Whole-genome sequencing of Mesorhizobium huakuii 7653R provides molecular insights into host specificity and symbiosis island dynamics
- Shanming Wang1,
- Baohai Hao2,
- Jiarui Li3,
- Huilin Gu1,
- Jieli Peng1,
- Fuli Xie1,
- Xinyin Zhao3,
- Christian Frech3,
- Nansheng Chen†1, 3Email author,
- Binguang Ma†2Email author and
- Youguo Li†1Email author
© Wang et al.; licensee BioMed Central Ltd. 2014
Received: 15 November 2013
Accepted: 20 May 2014
Published: 6 June 2014
Evidence based on genomic sequences is urgently needed to confirm the phylogenetic relationship between Mesorhizobium strain MAFF303099 and M. huakuii. To define underlying causes for the rather striking difference in host specificity between M. huakuii strain 7653R and MAFF303099, several probable determinants also require comparison at the genomic level. An improved understanding of mobile genetic elements that can be integrated into the main chromosomes of Mesorhizobium to form genomic islands would enrich our knowledge of how genome dynamics may contribute to Mesorhizobium evolution in general.
In this study, we sequenced the complete genome of 7653R and compared it with five other Mesorhizobium genomes. Genomes of 7653R and MAFF303099 were found to share a large set of orthologs and, most importantly, a conserved chromosomal backbone and even larger perfectly conserved synteny blocks. We also identified candidate molecular differences responsible for the different host specificities of these two strains. Finally, we reconstructed an ancestral Mesorhizobium genomic island that has evolved into diverse forms in different Mesorhizobium species.
Our ortholog and synteny analyses firmly establish MAFF303099 as a strain of M. huakuii. Differences in nodulation factors and secretion systems T3SS, T4SS, and T6SS may be responsible for the unique host specificities of 7653R and MAFF303099 strains. The plasmids of 7653R may have arisen by excision of the original genomic island from the 7653R chromosome.
KeywordsMesorhizobium huakuii 7653R Genome sequencing Comparative analysis Host specificity Symbiosis island
Rhizobia are nitrogen-fixing soil bacteria constituting around 100 known species classified into 13 genera [1, 2]. Mesorhizobium, whose growth rate is intermediate between that of genera Rhizobium and Bradyrhizobium, is one of the largest genera; it presently comprises 24 species found primarily in Asia, Europe, the Mediterranean region, and Africa [2, 3]. Mesorhizobium huakuii and M. loti were two of the first species identified in the genus. The first known strain of M. huakuii was isolated from a winter-growing green manure crop, Astragalus sinicus, in Hubei, China in the 1940s by Huakui Chen , and was initially named Rhizobium huakuii by Wenxin Chen . Rhizobium huakuii was later classified into Mesorhizobium gen. nov. and consequently renamed M. huakuii. M. huakuii is a narrow-host-range rhizobium: it only induces indeterminate-type nitrogen-fixing nodules on the roots of A. sinicus, an economically important forage and green manure crop grown throughout eastern Asia in winter. The M. huakuii strain 7653R has been studied extensively and has been applied in sustainable agriculture for many years [7–9]. To facilitate comparative genomic investigation of the mechanism underlying this strain’s symbiosis and its host-plant molecular interactions, the first specific aim of our research was to sequence, assemble, and annotate the entire genome of 7653R.
The first completely sequenced Mesorhizobium strain was M. huakuii bv. loti MAFF303099, initially considered a strain of M. loti. Comparative sequence analysis of additional conserved genes (including 16S rRNA, glnA, glnII, and recA) have suggested instead a closer phylogenetic relationship with strains of a different species, M. huakuii, prompting the hypothesis that MAFF303099 is a strain of M. huakuii. Whole-genome sequencing of native M. loti strain R7A by the JGI GEBA project and various research findings related to R7A, such as genomic island mobility , the NifA-RpoN regulon and its symbiotic activation , and the role of the type-IV secretion system in genomic islands [14, 15], have provided a suitable reference strain and basis for the genomic comparison in this study. Consequently, our second goal was to determine whether genome-wide evidence supports the hypothesized assignment of MAFF303099 to M. huakuii.
