ibañez, F.; Fabra, A.; DasGupta, M. Nod factor-independent ‘crack-entry’ symbiosis in dalbergoid legume Arachis hypogaea. Environ. Microbiol. 2022, 24, 2732–2746. [Google Scholar] [CrossRef]
  21. Busset, N.; Di Lorenzo, F.; Palmigiano, A.; Sturiale, L.; Gressent, F.; Fardoux, J.; Gully, D.; Chaintreuil, C.; Molinaro, A.; Silipo, A.; et al. The Very Long Chain Fatty Acid (C26:25OH) Linked to the Lipid A Is Important for the Fitness of the Photosynthetic Bradyrhizobium Strain ORS278 and the Establishment of a Successful Symbiosis with Aeschynomene Legumes. Front. Microbiol. 2017, 8, 1821. [Google Scholar] [CrossRef]
  22. Teulet, A.; Busset, N.; Fardoux, J.; Gully, D.; Chaintreuil, C.; Cartieaux, F.; Jauneau, A.; Comorge, V.; Okazaki, S.; Kaneko, T.; et al. The rhizobial type III effector ErnA confers the ability to form nodules in legumes. Proc. Natl. Acad. Sci. USA 2019, 116, 21758–21768. [Google Scholar] [CrossRef] [PubMed]
  23. Teulet, A.; Gully, D.; Rouy, Z.; Camuel, A.; Koebnik, R.; Giraud, E.; Lassalle, F. Phylogenetic distribution and evolutionary dynamics of nod and T3SS genes in the genus Bradyrhizobium. Microb. Genom. 2020, 6, e000407. [Google Scholar] [CrossRef] [PubMed]
  24. Camuel, A.; Teulet, A.; Carcagno, M.; Haq, F.; Pacquit, V.; Gully, D.; Pervent, M.; Chaintreuil, C.; Fardoux, J.; Horta-Araujo, N.; et al. Widespread Bradyrhizobium distribution of diverse Type III effectors that trigger legume nodulation in the absence of Nod factor. ISME J. 2023, 17, 1416–1429. [Google Scholar] [CrossRef] [PubMed]
  25. Weston, L.A.; Mathesius, U. Flavonoids: Their structure, biosynthesis and role in the rhizosphere, including allelopathy. J. Chem. Ecol. 2013, 39, 283–297. [Google Scholar] [CrossRef] [PubMed]
  26. Bosse, M.A.; Silva, M.B.D.; Oliveira, N.; Araujo, M.A.; Rodrigues, C.; Azevedo, J.P.; Reis, A.R.D. Physiological impact of flavonoids on nodulation and ureide metabolism in legume plants. Plant Physiol. Biochem. 2021, 166, 512–521. [Google Scholar] [CrossRef] [PubMed]
  27. Wassem, R.; Kobayashi, H.; Kambara, K.; Le Quere, A.; Walker, G.C.; Broughton, W.J.; Deakin, W.J. TtsI regulates symbiotic genes in Rhizobium species NGR234 by binding to tts boxes. Mol. Microbiol. 2008, 68, 736–748. [Google Scholar] [CrossRef] [PubMed]
  28. Pérez-Montaño, F.; Jiménez-Guerrero, I.; Acosta-Jurado, S.; Navarro-Gómez, P.; Ollero, F.J.; Ruiz-Sainz, J.E.; López-Baena, F.J.; Vinardell, J.M. A transcriptomic analysis of the effect of genistein on Sinorhizobium fredii HH103 reveals novel rhizobial genes putatively involved in symbiosis. Sci. Rep. 2016, 6, 31592. [Google Scholar] [CrossRef] [PubMed]
  29. Arashida, H.; Odake, H.; Sugawara, M.; Noda, R.; Kakizaki, K.; Ohkubo, S.; Mitsui, H.; Sato, S.; Minamisawa, K. Evolution of rhizobial symbiosis islands through insertion sequence-mediated deletion and duplication. ISME J. 2022, 16, 112–121. [Google Scholar] [CrossRef]
  30. Dagan, T.; Martin, W. Ancestral genome sizes specify the minimum rate of lateral gene transfer during prokaryote evolution. Proc. Natl. Acad. Sci. USA 2007, 104, 870–875. [Google Scholar] [CrossRef]
  31. Arnold, B.J.; Huang, I.T.; Hanage, W.P. Horizontal gene transfer and adaptive evolution in bacteria. Nat. Rev. Microbiol. 2022, 20, 206–218. [Google Scholar] [CrossRef] [PubMed]
  32. Liu, S.; Jiao, J.; Tian, C.F. Adaptive Evolution of Rhizobial Symbiosis beyond Horizontal Gene Transfer: From Genome Innovation to Regulation Reconstruction. Genes 2023, 14, 274. [Google Scholar] [CrossRef] [PubMed]
  33. Prjibelski, A.; Antipov, D.; Meleshko, D.; Lapidus, A.; Korobeynikov, A. Using SPAdes De Novo Assembler. Curr. Protoc. Bioinform. 2020, 70, e102. [Google Scholar] [CrossRef] [PubMed]
