BACKGROUND
rhizobia are nitrogen-fixing soil bacteria constituting around  <dig> known species classified into  <dig> genera  <cit> .
mesorhizobium, whose growth rate is intermediate between that of genera rhizobium and bradyrhizobium, is one of the largest genera; it presently comprises  <dig> species found primarily in asia, europe, the mediterranean region, and africa  <cit> .
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  <cit> , and was initially named rhizobium huakuii by wenxin chen  <cit> .
rhizobium huakuii was later classified into mesorhizobium gen. nov. and consequently renamed m. huakuii <cit> .
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 .
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 maff <dig>  initially considered a strain of m. loti <cit> .
comparative sequence analysis of additional conserved genes  have suggested instead a closer phylogenetic relationship with strains of a different species, m. huakuii, prompting the hypothesis that maff <dig> is a strain of m. huakuii <cit> .
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  <cit> , the nifa-rpon regulon and its symbiotic activation  <cit> , and the role of the type-iv secretion system in genomic islands  <cit> , 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 maff <dig> to m. huakuii.
although maff <dig> 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 maff <dig> forms determinant-type globular nodules and performs nitrogen fixing on several host plants of lotus, including l. japonicus and l. corniculatus <cit> .
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 maff <dig> 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  <cit> , the corresponding genes in 7653r are located primarily on plasmids  <cit> .
interestingly, nodulation and nitrogen-fixation gene locations of m. ciceri bv.
biserrulae wsm <dig>  <cit> , m. australicum wsm <dig>  <cit>  and m. opportunistum wsm <dig>  <cit> , show patterns similar to those found in maff <dig>  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
complete sequencing of the m. huakuii 7653r genome
our  <dig> ,675-bp assembly of the m. huakuii 7653r genome consisted of three circular replicons of  <dig> ,365 bp ,  <dig>  bp , and  <dig>  bp  .
the average gc content of the whole genome was calculated to be  <dig> %, while plasmids pmhu7653ra and pmhu7653rb showed slightly lower gc levels .
an overview of the gc content of the three replicons is shown on the 7653r genome physical maps .
the main genome characteristics of 7653r and four other mesorhizobium genomes  are summarized in table  <dig>  although the five strains all belong to the same genus, they possess different numbers of plasmids: 7653r and maff <dig> have two plasmids each, wsm <dig> has only one, and wsm <dig> and wsm <dig> have none.figure  <dig>
physical maps of the complete
mesorhizobium huakuii
7653r genome.
physical maps of three replicons were drawn using circos  <cit> .
displayed circles from the inside outwards represent: g-c skew using a 1-kb window , codon adaptation index , clusters of orthologous groups  of proteins in a counterclockwise/clockwise direction , predicted coding sequences transcribed in both directions , and scale in mb .
the position  <dig> represents the origin of replication in each replicon.
general genomic features of
mesorhizobium huakuii
7653rand four other mesorhizobial genomes
size
g+c content
total no.
of cds
cds coverage
trna genes
rrna operons
nb  cds with assigned functions
average length of genes
putative transposases
tandem repeat sequences
transcriptional regulators
putative abc transporters-related proteins
putative two component systems-related proteins
putative sigma factor-related proteins
cds: coding sequences.
statistics for maff <dig>  wsm <dig>  wsm <dig>  and wsm <dig> genomic features are based on ncbi annotations.
we predicted  <dig>  protein-coding genes in the 7653r genome, a number essentially identical despite the different genome sizes to the number predicted in maff <dig>   <cit> .
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 .
however, the numbers of putative transposase genes predicted in these genomes were dramatically different .
as discussed later, this variation may have a profound differential impact on genome stability and horizontal gene transfer  events.
genomic evidence supporting maf <dig> as a strain of m. huakuii
maff <dig> has been hypothesized to be a strain of m. huakuii on the basis of comparative analysis of a few conserved genes in maff <dig> and m. huakuii strains  <cit> .
the availability of genome sequences of both strains has enabled us to re-examine their phylogenetic relationship.
genome-wide orthologs
we identified a set of  <dig>  orthologous groups among five mesorhizobium genomes .
of these groups,  <dig>   were found to be conserved across all five genomes, with each group represented by at least one gene in each of the five genomes.
we termed this subset of orthologous groups the core genome of mesorhizobium.
an additional  <dig>  were observed to be present in four of the five genomes .
the remaining orthologous groups  occurred in two or three genomes.
similar numbers and proportions of proteins predicted in 7653r  and maff <dig>  were present in the core genome, whereas  <dig> %  of predicted proteins in r7a were present in the core genome.
among all pair-wise comparisons, the 7653r-maff <dig> pair was found to share the most abundant orthologous proteins, followed by the r7a-maff <dig> and wsm1271-wsm <dig> pairs; this ordering suggests that maff <dig> has a closer phylogenetic relationship to 7653r than to r7a, wsm <dig>  and wsm <dig>  from the  <dig>  7653r core genes, we randomly chose  <dig> single-copy genes  and performed hierarchical clustering analysis  <cit>  based on their presence or absence in  <dig> representative rhizobial species.