Although MAFF303099 and 7653R may both be strains of the same species—M. huakuii, they display drastically different host preferences. Strain 7653R forms specific symbiosis with A. sinicus, whereas MAFF303099 forms determinant-type globular nodules and performs nitrogen fixing on several host plants of Lotus, including L. japonicus and L. corniculatus. Our third aim was thus to identify genomic signatures possibly accounting for these differential host preferences.
Nodulation and nitrogen-fixation genes show remarkably different genomic locations in different genomes. While MAFF303099 and M. loti R7A have their nodulation and nitrogen-fixation genes concentrated in a long DNA region called a symbiosis island on their main chromosomes , the corresponding genes in 7653R are located primarily on plasmids . Interestingly, nodulation and nitrogen-fixation gene locations of M. ciceri bv. biserrulae WSM1271 , M. australicum WSM2073 [19, 20] and M. opportunistum WSM2075 [19, 21], show patterns similar to those found in MAFF303099. These similarities suggest that genome recombination events and horizontal gene transfer are frequent in rhizobia. Our final objective was to define these genomic differences with the aim of elucidating their origin.
Results and discussion
Complete sequencing of the M. huakuii7653R genome
General genomic features of Mesorhizobium huakuii 7653Rand four other mesorhizobial genomes
G+C content (%)
Total no. of CDS
CDS coverage (%)
Nb (%) CDS with assigned functions
Average length of genes (bp)
Tandem repeat sequences
Putative ABC transporters-related proteins
Putative two component systems-related proteins
Putative sigma factor-related proteins
We predicted 7,205 protein-coding genes in the 7653R genome, a number essentially identical despite the different genome sizes to the number predicted in MAFF303099 (7,281 genes) . 7653R was found to have the highest gene density among the five genomes, but have a lower ratio of genes with annotated functions, suggesting that it contains a higher ratio of genes with undefined functions. We examined the numbers and types of rRNAs and tRNAs of all five genomes predicted using the same strategy. We found that these five genomes had essentially identical numbers of rRNAs and tRNAs (Additional file 1: Table S1). However, the numbers of putative transposase genes predicted in these genomes were dramatically different (Table 1). As discussed later, this variation may have a profound differential impact on genome stability and horizontal gene transfer (HGT) events.
Genomic evidence supporting MAF303099 as a strain of M. huakuii
MAFF303099 has been hypothesized to be a strain of M. huakuii on the basis of comparative analysis of a few conserved genes in MAFF303099 and M. huakuii strains . The availability of genome sequences of both strains has enabled us to re-examine their phylogenetic relationship.
Thus, both ortholog and synteny analyses support a closer phylogenetic relationship between 7653R and MAFF303099 than with the other Mesorhizobium strains. These results provide further evidence that MAFF303099 is a strain of M. huakuii.
Although 7653R and MAFF303099 are both strains of the same species, M. huakuii, they display drastically different host specificities. While the strain 7653R forms a specific symbiosis with A. sinicus, MAFF303099 forms symbioses with several Lotus species host plants, including L. japonicus and L. corniculatus[11, 16]. We aimed to determine what genomic features are responsible for such unique host preferences. Host specificity, an important trait underlying the interaction of rhizobia with their hosts, is still poorly understood . Host switching or host jumping can often be traced to the modification of key microbial genes that facilitate the formation of particular host associations . Because the determinants of host specificity of a bacterium mainly depend on three groups of signaling molecules—nodulation factors (NFs), surface polysaccharides, and secreted proteins , we explored genes that affect the biological synthesis of these signaling molecules in the genomes of these two strains and compared them with those of native M. loti strain R7A.