  34. Walker, B.J.; Abeel, T.; Shea, T.; Priest, M.; Abouelliel, A.; Sakthikumar, S.; Cuomo, C.A.; Zeng, Q.; Wortman, J.; Young, S.K.; et al. Pilon: An integrated tool for comprehensive microbial variant detection and genome assembly improvement. PLoS ONE 2014, 9, e112963. [Google Scholar] [CrossRef]
  35. Du, Z.; Wu, Q.; Wang, T.; Chen, D.; Huang, X.; Yang, W.; Luo, W. BlastGUI: A Python-based Cross-platform Local BLAST Visualization Software. Mol. Inform. 2020, 39, e1900120. [Google Scholar] [CrossRef] [PubMed]
  36. Emms, D.M.; Kelly, S. OrthoFinder: Phylogenetic orthology inference for comparative genomics. Genome Biol. 2019, 20, 238. [Google Scholar] [CrossRef]
  37. Letunic, I.; Bork, P. Interactive Tree Of Life (iTOL) v5: An online tool for phylogenetic tree display and annotation. Nucleic Acids Res. 2021, 49, W293–W296. [Google Scholar] [CrossRef] [PubMed]
  38. Hyatt, D.; Chen, G.L.; Locascio, P.F.; Land, M.L.; Larimer, F.W.; Hauser, L.J. Prodigal: Prokaryotic gene recognition and translation initiation site identification. BMC Bioinform. 2010, 11, 119. [Google Scholar] [CrossRef] [PubMed]
  39. Buchfink, B.; Xie, C.; Huson, D.H. Fast and sensitive protein alignment using DIAMOND. Nat. Methods 2015, 12, 59–60. [Google Scholar] [CrossRef]
  40. Reau, M.; Lagarde, N.; Zagury, J.F.; Montes, M. Nuclear Receptors Database Including Negative Data (NR-DBIND): A Database Dedicated to Nuclear Receptors Binding Data Including Negative Data and Pharmacological Profile. J. Med. Chem. 2019, 62, 2894–2904. [Google Scholar] [CrossRef]
  41. Gasteiger, E.; Jung, E.; Bairoch, A. SWISS-PROT: Connecting biomolecular knowledge via a protein database. Curr. Issues Mol. Biol. 2001, 3, 47–55. [Google Scholar] [CrossRef] [PubMed]
  42. Rasche, H.; Hiltemann, S. Galactic Circos: User-friendly Circos plots within the Galaxy platform. Gigascience 2020, 9, giaa065. [Google Scholar] [CrossRef]
  43. Kanehisa, M.; Furumichi, M.; Sato, Y.; Kawashima, M.; Ishiguro-Watanabe, M. KEGG for taxonomy-based analysis of pathways and genomes. Nucleic Acids Res. 2022, 51, D587–D592. [Google Scholar] [CrossRef] [PubMed]
  44. Chauhan, A.; Rathore, R.S.; Agarwal, M.; Chauhan, A.J.; Taywade, A. Friend or Foe: Draft Genome Sequence of Bradyrhizobium sp. Strain SRS-191. Microbiol. Resour. Announc. 2022, 11, e0075322. [Google Scholar] [CrossRef]
  45. Toniutti, M.A.; Albicoro, F.J.; Castellani, L.G.; Lopez Garcia, S.L.; Fornasero, L.V.; Zuber, N.E.; Vera, L.M.; Vacca, C.; Cafiero, J.H.; Winkler, A.; et al. Genome sequence of Bradyrhizobium yuanmingense strain P10 130, a highly efficient nitrogen-fixing bacterium that could be used for Desmodium incanum inoculation. Gene 2021, 768, 145267. [Google Scholar] [CrossRef]
  46. Krishnan, H.B.; Kim, W.S.; Givan, S.A. Draft Genome Sequence of Bradyrhizobium sp. Strain LVM 105, a Nitrogen-Fixing Symbiont of Chamaecrista fasciculata (Michx.) Greene. Microbiol. Resour. Announc. 2019, 8, e00132-19. [Google Scholar] [CrossRef]
  47. Bromfield, E.S.P.; Cloutier, S.; Nguyen, H.D.T. Description and complete genome sequences of Bradyrhizobium symbiodeficiens sp. nov., a non-symbiotic bacterium associated with legumes native to Canada. Int. J. Syst. Evol. Microbiol. 2020, 70, 442–449. [Google Scholar] [CrossRef] [PubMed]
  48. Sun, L.; Zhang, Z.; Dong, X.; Tang, Z.; Ju, B.; Du, Z.; Wang, E.; Xie, Z. Bradyrhizobium aeschynomenes sp. nov., a root and stem nodule microsymbiont of Aeschynomene indica. Syst. Appl. Microbiol. 2022, 45, 126337. [Google Scholar] [CrossRef] [PubMed]