the clustering results also revealed a closer phylogenetic relationship between 7653r and maff <dig> , further supporting maff <dig> as a strain of m. huakuii.figure  <dig>
core and accessory genome analyses of
mesorhizobium
strains.
the numbers of orthologous groups and related genes found in each intersection are shown.
the numbers of genes found in related strains for each intersection are shown in parentheses.
the numbers of transposase genes are shown in square brackets.
areas are not in scale.
hierarchical clustering analysis of rhizobia based on a heat map of  <dig> genes chosen from
mesorhizobium
core genes.
gene homologs were chosen on the basis of blastp results .
homolog presence and absence are indicated by yellow and red, respectively.
the five genera for which multiple genomes were available are indicated in different colors.
the numbers on the dendrogram represent bootstrap values.
synteny analysis
the above analysis of orthologs suggested that maff <dig> is phylogenetically more closely related to m. huakuii strain 7653r than to strains of other species.
we further hypothesized that m. huakuii strain 7653r and maff <dig> share larger synteny blocksbetween them than with the other three strains.
to test this hypothesis, we carried out a synteny analysis of five strains  using a few complementary approaches.
for convenience of comparison, we considered dnaa to be the start position of the main chromosome and repabc to be the start position of plasmids in all five genomes.
mauve alignment  <cit>  of chromosomes of the five strains revealed a remarkably consistent backbone .
compared with wsm <dig>  wsm <dig>  and wsm <dig>  synteny blocks of the 7653r chromosome shared a longer average length and more consistent relative positions with maff <dig>  additionally, fewer sequence inversions were observed between the chromosomes of 7653r and maff <dig> than between 7653r and wsm <dig>  wsm <dig>  or wsm <dig>  we used orthocluster  <cit>  to identify synteny blocks perfectly conserved between each pair of these five genomes, and assigned a score to each 7653r gene according to the length of the synteny .
we found that the mean score of all genes in synteny between 7653r and maff <dig> was larger  than the mean scores between 7653r and wsm <dig> , wsm <dig> , or wsm <dig> .
additionally, we performed a statistical test of significance, the results of which are shown additional file 3: table s <dig>  moreover, analyses of phylogenetic relationships based on the consistency of dna sequences using mauve and the composition vector using cvtree  <cit>  both indicated that 7653r has a closer relationship to maff <dig> than to the other three mesorhizobium strains .figure  <dig>
chromosomal alignment of five mesorhizobial species  visualized using mauve and a phylogenetic tree .
chromosomal alignments for 7653r and maff <dig> , 7653r and wsm <dig> , 7653r and wsm <dig> , and 7653r and wsm <dig>  were created using mauve software.
each colored block represents a synteny block and is internally independent from genomic rearrangement.
white regions correspond to unaligned sequences that likely contain sequence elements specific to a particular genome.
blocks below the center line indicate regions that are aligned in the reverse complement  orientation.
phylogenetic tree  created by mauve based on dna sequence consistency.
thus, both ortholog and synteny analyses support a closer phylogenetic relationship between 7653r and maff <dig> than with the other mesorhizobium strains.
these results provide further evidence that maff <dig> is a strain of m. huakuii.
host specificity
although 7653r and maff <dig> 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, maff <dig> forms symbioses with several lotus species host plants, including l. japonicus and l. corniculatus <cit> .
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  <cit> .
host switching or host jumping can often be traced to the modification of key microbial genes that facilitate the formation of particular host associations  <cit> .
because the determinants of host specificity of a bacterium mainly depend on three groups of signaling molecules—nodulation factors , surface polysaccharides, and secreted proteins  <cit> , 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.
nodulation factors
nfs, which are signaling molecules between symbiotic bacteria and plants, are produced by bacteria in response to flavonoids secreted by legume root hairs  <cit> .
in most rhizobia, expression of nodulation genes  is needed for the biosynthesis and transport of nfs that induce nodule organogenesis.
a total of  <dig> nodulation genes  were identified in the 7653r chromosome and plasmids, while  <dig> nodulation genes were located in maff <dig> .
in contrast, in r7a,  <dig> nodulation genes were found to be present and all were found to be homologous with very high similarities to genes in maff <dig>  comparison of nodulation genes between these three strains not only revealed some genes with high sequence similarity but also uncovered two striking differences likely related to nf synthesis.figure  <dig>
similarities  between maff <dig>  wsm <dig>  wsm <dig>  and wsm <dig> nodulation genes and those of 7653r.
first, genomic distribution of these nodulation genes was different between 7653r and maff <dig>  while all  <dig> nodulation genes in maff <dig> were found on its chromosome, only  <dig> nodulation genes were present on the chromosome of 7653r, with  <dig> found on its plasmids .
specifically, the  <dig> nod genes  were identified in a 140-kb genomic region of the pmhu7653rb plasmid of 7653r .