Rhizobial cell-surface polysaccharides, including cyclic-β-glucans (CβGs), exopolysaccharides (EPSs), lipopolysaccharides (LPSs), and capsular polysaccharides (KPSs or K-antigens), are necessary for establishing successful symbiosis with their hosts to form effective root nodules . Comparative genomics analysis revealed that the genes needed for the biosynthesis of CβGs (ndvA and ndvB), EPSs (26 exo/exs genes; in Additional file 1: Table S5), and LPSs (Additional file 4: Figure S2 and Additional file 1: Table S6) are well conserved in all six genomes, suggesting that genes involved in the biosynthesis of surface polysaccharides are unlikely to contribute substantially to host preference differentiation.
Numbers and distributions of genes associated with different types of secretion systems in mesorhizobial genomes
Secretion system and characteristics
Gene numbers associated with the formation of different types of secretion systems
GSP (general secretion pathway)
P-type (Flp and attachment)
TAT (twin arginine)
Our comparative analysis of these secretion systems in the genomes of the two M. huakuii strains revealed important differences in three secretion systems: T3SS, T4SS, and T6SS. Gene clusters encoding the major and conserved components of T3SSs are present in diverse and distantly related rhizobia [34, 35]. The 7653R genome was found to contain a complete T3SS on the pMhu7653Rb plasmid, with gene organization conserved with respect to MAFF303099. Proteins secreted by rhizobial T3SS are called nodulation outer proteins (Nops) and can be divided into two types: effectors and helper proteins. T3SSs of both 7653R and MAFF303099 have three helper proteins, NopA, NopB, and NopX, but different candidate effectors: NopP in 7653R and NopC in MAFF303099 (Additional file 5: Table S7). Although T3SS and its secreted effectors are dispensable for rhizobial infection and nodulation, they may function as facilitators superimposed on the Nod-factor signaling pathway and modulate host range in a genotype-specific manner . Thus, T3SS might be one determinant of host range variation in 7653R and MAFF303099. The Vir system, an important example of a T4SS, is usually formed by 12 proteins, VirB1–VirB11 and VirD4. Except for VirB1 and VirB7, these proteins are encoded by genes on plasmid pMhu7653Ra. Interestingly, neither VirB1 nor VirB7 are present in MAFF303099 and R7A . The Vir systems of 7653R and MAFF303099 are thus essentially identical. In contrast, the T4SS Trb system was found to differ between 7653R and the other five Mesorhizobium strains; in particular, 7653R has no trb gene, whereas MAFF303099 has 19 trb genes (Table 2). The T6SS apparatus is assembled by a conserved set of proteins whose functions are closely related to bacterial pathogenesis and host cell survival . Two T6SSs were found in the 7653R genome, while one each was identified in MAFF303099 and R7A genomes (Table 2).
Taken together, our analysis revealed that the two M. huakuii strains 7653R and MAFF303099 have substantial differences in the number and arrangement of genes responsible for synthesizing NFs, and also differ with respect to secretion systems T3SS, T4SS, and T6SS. These differences may contribute to the establishment of differential host specificity.
Changes in host specificity determinants—for example, by acquisition of new genetic elements that grant a selective advantage in a particular host environment—can have a great impact on host range and may lead to host jumps . Both intrageneric and intergeneric HGT have been reported as important mechanisms for the spread of symbiotic capacity in the Salado River Basin . Intrageneric HGT might be a main pathway to change symbiotic capacity in MAFF303099. Mesorhizobium strains isolated from A. sinicus in Japan, designated as M. huakuii subsp. rengei, are able to coexist with M. loti strains and thus have a chance to exchange genetic information through conjugation. The ancestral strain of M. huakuii presumably derived some genetic information from native M. loti strains, thereby introducing genetic variation in host specificity determination. The ancestral strain eventually evolved into strain MAFF303099, which can form an effective symbiotic relationship with Lotus corniculatus. The introduction of novel genetic variation by HGT is typically accompanied by the acquisition and incorporation of genetic fragments or intact transcriptional units into the genome . Although NFs and secreted effectors of T3SS in MAFF303099 are associated with genetic fragments and intact transcriptional units, we still cannot confirm the underlying causes of the host specificity changes: there may be a continuum that ranges from changes in single residues to gene domains, whole genes, and eventually entire genomic islands (GEIs) . Consequently, much remains to be learned about whether many or only a few gene loci are involved in the determination of nodulation specificity. Moreover, genes from leguminous plants, such as the R-gene from soybean , can also participate in the control of genotype-specific infection and nodulation.