  49. Rejili, M.; Off, K.; Brachmann, A.; Marin, M. Bradyrhizobium hipponense sp. nov., isolated from Lupinus angustifolius growing in the northern region of Tunisia. Int. J. Syst. Evol. Microbiol. 2020, 70, 5539–5550. [Google Scholar] [CrossRef]
  50. Simoes-Araujo, J.L.; Leite, J.; Passos, S.R.; Xavier, G.R.; Rumjanek, N.G.; Zilli, J.E. Draft genome sequence of Bradyrhizobium sp. strain BR 3267, an elite strain recommended for cowpea inoculation in Brazil. Braz. J. Microbiol 2016, 47, 781–782. [Google Scholar] [CrossRef]
  51. Zhang, N.; Jin, C.Z.; Zhuo, Y.; Li, T.; Jin, F.J.; Lee, H.G.; Jin, L. Genetic diversity into a novel free-living species of Bradyrhizobium from contaminated freshwater sediment. Front. Microbiol. 2023, 14, 1295854. [Google Scholar] [CrossRef] [PubMed]
  52. Tomkins, J.P.; Wood, T.C.; Stacey, M.G.; Loh, J.T.; Judd, A.; Goicoechea, J.L.; Stacey, G.; Sadowsky, M.J.; Wing, R.A. A marker-dense physical map of the Bradyrhizobium japonicum genome. Genome Res. 2001, 11, 1434–1440. [Google Scholar] [CrossRef] [PubMed]
  53. Blommaert, J. Genome size evolution: Towards new model systems for old questions. Proc. Biol. Sci. 2020, 287, 20201441. [Google Scholar] [CrossRef] [PubMed]
  54. Nouwen, N.; Chaintreuil, C.; Fardoux, J.; Giraud, E. A glutamate synthase mutant of Bradyrhizobium sp. strain ORS285 is unable to induce nodules on Nod factor-independent Aeschynomene species. Sci. Rep. 2021, 11, 20910. [Google Scholar] [CrossRef] [PubMed]
  55. Stępkowski, T.; Watkin, E.; McInnes, A.; Gurda, D.; Gracz, J.; Steenkamp, E.T. Distinct Bradyrhizbium communities nodulate legumes native to temperate and tropical monsoon Australia. Mol. Phylogenet. Evol. 2012, 63, 265–277. [Google Scholar] [CrossRef] [PubMed]
  56. Sprent, J.I.; Ardley, J.; James, E.K. Biogeography of nodulated legumes and their nitrogen-fixing symbionts. New Phytol. 2017, 215, 40–56. [Google Scholar] [CrossRef] [PubMed]
  57. Beukes, C.W.; Stępkowski, T.; Venter, S.N.; Cłapa, T.; Phalane, F.L.; le Roux, M.M.; Steenkamp, E.T. Crotalarieae and Genisteae of the South African Great Escarpment are nodulated by novel Bradyrhizobium species with unique and diverse symbiotic loci. Mol. Phylogenet. Evol. 2016, 100, 206–218. [Google Scholar] [CrossRef] [PubMed]
  58. Cloutier, S.; Bromfield, E.S.P. Analysis of the Complete Genome Sequence of the Widely Studied Strain Bradyrhizobium betae PL7HG1(T) Reveals the Presence of Photosynthesis Genes and a Putative Plasmid. Microbiol. Resour. Announc. 2019, 8, 01282-19. [Google Scholar] [CrossRef] [PubMed]
  59. Brown, S.D.; Utturkar, S.M.; Klingeman, D.M.; Johnson, C.M.; Martin, S.L.; Land, M.L.; Lu, T.Y.; Schadt, C.W.; Doktycz, M.J.; Pelletier, D.A. Twenty-one genome sequences from Pseudomonas species and 19 genome sequences from diverse bacteria isolated from the rhizosphere and endosphere of Populus deltoides. J. Bacteriol. 2012, 194, 5991–5993. [Google Scholar] [CrossRef]
  60. Fabra, A.; Castro, S.; Taurian, T.; Angelini, J.; Ibañez, F.; Dardanelli, M.; Tonelli, M.; Bianucci, E.; Valetti, L. Interaction among Arachis hypogaea L. (peanut) and beneficial soil microorganisms: How much is it known? Crit. Rev. Microbiol. 2010, 36, 179–194. [Google Scholar] [CrossRef]
  61. Stecher, B.; Maier, L.; Hardt, W.D. ‘Blooming’ in the gut: How dysbiosis might contribute to pathogen evolution. Nat. Rev. Microbiol. 2013, 11, 277–284. [Google Scholar] [CrossRef]
  62. Doucet-Populaire, F.; Trieu-Cuot, P.; Dosbaa, I.; Andremont, A.; Courvalin, P. Inducible transfer of conjugative transposon Tn1545 from Enterococcus faecalis to Listeria monocytogenes in the digestive tracts of gnotobiotic mice. Antimicrob. Agents Chemother. 1991, 35, 185–187. [Google Scholar] [CrossRef]