this genomic region also contained  <dig> fix genes  and  <dig> nif genes  .
the  <dig> nod genes were well conserved across all six genomes , as were the  <dig> fix genes and  <dig> nif genes .
although these nod genes individually exhibited strong conservation, major differences were observed in regard to their arrangement in the genomes.
for example, the  <dig> nod genes on the pmhu7653rb plasmid of 7653r were found to be segregated into two independent operons preceded by two nod-boxes , with noda separated from nodbc by a 22-kb genome region containing  <dig> genes  <cit> .
in sharp contrast, orthologs of noda and nodbc in other mesorhizobium strains, including maff <dig> and r7a  <cit> , are adjacent and localized on the same strand .figure  <dig>
arrangement of nodulation genes , nitrogen fixation genes , and gene clusters in the 7653r plasmid pmhu7653rb .
double slash marks represent genome regions that are not shown.
in the clusters, nitrogen-fixation genes  conserved among the six strains are represented by white arrows, while those varying in copy number, location, or transcriptional orientation are shown in different colors.
nodulation and nitrogen-fixation gene clusters  in 7653r plasmid pmhu7653rb.
genes are colored according to their names.
double slash marks represent genome regions that are not shown.
arrows indicate the location of potential nod-boxes.
second, the numbers of nodulation genes putatively participating in nf synthesis were found to be different between these two strains.
the nod gene nodh, required for nf synthesis in 7653r  <cit> , had no ortholog in maff <dig> and r7a .
each of the four nod genes in maff <dig>  had a substantially higher percentage identity  with its ortholog in r7a than with its ortholog in 7653r .
for example, nodc in maff <dig> had a pid of 99% with its ortholog in r7a and a pid of 74% with its ortholog in 7653r .
interestingly, seven nodulation genes in maff <dig> with orthologs in both 7653r and r7a were found to have substantially higher pids with their orthologs in r7a than with those in 7653r; nine nodulation genes in maff <dig> had high pids with their orthologs in r7a but had no orthologs in 7653r.
these results suggest that maff <dig> may have obtained these  <dig> nodulation genes from an ancestor of r7a.
thus, although maff <dig> shares  <dig> nodulation genes with high pids  with 7653r, it shares  <dig> nodulation genes with high pids with r7a.
furthermore, maff <dig> was found to have an additional five nodulation genes .
taken together, 7653r and maff <dig> have very different numbers of nodulation genes.
indeed, different nodulation genes are required for nf synthesis in these two strains.
of the  <dig> nod genes identified in 7653r,  <dig>  are possibly key elements involved in the synthesis of the core nfs of 7653r  <cit>  .
in the m. loti strain r7a, nod genes organized in seven transcriptional units—noekj, nodznoelnolk, nods, nodacijnolo, nodb, noll, and nodm—are needed for nf synthesis  <cit>  .
considering that many of the maff <dig> nodulation genes showed higher pids with r7a genes, we further propose that nodulation genes required for the synthesis of nfs in maff <dig> are closely related to those in r7a.
this inference is consistent with a report that maff <dig> and r7a may share the same steps of nf synthesis  <cit> .figure  <dig>
nodulation factor  biosynthesis pathway in
m. huakuii
7653r.
biosynthesis pathway of the core nfs of strain 7653r and involved nod proteins are presented.
two variable ends of the chemical structure of the core nfs, r <dig> and r <dig>  are shown in the figure.
r <dig> thus far seems to be represented only by h in the 7653r nf structures.
surface polysaccharides
rhizobial cell-surface polysaccharides, including cyclic-β-glucans , exopolysaccharides , lipopolysaccharides , and capsular polysaccharides , are necessary for establishing successful symbiosis with their hosts to form effective root nodules  <cit> .
comparative genomics analysis revealed that the genes needed for the biosynthesis of cβgs , epss , and lpss  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.
secretion system
proteins secreted by some rhizobial strains play an important role in infection of leguminous plant roots and establishment of a mutually beneficial symbiosis.
different types and numbers of protein secretion systems are present in almost all rhizobial strains.
by means of similarity searches using protein secretion genes identified in other gram-negative bacteria as queries, we identified  <dig> genes related to secretory processes in the 7653r genome.
these genes and proteins are involved in  <dig> putative protein secretion systems: a general export pathway, four separate type-i systems, a twin-arginine translocase secretion system, one functional type-iii system , two type-iv systems , one type-v autotransporter, and two putative type-vi secretion systems  .table  <dig>
numbers and distributions of genes associated with different types of secretion systems in mesorhizobial genomes
type i
type ii
type iii
type iv
type v
type vi
srp
mechanosensitive channels
total
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  <cit> .
the 7653r genome was found to contain a complete t3ss on the pmhu7653rb plasmid, with gene organization conserved with respect to maff <dig>  proteins secreted by rhizobial t3ss are called nodulation outer proteins  and can be divided into two types: effectors and helper proteins.
t3sss of both 7653r and maff <dig> have three helper proteins, nopa, nopb, and nopx, but different candidate effectors: nopp in 7653r and nopc in maff <dig> .