Symbiosis island dynamics and the origin of symbiotic plasmids
Of the five Mesorhizobium strains whose genomes have been completely sequenced (excluding R7A with its incomplete genome data), only 7653R has symbiotic plasmids. In contrast, all other strains either have no plasmids, or their plasmids do not contain genes involved in symbiosis. Thus, while the nodulation and nitrogen-fixation genes are localized on the plasmids as a symbiosis island in 7653R, they are localized on the main chromosomes of the other four strains. Global genome alignment between 7653R and the other genomes revealed that the symbiosis islands are positioned in a synteny gap region that corresponds to the genome-specific region in MAFF303099 and the gap in 7653R (Figure 8 and Additional file 4: Figure S3), suggesting that the plasmids were excised from the main 7653R chromosome. Plasmids of 7653R and these genome-specific regions found in the other four genomes are thus likely GEIs. To test this hypothesis, we examined these genome-specific regions i.e., symbiosis islands, using IslandViewer, a program for finding GEIs . As expected, IslandViewer identified these MAFF303099, WSM1271, WSM2073, and WSM2075 symbiosis islands as typical GEIs (Additional file 4: Figure S4). These predictions are supported by the results of further analysis of genomic features. First, plasmids of 7653R and the other four GEIs have similar sizes (514–611 kb) and similar GC content (58–59%), which is strikingly lower than that of the corresponding genome (62.51–62.87%). Second, codon usage of 7653R plasmid ORFs is significantly different from that of the chromosome but surprisingly consistent with those of the other four GEIs (Additional file 4: Figure S5). Third, T3SSs and/or T4SSs of the five strains are all located in the corresponding candidate GEIs. Fourth, a highly conserved tRNA(Gly) gene is found in the vicinity of the candidate GEI in all five Mesorhizobium strains except for 7653R. In 7653R, plasmids possess the same characteristics as the other four GEIs located in specific genome regions. We propose that the plasmids of 7653R were formed during evolution by the excision of the GEI from the 7653R chromosome, as described previously in other systems .
Because the five GEIs likely share a common ancestor, we expected them to maintain well-conserved syntenic relationships. Although the GEIs in WSM1271, WSM2075, and WSM2073 displayed conserved synteny, the GEIs in these three strains and two other strains surprisingly showed little resemblance in regard to gene organization. We noticed that 80% of all transposase genes in the entire 7653R genome are concentrated on its plasmids. This enrichment of transposase genes on the plasmids of 7653R resembles that of the MAFF303099 GEI, which possesses 89 predicted transposase genes—86% of all transposase genes in the entire MAFF303099 genome. Similarly, 85% (41) of all transposase genes identified in the entire contigs of R7A are found in the symbiosis island of contig 3. In contrast, the GEIs of the other three Mesorhizobium strains harbor only a few transposase genes, and they show highly conserved synteny. On the basis of this observation, we propose that the enrichment of transposase genes in the GEIs of 7653R and MAFF303099 caused a disruption in gene order within their GEIs, whereas the lack of transposase genes in the other three Mesorhizobium strains helped to maintain their GEI synteny. The question then arises: what is the source of these transposase genes in the GEIs of 7653R and MAFF303099? One likely source is HGT. Previous analysis of nodulation genes has proved that the GEI of MAFF303099 has acquired many foreign genes by HGT . Our clustering analysis of transposase genes in the plasmids of 7653R and the MAFF303099 GEI revealed that most of them belong to different families, suggesting that these transposase genes were likely acquired via HGT. Thus, these five Mesorhizobium strains may have inherited their GEIs from a common ancestral GEI, which later underwent various degrees of change.