  63. Sun, J.; Qiu, Y.; Ding, P.; Peng, P.; Yang, H.; Li, L. Conjugative Transfer of Dioxin-Catabolic Megaplasmids and Bioaugmentation Prospects of a Rhodococcus sp. Environ. Sci. Technol. 2017, 51, 6298–6307. [Google Scholar] [CrossRef]
  64. Kim, M.K.; Ha, H.S.; Choi, S.U. Conjugal transfer using the bacteriophage phiC31 att/int system and properties of the attB site in Streptomyces ambofaciens. Biotechnol. Lett. 2008, 30, 695–699. [Google Scholar] [CrossRef] [PubMed]
  65. Hulin, M.T.; Rabiey, M.; Zeng, Z.; Vadillo Dieguez, A.; Bellamy, S.; Swift, P.; Mansfield, J.W.; Jackson, R.W.; Harrison, R.J. Genomic and functional analysis of phage-mediated horizontal gene transfer in Pseudomonas syringae on the plant surface. New Phytol. 2023, 237, 959–973. [Google Scholar] [CrossRef]
  66. González-Villalobos, E.; Balcázar, J.L. Does phage-mediated horizontal gene transfer represent an environmental risk? Trends Microbiol. 2022, 30, 1022–1024. [Google Scholar] [CrossRef]
  67. Kenzaka, T.; Tani, K.; Nasu, M. High-frequency phage-mediated gene transfer in freshwater environments determined at single-cell level. ISME J. 2010, 4, 648–659. [Google Scholar] [CrossRef] [PubMed]
  68. Baev, N.; Schultze, M.; Barlier, I.; Ha, D.C.; Virelizier, H.; Kondorosi, E.; Kondorosi, A. Rhizobium nodM and nodN genes are common nod genes: nodM encodes functions for efficiency of nod signal production and bacteroid maturation. J. Bacteriol. 1992, 174, 7555–7565. [Google Scholar] [CrossRef] [PubMed]
  69. Yan, H.; Xie, J.B.; Ji, Z.J.; Yuan, N.; Tian, C.F.; Ji, S.K.; Wu, Z.Y.; Zhong, L.; Chen, W.X.; Du, Z.L.; et al. Evolutionarily Conserved nodE, nodO, T1SS, and Hydrogenase System in Rhizobia of Astragalus membranaceus and Caragana intermedia. Front. Microbiol. 2017, 8, 2282. [Google Scholar] [CrossRef]
  70. Ke, X.; Xiao, H.; Peng, Y.; Wang, J.; Lv, Q.; Wang, X. Phosphoenolpyruvate reallocation links nitrogen fixation rates to root nodule energy state. Science 2022, 378, 971–977. [Google Scholar] [CrossRef]
  71. Lindström, K.; Mousavi, S.A. Effectiveness of nitrogen fixation in rhizobia. Microb. Biotechnol. 2020, 13, 1314–1335. [Google Scholar] [CrossRef]
  72. Okubo, T.; Tsukui, T.; Maita, H.; Okamoto, S.; Oshima, K.; Fujisawa, T.; Saito, A.; Futamata, H.; Hattori, R.; Shimomura, Y.; et al. Complete genome sequence of Bradyrhizobium sp. S23321: Insights into symbiosis evolution in soil oligotrophs. Microbes Environ. 2012, 27, 306–315. [Google Scholar] [CrossRef]
  73. Sun, T.; Wang, P.; Rao, S.; Zhou, X.; Wrightstone, E.; Lu, S.; Yuan, H.; Yang, Y.; Fish, T.; Thannhauser, T.; et al. Co-chaperoning of chlorophyll and carotenoid biosynthesis by ORANGE family proteins in plants. Mol. Plant 2023, 16, 1048–1065. [Google Scholar] [CrossRef]
  74. Okazaki, S.; Tittabutr, P.; Teulet, A.; Thouin, J.; Fardoux, J.; Chaintreuil, C.; Gully, D.; Arrighi, J.F.; Furuta, N.; Miwa, H.; et al. Rhizobium-legume symbiosis in the absence of Nod factors: Two possible scenarios with or without the T3SS. ISME J. 2016, 10, 64–74. [Google Scholar] [CrossRef]
  75. Teulet, A.; Camuel, A.; Perret, X.; Giraud, E. The Versatile Roles of Type III Secretion Systems in Rhizobium-Legume Symbioses. Annu. Rev. Microbiol. 2022, 76, 45–65. [Google Scholar] [CrossRef]
  76. Okazaki, S.; Kaneko, T.; Sato, S.; Saeki, K. Hijacking of leguminous nodulation signaling by the rhizobial type III secretion system. Proc. Natl. Acad. Sci. USA 2013, 110, 17131–17136. [Google Scholar] [CrossRef]
  77. Periwal, V.; Scaria, V. Insights into structural variations and genome rearrangements in prokaryotic genomes. Bioinformatics 2015, 31, 1–9. [Google Scholar] [CrossRef]