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  <cit> .
thus, t3ss might be one determinant of host range variation in 7653r and maff <dig>  the vir system, an important example of a t4ss, is usually formed by  <dig> proteins, virb1–virb <dig> and vird <dig>  except for virb <dig> and virb <dig>  these proteins are encoded by genes on plasmid pmhu7653ra.
interestingly, neither virb <dig> nor virb <dig> are present in maff <dig> and r7a  <cit> .
the vir systems of 7653r and maff <dig> 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 maff <dig> has  <dig> trb genes .
the t6ss apparatus is assembled by a conserved set of proteins whose functions are closely related to bacterial pathogenesis and host cell survival  <cit> .
two t6sss were found in the 7653r genome, while one each was identified in maff <dig> and r7a genomes .
taken together, our analysis revealed that the two m. huakuii strains 7653r and maff <dig> 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  <cit> .
both intrageneric and intergeneric hgt have been reported as important mechanisms for the spread of symbiotic capacity in the salado river basin  <cit> .
intrageneric hgt might be a main pathway to change symbiotic capacity in maff <dig>  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 maff <dig>  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  <cit> .
although nfs and secreted effectors of t3ss in maff <dig> 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   <cit> .
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  <cit> , can also participate in the control of genotype-specific infection and nodulation.
symbiosis island dynamics and the origin of symbiotic plasmids
although the chromosomes of 7653r and maff <dig> showed good overall co-linearity, a large, approximately 610-kb genomic region unique to maff <dig> was identified .
comparison of 7653r genomic structures to genomes of r7a and maff <dig> using the act   <cit>  confirmed this observation .
such genome-specific sequences were also noticed in similar positions in the other three genomes  , which was verified through genome alignment using promer   <cit>  .
these genome-specific regions harbor most nodulation and nitrogen-fixation genes.
interestingly, homologs of these nodulation and nitrogen-fixation genes can be traced to the two 7653r plasmids, suggesting a connection between the ‘missing’ dna fragment and these symbiotic plasmids .figure  <dig>
four mesorhizobial genomes plotted against the 7653r genome using promer .
a sequence aligned according to the maff <dig> replication origin and reverse-complement sequences of wsm <dig>  wsm <dig>  and wsm <dig> were used.
line figures depict the results of promer analysis.
red dots represent similar sequences in the forward direction in each genome pair, whereas blue dots indicate that the similarity is in the opposite orientation.
green arrows show the location of fragments missing on the 7653r chromosome.
of the five mesorhizobium strains whose genomes have been completely sequenced , 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 maff <dig> and the gap in 7653r , 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  <cit> .
as expected, islandviewer identified these maff <dig>  wsm <dig>  wsm <dig>  and wsm <dig> symbiosis islands as typical geis .
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  and similar gc content , which is strikingly lower than that of the corresponding genome .
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 .
third, t3sss and/or t4sss of the five strains are all located in the corresponding candidate geis.
fourth, a highly conserved trna 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  <cit> .
because the five geis likely share a common ancestor, we expected them to maintain well-conserved syntenic relationships.
although the geis in wsm <dig>  wsm <dig>  and wsm <dig> 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 maff <dig> gei, which possesses  <dig> predicted transposase genes—86% of all transposase genes in the entire maff <dig> genome.
similarly, 85%  of all transposase genes identified in the entire contigs of r7a are found in the symbiosis island of contig  <dig>  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 maff <dig> 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 maff <dig> has acquired many foreign genes by hgt  <cit> .
our clustering analysis of transposase genes in the plasmids of 7653r and the maff <dig> 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.
it has been speculated that geis may be derived from integrating mobile genetic elements such as plasmids or phages.
their acquisition by hgt and integration with the host chromosome by site-specific recombination might lead to the formation of a new gei  <cit> .
slater et al.
have proposed that integration of an ancestral intragenomic translocation recipient  plasmid into the main chromosome is an important evolutionary pathway in rhizobiales <cit> .
bradyrhizobium and mesorhizobium strains with a few or no relatively small plasmids are typically cited as examples, although the sole evidence for this viewpoint is the presence of itr plasmid gene clusters and other plasmid genes on the chromosomes of these species.
aside from plasmid genes shown on chromosomes, further evidence based on genomic structure, nucleotide composition, and transposase genes was used in this study to infer a possible evolutionary pathway explaining gei formation .
in our proposed scenario, integration of an itr plasmid into an ancestral mesorhizobium main chromosome would be followed by the formation of a new gei—the original parent of the present-day chromosomal geis of the four fully sequenced mesorhizobium strains.
because the evolving strains lived under different environmental conditions and experienced different selection pressures, the new geis underwent various changes at different rates.
geis of some strains, such as wsm <dig>  wsm <dig>  and wsm <dig>  maintained high conservation under weak selection pressures.
geis of strains such as 7653r and maff <dig>  however, underwent frequent recombination events that created high levels of instability.