Many transposase genes exist within GEIs of 7653R and MAFF303099. Except for several conserved but inactive genes, these genes were acquired from foreign species. The transposases encoded by foreign genes have retained high activity, indicating a continuous exchange of 7653R and MAFF303099 genetic information with other species. How rhizobial genomes are able to select the proper foreign genes while still maintaining structural stability and gene function despite the disruption remains unknown. Complex cellular programs associated with some bacterial traits, such as symbiosis, must exist to ensure adaption to the surrounding environment and to maintain competitiveness. A large body of research has confirmed this point. In one recent case, genes on a genomic island were reported to confer an adaptive advantage to specific stresses in marine Synechococcus. For better survival and growth in various habitats, GEIs from MAFF303099 acquired some foreign nodulation genes by HGT during the genetic information exchange process, enabling functional symbiosis between MAFF303099 and a new host plant. Furthermore, the acquisition of foreign genetic elements is frequently accompanied by the loss of native genes. As to the argument that the lost genes are randomly selected or under special selection, increasing evidence inclines to the view that loss of functionality can be a selective advantage in some specific situations .
In Legionella pneumophila, a newly identified conjugation/type-IVA secretion system (trb/tra) composed of clusters of tra and trb genes (related to the Vir system and conjugal transformation) seems to be necessary for integrase-dependent excision and horizontal transfer of GEIs . A similar system has been identified on the other four GEIs, excluding 7653R, with different sets of tra and trb genes scattered on them. The existence of the same set of tra and trb genes with high similarities in strains MAFF303099, WSM1271, WSM2075, WSM2073, and R7A  indicates that the ancestral ITR plasmid that integrated into chromosomes of ancestral Mesorhizobium strains contained a functional conjugation/type-IVA secretion system. Plasmid pMhu7653Ra of 7653R, however, has only a few tra genes and no trb gene. Integrated mobile elements should theoretically be inactivated or lose genes related to plasmid mobilization or transfer, such as tra and trb. It is difficult to judge whether the IVA systems are inactive or if some of the key tra-trb genes have already been deleted from the GEIs of MAFF303099, WSM1271, WSM2075, and WSM2073. To determine what happened to the tra-trb genes on the GEI of 7653R chromosome before excision, further bioinformatics analysis and experimental evidence are needed.
Whole-genome sequencing has proven valuable and critical for refining the phylogenetic positions of a series of rhizobial strains . In this study, we sequenced, assembled, and annotated the M. huakuaii 7653R genome. We used this genome sequence to examine the phylogenetic position of MAFF303099, a strain whose phylogenetic position has been debated. These two strains share a large set of orthologs and, most importantly, a conserved chromosomal backbone and even larger perfectly conserved synteny blocks. Our ortholog and synteny analyses have firmly placed MAFF303099 as a strain of M. huakuii, as is 7653R.
Although 7653R and MAFF303099 are both strains of M. huakuii, they exhibit important differences in symbiotic phenotypes and thus belong to different symbiosis variants (also known as symbiovars) . This placement is supported by our analysis of nodulation and fixation genes, which revealed notable differences in several nodulation genes, mostly related to NF generation. Such differences have a profound impact on host specificity. In a few rhizobium strains, mutations of some specific genes related to NFs and T3SS have been found to alter host specificity; additionally, the distribution of nodulation genes is reportedly related to requirements for effective symbiosis with some legume hosts [49–51]. Furthermore, our analysis of the three groups of signaling molecules revealed substantial differences between the two M. huakuii strains 7653R and MAFF303099 that were focused on the number and arrangement of genes responsible for synthesizing NFs and secretion systems T3SS, T4SS, and T6SS. In conjunction with NFs, these secretion systems may contribute to the establishment of differential host specificity.