  78. Polz, M.F.; Alm, E.J.; Hanage, W.P. Horizontal gene transfer and the evolution of bacterial and archaeal population structure. Trends Genet. 2013, 29, 170–175. [Google Scholar] [CrossRef]
  79. Kelly, S. The economics of organellar gene loss and endosymbiotic gene transfer. Genome Biol. 2021, 22, 345. [Google Scholar] [CrossRef] [PubMed]
  80. de Mattos, K.; Viger, R.S.; Tremblay, J.J. Transcription Factors in the Regulation of Leydig Cell Gene Expression and Function. Front. Endocrinol. 2022, 13, 881309. [Google Scholar] [CrossRef] [PubMed]
  81. Chaintreuil, C.; Perrier, X.; Martin, G.; Fardoux, J.; Lewis, G.P.; Brottier, L.; Rivallan, R.; Gomez-Pacheco, M.; Bourges, M.; Lamy, L.; et al. Naturally occurring variations in the nod-independent model legume Aeschynomene evenia and relatives: A resource for nodulation genetics. BMC Plant Biol. 2018, 18, 54. [Google Scholar] [CrossRef] [PubMed]
  82. Schüler, L.M.; Bombo, G.; Duarte, P.; Santos, T.F.; Maia, I.B.; Pinheiro, F.; Marques, J.; Jacinto, R.; Schulze, P.S.C.; Pereira, H.; et al. Carotenoid biosynthetic gene expression, pigment and n-3 fatty acid contents in carotenoid-rich Tetraselmis striata CTP4 strains under heat stress combined with high light. Bioresour. Technol. 2021, 337, 125385. [Google Scholar] [CrossRef] [PubMed]
  83. Zhou, X.; Rao, S.; Wrightstone, E.; Sun, T.; Lui, A.C.W.; Welsch, R.; Li, L. Phytoene Synthase: The Key Rate-Limiting Enzyme of Carotenoid Biosynthesis in Plants. Front. Plant Sci. 2022, 13, 884720. [Google Scholar] [CrossRef] [PubMed]
  84. Koschmieder, J.; Fehling-Kaschek, M.; Schaub, P.; Ghisla, S.; Brausemann, A.; Timmer, J.; Beyer, P. Plant-type phytoene desaturase: Functional evaluation of structural implications. PLoS ONE 2017, 12, e0187628. [Google Scholar] [CrossRef] [PubMed]
  85. Wang, S.; Zhuang, K.; Zhang, S.; Yang, M.; Kong, F.; Meng, Q. Overexpression of a tomato carotenoid ε-hydroxylase gene (SlLUT1) improved the drought tolerance of transgenic tobacco. J. Plant Physiol. 2018, 222, 103–112. [Google Scholar] [CrossRef]
  86. Lu, S.; Zhang, Y.; Zheng, X.; Zhu, K.; Xu, Q.; Deng, X. Isolation and Functional Characterization of a Lycopene β-cyclase Gene Promoter from Citrus. Front. Plant Sci. 2016, 7, 1367. [Google Scholar] [CrossRef]
Agronomy 14 01295 g001
Figure 1. The phylogenetic tree based on homologous gene families of Bradyrhizobium strains.
Figure 1. The phylogenetic tree based on homologous gene families of Bradyrhizobium strains.
Agronomy 14 01295 g001
Agronomy 14 01295 g002
Figure 2. Comparative genomic analysis of Bradyrhizobium strains associated with A. indica from diverse geographical origins. (A). The circles from inside to outside correspond to strains Bradyrhizobium sp. VIII 83002, GC%, sp. III 80001, sp. IV 81033, sp. V 80016, sp. VI 80004, sp. VII 82079, B. denitrificans 80005, B. oligotrophicum 80025, sp. DOA9, sp. I 82044, B. yuanmingense CCBAU10071, sp. II 80013, B. betae PL7HG1T, sp. S23321. (B). The circles from inside to outside are Bradyrhizobium sp. 80013, GC%, B. betae PL7HG1T, sp. S23321, sp. AT1, sp. G22, sp. LTSP849, sp. YR681, sp. I 82044, sp. VII 82079, sp. VIII 83002, B. cytisi CTAW11T, B. japonicum USDA6T. (C). The circles from inside to outside are Bradyrhizobium sp. II 82044, GC%, B. liaoningense CCBAU05525T, B. yuanmingense CCBAU10071T, B. daqingense CGMCC1.10947T, B. ottawaense OO99T, B. stylosanthis BR446T, B. guangxiense CCBAU53363T, sp. II 80013, B. betae PL7HG1T, sp. S23321, sp. VII 82079, sp. VIII 83002. Black arrow means the GC content of this region is very low.