in particular, geis of 7653r and maff <dig> both encode mobility enzymes, such as integrase, that allow excision from the host chromosome.
nevertheless, only the original gei of 7653r can excise itself spontaneously from the chromosome and form replicable plasmids.
the gei of maff <dig> may have become immobilized because of failure to regain the origins of plasmid replication or the genes involved in mobilization  <cit> .figure  <dig>
the contribution of genome dynamics to general
mesorhizobium
evolution.
chromosomal, itr plasmid, and foreign genes are shown in different colors.
many transposase genes exist within geis of 7653r and maff <dig>  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 maff <dig> 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 <cit> .
for better survival and growth in various habitats, geis from maff <dig> acquired some foreign nodulation genes by hgt during the genetic information exchange process, enabling functional symbiosis between maff <dig> 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  <cit> .
in legionella pneumophila, a newly identified conjugation/type-iva secretion system  composed of clusters of tra and trb genes  seems to be necessary for integrase-dependent excision and horizontal transfer of geis  <cit> .
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 maff <dig>  wsm <dig>  wsm <dig>  wsm <dig>  and r7a  <cit>  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 maff <dig>  wsm <dig>  wsm <dig>  and wsm <dig>  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.
CONCLUSIONS
whole-genome sequencing has proven valuable and critical for refining the phylogenetic positions of a series of rhizobial strains  <cit> .
in this study, we sequenced, assembled, and annotated the m. huakuaii 7653r genome.
we used this genome sequence to examine the phylogenetic position of maff <dig>  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 maff <dig> as a strain of m. huakuii, as is 7653r.
although 7653r and maff <dig> are both strains of m. huakuii, they exhibit important differences in symbiotic phenotypes and thus belong to different symbiosis variants   <cit> .
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 .
furthermore, our analysis of the three groups of signaling molecules revealed substantial differences between the two m. huakuii strains 7653r and maff <dig> 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 usda <dig>  and comparative genomic hybridization profiles show that geis may be highly dynamic entities in b. japonicum genomes  <cit> .
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  <cit> .
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  or can be transferred to other suitable recipients  <cit> .
we therefore conclude that 7653r plasmids may have arisen by the excision of the original gei from the 7653r chromosome.

 rhizobia are nitrogen-fixing soil bacteria constituting around known species classified into genera. mesorhizobium, whose growth rate is intermediate between that of genera rhizobium and bradyrhizobium, is one of the largest genera; it presently comprises species found primarily in asia, europe, the mediterranean region, and africa. 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. 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 maff initially considered a strain of m. loti. comparative sequence analysis of additional conserved genes have suggested instead a closer phylogenetic relationship with strains of a different species, m. huakuii, prompting the hypothesis that maff 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, 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 maff to m. huakuii. although maff 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 maff 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 maff 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 wsm, m. australicum wsm and m. opportunistum wsm, show patterns similar to those found in maff 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.  complete sequencing of the m. huakuii 7653r genome our,675-bp assembly of the m. huakuii 7653r genome consisted of three circular replicons of,365 bp,  bp, and  bp. the average gc content of the whole genome was calculated to be %, while plasmids pmhu7653ra and pmhu7653rb showed slightly lower gc levels. an overview of the gc content of the three replicons is shown on the 7653r genome physical maps. the main genome characteristics of 7653r and four other mesorhizobium genomes are summarized in table  although the five strains all belong to the same genus, they possess different numbers of plasmids: 7653r and maff have two plasmids each, wsm has only one, and wsm and wsm have none.figure physical maps of the complete mesorhizobium huakuii 7653r genome. physical maps of three replicons were drawn using circos. displayed circles from the inside outwards represent: g-c skew using a 1-kb window, codon adaptation index, clusters of orthologous groups of proteins in a counterclockwise/clockwise direction, predicted coding sequences transcribed in both directions, and scale in mb. the position represents the origin of replication in each replicon. general genomic features of mesorhizobium huakuii 7653rand four other mesorhizobial genomes size g+c content total no. of cds cds coverage trna genes rrna operons nb cds with assigned functions average length of genes putative transposases tandem repeat sequences transcriptional regulators putative abc transporters-related proteins putative two component systems-related proteins putative sigma factor-related proteins cds: coding sequences. statistics for maff wsm wsm and wsm genomic features are based on ncbi annotations. we predicted protein-coding genes in the 7653r genome, a number essentially identical despite the different genome sizes to the number predicted in maff. 