Our results strongly suggest a common site-specific GEI localization mechanism in the ancestral Mesorhizobium chromosome, with the GEIs of the genus showing different degrees of variability after divergence from the mesorhizobial ancestor. A similar phenomenon has been observed in Bradyrhizobium japonicum strains. Various lines of evidence support past horizontal insertion of GEIs into the ancestral genome of B. japonicum USDA110, and comparative genomic hybridization profiles show that GEIs may be highly dynamic entities in B. japonicum genomes . The ability of integrating mobile genetic elements to enlarge chromosomes may be due to the fact that Bradyrhizobium and Mesorhizobium species have very large chromosomes with few plasmids . The recent completion of genome-sequencing projects for several Mesorhizobium species has enabled analysis of the global changes between them after the acquisition and integration of the ancestral ITR plasmid. An improved understanding of these variations should improve our understanding of how genome dynamics can contribute to bacterial evolution in general.
7653R plasmids possess the same characteristics as the GEIs of the other four Mesorhizobium genomes. Additionally, homologs of nodulation and nitrogen-fixation genes on the other four GEIs are found on the two plasmids of 7653R. Moreover, it has been reported that GEIs can excise themselves spontaneously from the chromosome and form plasmids with the acquisition of functions for autonomous replication (e.g., repABC genes) or can be transferred to other suitable recipients . We therefore conclude that 7653R plasmids may have arisen by the excision of the original GEI from the 7653R chromosome.
Bacterial strains and DNA preparation
Mesorhizobium huakuii 7653R was cultured for 3 days at 28°C in trypticase-yeast extract medium. Cells of 7653R were harvested by centrifugation, with total DNA prepared using a Genomic DNA Mini Preparation kit.
Sequencing and annotation
For de novo sequencing of the 7653R genome, a combined strategy comprising Solexa sequencing on an Illumina GAIIx platform was carried out by BGI (Beijing Genomics Institute, Beijing, China). As a result, 367 contigs were generated with a 29-fold median coverage depth.
Sequence assembly was performed using SOAPdenovo , with PCR-based amplicon sequencing used for gap closure. Glimmer 3.0 , RNAmmer 1.2 , and tRNAscan-SE  were used respectively for de novo prediction of genes, rRNA genes, and tRNAs. Clusters of Orthologous Groups (COG) annotation was performed using RPS-BLAST against the CDD database , and Gene Ontology annotation was carried out with InterProScan V4 . A bidirectional best hit approach (E-value < 1 × 10−5, identity > 30%, coverage > 70%, and bit score > 60) was used for KEGG  and SWISS-PROT  annotations.
The complete nucleotide sequences of strains MAFF303099, WSM1271, WSM2075, and WSM2073 were obtained from GenBank (accession numbers: M. huakuii bv. loti, NC_002678, NC_002679, and NC_002682; M. ciceri, NC_014923 and NC_014918; M. opportunistum, NC_015675; M. australicum, NC_019973). The sequences were organized according to their chromosomal origins of replication for intuitive comparison. Sequences of three contigs from R7A were obtained from the JGI Genome Portal (Project ID: 404030). Genome sequence alignments were created using MUMmer, ACT, and Mauve software.
The OrthoMCL  approach was adopted to construct gene families for all coding sequences in the five Mesorhizobium genomes. Quartets of orthologous proteins (quartops) in all pairwise genome comparisons were considered to constitute the ‘core’ genome. Proteins with no homologs in the other four Mesorhizobium genomes were defined as differential genes.
Nucleotide sequence accession numbers
Complete genome sequences of M. huakuii 7653R have been submitted to GenBank under the following assigned accession numbers: Mesorhizobium CP006581; Mesorhizobium_1 CP006582; Mesorhizobium_2 CP006583.
Horizontal gene transfer
Intragenomic translocation recipient
This work was supported by funds from the National Basic Research Program of China (973 Program; 2010CB126502), the Research Fund for the Doctoral Program of Higher Education of China (20110146110012), the Fundamental Research Funds for the Central Universities (2009PY020 and 2010QC016), the National Natural Science Foundation of China (30970074 and 31100602), and the Natural Sciences and Engineering Research Council of Canada to NC. NC is also a Michael Smith Foundation for Health Research Scholar and a Canadian Institutes of Health Research New Investigator.
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