Figure 2. Comparative genomic analysis of Bradyrhizobium strains associated with A. indica from diverse geographical origins. (A). The circles from inside to outside correspond to strains Bradyrhizobium sp. VIII 83002, GC%, sp. III 80001, sp. IV 81033, sp. V 80016, sp. VI 80004, sp. VII 82079, B. denitrificans 80005, B. oligotrophicum 80025, sp. DOA9, sp. I 82044, B. yuanmingense CCBAU10071, sp. II 80013, B. betae PL7HG1T, sp. S23321. (B). The circles from inside to outside are Bradyrhizobium sp. 80013, GC%, B. betae PL7HG1T, sp. S23321, sp. AT1, sp. G22, sp. LTSP849, sp. YR681, sp. I 82044, sp. VII 82079, sp. VIII 83002, B. cytisi CTAW11T, B. japonicum USDA6T. (C). The circles from inside to outside are Bradyrhizobium sp. II 82044, GC%, B. liaoningense CCBAU05525T, B. yuanmingense CCBAU10071T, B. daqingense CGMCC1.10947T, B. ottawaense OO99T, B. stylosanthis BR446T, B. guangxiense CCBAU53363T, sp. II 80013, B. betae PL7HG1T, sp. S23321, sp. VII 82079, sp. VIII 83002. Black arrow means the GC content of this region is very low.
Agronomy 14 01295 g002
Agronomy 14 01295 g003
Figure 3. Analysis of nodulation gene components in Bradyrhizobium.
Figure 3. Analysis of nodulation gene components in Bradyrhizobium.
Agronomy 14 01295 g003
Agronomy 14 01295 g004
Figure 4. Analysis of nitrogen fixation gene components of Bradyrhizobium.
Figure 4. Analysis of nitrogen fixation gene components of Bradyrhizobium.
Agronomy 14 01295 g004
Agronomy 14 01295 g005
Figure 5. Phylogenetic tree of Bradyrhizobium nifDKNEX gene cluster.
Figure 5. Phylogenetic tree of Bradyrhizobium nifDKNEX gene cluster.
Agronomy 14 01295 g005
Agronomy 14 01295 g006
Figure 6. Collinearity analysis of nitrogen fixation gene cluster in Bradyrhizobium. The circles from inside to outside in circus figure mean: B. oligotrophicum S58T nif gene cluster, S58T GC%, B. oligotrophicum 80025, Bradyrhizobium sp. III 80001, Bradyrhizobium sp. IV 81033, Bradyrhizobium sp. V 80016, Bradyrhizobium sp. VI 80004, Bradyrhizobium sp. VII 82079, Bradyrhizobium sp. VIII 83002, Bradyrhizobium sp. II 80013, B. betae PL7HG1, Bradyrhizobium sp. S23321, Bradyrhizobium sp. I 82044, B. guangxiense CCBAU53363, B. japonicum USDA 6.
Figure 6. Collinearity analysis of nitrogen fixation gene cluster in Bradyrhizobium. The circles from inside to outside in circus figure mean: B. oligotrophicum S58T nif gene cluster, S58T GC%, B. oligotrophicum 80025, Bradyrhizobium sp. III 80001, Bradyrhizobium sp. IV 81033, Bradyrhizobium sp. V 80016, Bradyrhizobium sp. VI 80004, Bradyrhizobium sp. VII 82079, Bradyrhizobium sp. VIII 83002, Bradyrhizobium sp. II 80013, B. betae PL7HG1, Bradyrhizobium sp. S23321, Bradyrhizobium sp. I 82044, B. guangxiense CCBAU53363, B. japonicum USDA 6.
Agronomy 14 01295 g006
Agronomy 14 01295 g007
Figure 7. Analysis of photosynthetic gene components of Bradyrhizobium.
Figure 7. Analysis of photosynthetic gene components of Bradyrhizobium.
Agronomy 14 01295 g007
Agronomy 14 01295 g008
Figure 8. Phylogenetic tree of photosynthetic gene concatenated sequences. “T” indicates that the strain is a Type strain.
Figure 8. Phylogenetic tree of photosynthetic gene concatenated sequences. “T” indicates that the strain is a Type strain.