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. however, the numbers of putative transposase genes predicted in these genomes were dramatically different. as discussed later, this variation may have a profound differential impact on genome stability and horizontal gene transfer events. genomic evidence supporting maf as a strain of m. huakuii maff has been hypothesized to be a strain of m. huakuii on the basis of comparative analysis of a few conserved genes in maff and m. huakuii strains. the availability of genome sequences of both strains has enabled us to re-examine their phylogenetic relationship. genome-wide orthologs we identified a set of orthologous groups among five mesorhizobium genomes. of these groups, were found to be conserved across all five genomes, with each group represented by at least one gene in each of the five genomes. we termed this subset of orthologous groups the core genome of mesorhizobium. an additional were observed to be present in four of the five genomes. the remaining orthologous groups occurred in two or three genomes. similar numbers and proportions of proteins predicted in 7653r and maff were present in the core genome, whereas % of predicted proteins in r7a were present in the core genome. among all pair-wise comparisons, the 7653r-maff pair was found to share the most abundant orthologous proteins, followed by the r7a-maff and wsm1271-wsm pairs; this ordering suggests that maff has a closer phylogenetic relationship to 7653r than to r7a, wsm and wsm from the 7653r core genes, we randomly chose single-copy genes and performed hierarchical clustering analysis based on their presence or absence in representative rhizobial species. the clustering results also revealed a closer phylogenetic relationship between 7653r and maff, further supporting maff as a strain of m. huakuii.figure core and accessory genome analyses of mesorhizobium strains. the numbers of orthologous groups and related genes found in each intersection are shown. the numbers of genes found in related strains for each intersection are shown in parentheses. the numbers of transposase genes are shown in square brackets. areas are not in scale. hierarchical clustering analysis of rhizobia based on a heat map of genes chosen from mesorhizobium core genes. gene homologs were chosen on the basis of blastp results. homolog presence and absence are indicated by yellow and red, respectively. the five genera for which multiple genomes were available are indicated in different colors. the numbers on the dendrogram represent bootstrap values. synteny analysis the above analysis of orthologs suggested that maff is phylogenetically more closely related to m. huakuii strain 7653r than to strains of other species. we further hypothesized that m. huakuii strain 7653r and maff share larger synteny blocksbetween them than with the other three strains. to test this hypothesis, we carried out a synteny analysis of five strains using a few complementary approaches. for convenience of comparison, we considered dnaa to be the start position of the main chromosome and repabc to be the start position of plasmids in all five genomes. mauve alignment of chromosomes of the five strains revealed a remarkably consistent backbone. compared with wsm wsm and wsm synteny blocks of the 7653r chromosome shared a longer average length and more consistent relative positions with maff additionally, fewer sequence inversions were observed between the chromosomes of 7653r and maff than between 7653r and wsm wsm or wsm we used orthocluster to identify synteny blocks perfectly conserved between each pair of these five genomes, and assigned a score to each 7653r gene according to the length of the synteny. we found that the mean score of all genes in synteny between 7653r and maff was larger than the mean scores between 7653r and wsm, wsm, or wsm. additionally, we performed a statistical test of significance, the results of which are shown additional file 3: table s moreover, analyses of phylogenetic relationships based on the consistency of dna sequences using mauve and the composition vector using cvtree both indicated that 7653r has a closer relationship to maff than to the other three mesorhizobium strains.figure chromosomal alignment of five mesorhizobial species visualized using mauve and a phylogenetic tree. chromosomal alignments for 7653r and maff, 7653r and wsm, 7653r and wsm, and 7653r and wsm were created using mauve software. each colored block represents a synteny block and is internally independent from genomic rearrangement. white regions correspond to unaligned sequences that likely contain sequence elements specific to a particular genome. blocks below the center line indicate regions that are aligned in the reverse complement orientation. phylogenetic tree created by mauve based on dna sequence consistency. thus, both ortholog and synteny analyses support a closer phylogenetic relationship between 7653r and maff than with the other mesorhizobium strains. these results provide further evidence that maff is a strain of m. huakuii. host specificity although 7653r and maff 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, maff forms symbioses with several lotus species host plants, including l. japonicus and l. corniculatus. 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, 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. nodulation factors nfs, which are signaling molecules between symbiotic bacteria and plants, are produced by bacteria in response to flavonoids secreted by legume root hairs. in most rhizobia, expression of nodulation genes is needed for the biosynthesis and transport of nfs that induce nodule organogenesis. a total of nodulation genes were identified in the 7653r chromosome and plasmids, while nodulation genes were located in maff. in contrast, in r7a, nodulation genes were found to be present and all were found to be homologous with very high similarities to genes in maff comparison of nodulation genes between these three strains not only revealed some genes with high sequence similarity but also uncovered two striking differences likely related to nf synthesis.figure similarities between maff wsm wsm and wsm nodulation genes and those of 7653r. first, genomic distribution of these nodulation genes was different between 7653r and maff while all nodulation genes in maff were found on its chromosome, only nodulation genes were present on the chromosome of 7653r, with found on its plasmids. specifically, the nod genes were identified in a 140-kb genomic region of the pmhu7653rb plasmid of 7653r. this genomic region also contained fix genes and nif genes. the nod genes were well conserved across all six genomes, as were the fix genes and nif genes. although these nod genes individually exhibited strong conservation, major differences were observed in regard to their arrangement in the genomes. for example, the nod genes on the pmhu7653rb plasmid of 7653r were found to be segregated into two independent operons preceded by two nod-boxes, with noda separated from nodbc by a 22-kb genome region containing genes. in sharp contrast, orthologs of noda and nodbc in other mesorhizobium strains, including maff and r7a, are adjacent and localized on the same strand.figure arrangement of nodulation genes, nitrogen fixation genes, and gene clusters in the 7653r plasmid pmhu7653rb. double slash marks represent genome regions that are not shown. in the clusters, nitrogen-fixation genes conserved among the six strains are represented by white arrows, while those varying in copy number, location, or transcriptional orientation are shown in different colors. nodulation and nitrogen-fixation gene clusters in 7653r plasmid pmhu7653rb. genes are colored according to their names. double slash marks represent genome regions that are not shown. arrows indicate the location of potential nod-boxes. second, the numbers of nodulation genes putatively participating in nf synthesis were found to be different between these two strains. the nod gene nodh, required for nf synthesis in 7653r, had no ortholog in maff and r7a. each of the four nod genes in maff had a substantially higher percentage identity with its ortholog in r7a than with its ortholog in 7653r. for example, nodc in maff had a pid of 99% with its ortholog in r7a and a pid of 74% with its ortholog in 7653r. interestingly, seven nodulation genes in maff with orthologs in both 7653r and r7a were found to have substantially higher pids with their orthologs in r7a than with those in 7653r; nine nodulation genes in maff had high pids with their orthologs in r7a but had no orthologs in 7653r. these results suggest that maff may have obtained these nodulation genes from an ancestor of r7a. thus, although maff shares nodulation genes with high pids with 7653r, it shares nodulation genes with high pids with r7a. furthermore, maff was found to have an additional five nodulation genes. taken together, 7653r and maff have very different numbers of nodulation genes. indeed, different nodulation genes are required for nf synthesis in these two strains. of the nod genes identified in 7653r, are possibly key elements involved in the synthesis of the core nfs of 7653r. in the m. loti strain r7a, nod genes organized in seven transcriptional units—noekj, nodznoelnolk, nods, nodacijnolo, nodb, noll, and nodm—are needed for nf synthesis. considering that many of the maff nodulation genes showed higher pids with r7a genes, we further propose that nodulation genes required for the synthesis of nfs in maff are closely related to those in r7a. this inference is consistent with a report that maff and r7a may share the same steps of nf synthesis.figure nodulation factor biosynthesis pathway in m. huakuii 7653r. biosynthesis pathway of the core nfs of strain 7653r and involved nod proteins are presented. two variable ends of the chemical structure of the core nfs, r and r are shown in the figure. r thus far seems to be represented only by h in the 7653r nf structures. surface polysaccharides rhizobial cell-surface polysaccharides, including cyclic-β-glucans, exopolysaccharides, lipopolysaccharides, and capsular polysaccharides, 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, epss, and lpss 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. secretion system proteins secreted by some rhizobial strains play an important role in infection of leguminous plant roots and establishment of a mutually beneficial symbiosis. different types and numbers of protein secretion systems are present in almost all rhizobial strains. by means of similarity searches using protein secretion genes identified in other gram-negative bacteria as queries, we identified genes related to secretory processes in the 7653r genome. these genes and proteins are involved in putative protein secretion systems: a general export pathway, four separate type-i systems, a twin-arginine translocase secretion system, one functional type-iii system, two type-iv systems, one type-v autotransporter, and two putative type-vi secretion systems.table numbers and distributions of genes associated with different types of secretion systems in mesorhizobial genomes type i type ii type iii type iv type v type vi srp mechanosensitive channels total 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. the 7653r genome was found to contain a complete t3ss on the pmhu7653rb plasmid, with gene organization conserved with respect to maff proteins secreted by rhizobial t3ss are called nodulation outer proteins and can be divided into two types: effectors and helper proteins. t3sss of both 7653r and maff have three helper proteins, nopa, nopb, and nopx, but different candidate effectors: nopp in 7653r and nopc in maff. 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 maff the vir system, an important example of a t4ss, is usually formed by proteins, virb1–virb and vird except for virb and virb these proteins are encoded by genes on plasmid pmhu7653ra. interestingly, neither virb nor virb are present in maff and r7a. the vir systems of 7653r and maff 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 maff has trb genes. 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 maff and r7a genomes. taken together, our analysis revealed that the two m. huakuii strains 7653r and maff 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 maff 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 maff 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 maff 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. 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 although the chromosomes of 7653r and maff showed good overall co-linearity, a large, approximately 610-kb genomic region unique to maff was identified. comparison of 7653r genomic structures to genomes of r7a and maff using the act confirmed this observation. such genome-specific sequences were also noticed in similar positions in the other three genomes, which was verified through genome alignment using promer. these genome-specific regions harbor most nodulation and nitrogen-fixation genes. interestingly, homologs of these nodulation and nitrogen-fixation genes can be traced to the two 7653r plasmids, suggesting a connection between the ‘missing’ dna fragment and these symbiotic plasmids.figure four mesorhizobial genomes plotted against the 7653r genome using promer. a sequence aligned according to the maff replication origin and reverse-complement sequences of wsm wsm and wsm were used. line figures depict the results of promer analysis. red dots represent similar sequences in the forward direction in each genome pair, whereas blue dots indicate that the similarity is in the opposite orientation. green arrows show the location of fragments missing on the 7653r chromosome. of the five mesorhizobium strains whose genomes have been completely sequenced, 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 maff and the gap in 7653r, 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 maff wsm wsm and wsm symbiosis islands as typical geis. 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 and similar gc content, which is strikingly lower than that of the corresponding genome. 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. third, t3sss and/or t4sss of the five strains are all located in the corresponding candidate geis. fourth, a highly conserved trna 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 wsm wsm and wsm 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 maff gei, which possesses predicted transposase genes—86% of all transposase genes in the entire maff genome. similarly, 85% of all transposase genes identified in the entire contigs of r7a are found in the symbiosis island of contig 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 maff 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 maff has acquired many foreign genes by hgt. our clustering analysis of transposase genes in the plasmids of 7653r and the maff 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. it has been speculated that geis may be derived from integrating mobile genetic elements such as plasmids or phages. their acquisition by hgt and integration with the host chromosome by site-specific recombination might lead to the formation of a new gei. slater et al. have proposed that integration of an ancestral intragenomic translocation recipient plasmid into the main chromosome is an important evolutionary pathway in rhizobiales. bradyrhizobium and mesorhizobium strains with a few or no relatively small plasmids are typically cited as examples, although the sole evidence for this viewpoint is the presence of itr plasmid gene clusters and other plasmid genes on the chromosomes of these species. aside from plasmid genes shown on chromosomes, further evidence based on genomic structure, nucleotide composition, and transposase genes was used in this study to infer a possible evolutionary pathway explaining gei formation. in our proposed scenario, integration of an itr plasmid into an ancestral mesorhizobium main chromosome would be followed by the formation of a new gei—the original parent of the present-day chromosomal geis of the four fully sequenced mesorhizobium strains. because the evolving strains lived under different environmental conditions and experienced different selection pressures, the new geis underwent various changes at different rates. geis of some strains, such as wsm wsm and wsm maintained high conservation under weak selection pressures. geis of strains such as 7653r and maff however, underwent frequent recombination events that created high levels of instability. in particular, geis of 7653r and maff both encode mobility enzymes, such as integrase, that allow excision from the host chromosome. nevertheless, only the original gei of 7653r can excise itself spontaneously from the chromosome and form replicable plasmids. the gei of maff may have become immobilized because of failure to regain the origins of plasmid replication or the genes involved in mobilization.figure the contribution of genome dynamics to general mesorhizobium evolution. chromosomal, itr plasmid, and foreign genes are shown in different colors. many transposase genes exist within geis of 7653r and maff 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 maff 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 maff acquired some foreign nodulation genes by hgt during the genetic information exchange process, enabling functional symbiosis between maff 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 composed of clusters of tra and trb genes 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 maff wsm wsm wsm 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 maff wsm wsm and wsm 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 maff 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 maff as a strain of m. huakuii, as is 7653r. although 7653r and maff are both strains of m. huakuii, they exhibit important differences in symbiotic phenotypes and thus belong to different symbiosis variants. 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. furthermore, our analysis of the three groups of signaling molecules revealed substantial differences between the two m. huakuii strains 7653r and maff 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 usda 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 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. 
our ortholog and synteny analyses firmly establish maff as a strain of m. huakuii. evidence based on genomic sequences is urgently needed to confirm the phylogenetic relationship between mesorhizobium strain maff and m. huakuii. to define underlying causes for the rather striking difference in host specificity between m. huakuii strain 7653r and maff several probable determinants also require comparison at the genomic level. in this study, we sequenced the complete genome of 7653r and compared it with five other mesorhizobium genomes. genomes of 7653r and maff were found to share a large set of orthologs and, most importantly, a conserved chromosomal backbone and even larger perfectly conserved synteny blocks. 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. differences in nodulation factors and secretion systems t3ss, t4ss, and t6ss may be responsible for the unique host specificities of 7653r and maff strains. the plasmids of 7653r may have arisen by excision of the original genomic island from the 7653r chromosome. we also identified candidate molecular differences responsible for the different host specificities of these two strains. mesorhizobium huakuii 7653rgenome sequencingcomparative analysishost specificitysymbiosis islandissue-copyright-statement© the author 2014
 evidence based on genomic sequences is urgently needed to confirm the phylogenetic relationship between mesorhizobium strain maff and m. huakuii. to define underlying causes for the rather striking difference in host specificity between m. huakuii strain 7653r and maff 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 maff 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 maff 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 maff strains. the plasmids of 7653r may have arisen by excision of the original genomic island from the 7653r chromosome. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords mesorhizobium huakuii 7653rgenome sequencingcomparative analysishost specificitysymbiosis islandissue-copyright-statement© the author 2014 