Agronomy 14 01295 g008
Agronomy 14 01295 g009
Figure 9. Collinearity analysis of photosynthetic gene cluster in Bradyrhizobium. The circles from inside to outside in circus figure mean: B. oligotrophicum S58T Nif gene cluster, S58T GC%, B. denitrificans 80005, B. oligotrophicum LMG8443T, B. oligotrophicum 80025, Bradyrhizobium sp. II 80013, Bradyrhizobium sp. III 80001, Bradyrhizobium sp. IV 81033, Bradyrhizobium sp. V 80016, Bradyrhizobium sp. VII 82079, Bradyrhizobium sp. VIII 83002, Bradyrhizobium sp. VIII 83012, Bradyrhizobium sp. ORS 278, Bradyrhizobium sp. ORS 285, Bradyrhizobium sp. S23321, B. betae PL7HG1T, Rhodopseudomonas palustris BisA53, Rhodopseudomonas palustris BisB18.
Figure 9. Collinearity analysis of photosynthetic gene cluster in Bradyrhizobium. The circles from inside to outside in circus figure mean: B. oligotrophicum S58T Nif gene cluster, S58T GC%, B. denitrificans 80005, B. oligotrophicum LMG8443T, B. oligotrophicum 80025, Bradyrhizobium sp. II 80013, Bradyrhizobium sp. III 80001, Bradyrhizobium sp. IV 81033, Bradyrhizobium sp. V 80016, Bradyrhizobium sp. VII 82079, Bradyrhizobium sp. VIII 83002, Bradyrhizobium sp. VIII 83012, Bradyrhizobium sp. ORS 278, Bradyrhizobium sp. ORS 285, Bradyrhizobium sp. S23321, B. betae PL7HG1T, Rhodopseudomonas palustris BisA53, Rhodopseudomonas palustris BisB18.
Agronomy 14 01295 g009
Agronomy 14 01295 g010
Figure 10. Analysis of T3SS components of Bradyrhizobium.
Figure 10. Analysis of T3SS components of Bradyrhizobium.
Agronomy 14 01295 g010
Agronomy 14 01295 g011
Figure 11. T3SS effecting factor analysis of Bradyrhizobium.
Figure 11. T3SS effecting factor analysis of Bradyrhizobium.
Agronomy 14 01295 g011
Agronomy 14 01295 g012
Figure 12. Analysis of gene gain and loss in Bradyrhizobium. The red sector represents the ratio of gained genes, and the green sector represents the ratio of lost genes. The loss events of gene families are represented by arrows.
Figure 12. Analysis of gene gain and loss in Bradyrhizobium. The red sector represents the ratio of gained genes, and the green sector represents the ratio of lost genes. The loss events of gene families are represented by arrows.
Agronomy 14 01295 g012
Table 1. General data of Bradyrhizobium genomes employed in this study.
Table 1. General data of Bradyrhizobium genomes employed in this study.
StrainScaffoldsLongest ScaffoldGenome Size(G + C)%N90
Aeschynomene indica rhizobia
B. denitrificans 80005168478,4278.8 M64.5847,941
B. oligotrophicum 80025661,331,8887.9 M65.22175,316
B. oligotrophicum LMG8443T102491,1808.3 M64.7555,376
B. oligotrophicum S58T18,264,1658.3 M65.138,264,165
Bradyrhizobium sp. I 8204445880,5146.9 M64.55113,255
Bradyrhizobium sp. I 8205458887,8697.0 M64.6272,231
Bradyrhizobium sp. II 8001365819,2847.2 M64.83117,592
Bradyrhizobium sp. III 80001141712,7817.7 M65.6735,344
Bradyrhizobium sp. IV 8103359711,1467.5 M65.48113,462
Bradyrhizobium sp. V 8001675979,3627.8 M65.00136,370
Bradyrhizobium sp. V 80022542,692,9617.8 M65.02252,498
Bradyrhizobium sp. V 80029801,354,0287.9 M65.00247,781
Bradyrhizobium sp. VI 8000461671,4677.9 M64.77106,416
Bradyrhizobium sp. VII 81001112619,0926.6 M65.4845,649
Bradyrhizobium sp. VII 81035100769,6537.1 M65.6283,941
Bradyrhizobium sp. VII 8207969676,4857.4 M65.4280,746
Bradyrhizobium sp. VII 82084521,331,1537.5 M65.48125,140
Bradyrhizobium sp. VIII 81003461,209,9157.4 M65.78130,606
Bradyrhizobium sp. VIII 8101358768,8567.5 M65.51155,814
Bradyrhizobium sp. VIII 83002591,045,7257.5 M65.42150,740
Bradyrhizobium sp. VIII 83012581,487,7927.5 M65.54115,572
Bradyrhizobium sp. ORS 27817,456,5877.5 M65.517,456,587
Bradyrhizobium sp. ORS 285301212,7717.6 M65.2313,681
Bradyrhizobium sp. DOA927,114,4647.9 M64.497,114,464
Non-A. indica symbiotic rhizobia
B. arachidis LMG26795T98839,5899.8 M63.6370,254
B. cytisi CTAW11T186436,9328.9 M63.1735,919
B. daqingense CGMCC1.10947T107742,7977.9 M63.7338,665
B. diazoefficiens USDA110T19,105,8289.1 M64.069,105,828
B. elkanii USDA76T29,116,5059.5 M63.729,116,505
B. embrapense SEMIA6208T361,556,8358.3 M63.98139,684
B. guangdongense CCBAU51649T27,456,0458.4 M63.26981,946
B. guangxiense CCBAU53363T27,220,9488.2 M63.91979,173
B. icense LMTR13T18,322,7738.3 M62.038,322,773
B. japonicum USDA6T19,207,3849.2 M63.679,207,384
B. jicamae PAC68T235238,1438.7 M62.4022,567
B. lablabi CCBAU23086T135408,2388. M62.6333,805
B. liaoningense CCBAU05525T104062,7008.1 M63.804068
B. manausense BR3351T127850,5269.1 M62.8747,046
B. mercantei SEMIA6399T721,570,3738.8 M63.99259,995
B. neotropicale BR10247T125440,2298.7 M63.6042,996
B. ottawaense OO99T18,606,3288.6 M63.838,606,328
B. stylosanthis BR 446T222,454,0558.8 M64.56237,570
B. viridifuturi SEMIA690T152399,9268.8 M64.0350,014
B. yuanmingense CCBAU10071T1081,099,7638.2 M63.7737,101
Non-symbiotic Bradyrhizobium
B. betae PL7HG1T27,150,0957.41 M64.367,150,095
Bradyrhizobium sp. AT187,515,4457.5 M64.567,515,445
Bradyrhizobium sp. BF4917,547,6937.5 M63.777,547,693
Bradyrhizobium sp. G22209,022,9179.1 M63.739,022,917
Bradyrhizobium sp. LTSP84957599,4148.5 M63.24131,906
Bradyrhizobium sp. LTSP85750993,3298.4 M63.29106,609
Bradyrhizobium sp. LTSP885431,049,1297.9 M63.39116,147
Bradyrhizobium sp. LTSPM299100682,3989.1 M62.8259,442
Bradyrhizobium sp. OK095511,094,3267.8 M63.89157,651
Bradyrhizobium sp. S2332117,231,8417.2 M64.307,231,841
Bradyrhizobium sp. URHA0002144405,6187.0 M62.2026,273
Bradyrhizobium sp. YR681351167,8157.8M64.7211,443
“T” indicates that the strain is a Type strain.
Table 2. Enrichment and analysis of homologous gene family difference gene KEGG in Aeschynomene indica rhizobia and reference strain.
Table 2. Enrichment and analysis of homologous gene family difference gene KEGG in Aeschynomene indica rhizobia and reference strain.
TaxaNumber of Homologous Gene Families with DifferencesNumber of KEGG PathwaysNumber of Exclusive Access PointsTypical Pathways
Aeschynomene indica rhizobia187715916Bacterial infestation of epithelial cells, carotenoid synthesis, Nod-like receptor signaling pathway, O-glycan biosynthesis, photosynthesis, plant–pathogen interactions, protein transport, etc.
Reference strains521122168Bacterial secretion systems, biofilm synthesis, swarm-sensing effects, etc.
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Zhao, M.; Dong, J.; Zhang, Z.; Wang, E.; Wang, D.; Xie, H.; Wang, C.; Xie, Z. Nodulating Aeschynomene indica without Nod Factor Synthesis Genes: In Silico Analysis of Evolutionary Relationship. Agronomy 2024, 14, 1295. https://doi.org/10.3390/agronomy14061295

AMA Style

Zhao M, Dong J, Zhang Z, Wang E, Wang D, Xie H, Wang C, Xie Z. Nodulating Aeschynomene indica without Nod Factor Synthesis Genes: In Silico Analysis of Evolutionary Relationship. Agronomy. 2024; 14(6):1295. https://doi.org/10.3390/agronomy14061295

Chicago/Turabian Style

Zhao, Mengguang, Jingyi Dong, Zhenpeng Zhang, Entao Wang, Dandan Wang, Huijie Xie, Chao Wang, and Zhihong Xie. 2024. "Nodulating Aeschynomene indica without Nod Factor Synthesis Genes: In Silico Analysis of Evolutionary Relationship" Agronomy 14, no. 6: 1295. https://doi.org/10.3390/agronomy14061295

APA Style

Zhao, M., Dong, J., Zhang, Z., Wang, E., Wang, D., Xie, H., Wang, C., & Xie, Z. (2024). Nodulating Aeschynomene indica without Nod Factor Synthesis Genes: In Silico Analysis of Evolutionary Relationship. Agronomy, 14(6), 1295. https://doi.org/10.3390/agronomy14061295

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop