BACKGROUND
bacterial nitrogen fixation in legume nodules contributes to plant nutrition and allows plants to grow in nitrogen deficient soils.
genes for plant nodulation and nitrogen fixation are plasmid-borne in rhizobium spp.
and symbiovars define host specificity.
there are over twenty different symbiovars reported not only in rhizobium but also in bradyrhizobium and in other genera of nodule forming bacteria .
the term symbiovar was proposed as a counterpart to the term pathovar in pathogenic bacteria  <cit> .
a theoretical model proposes that a single species may exhibit alternative symbiovars depending on the presence of symbiotic plasmids or symbiotic islands  <cit> .
the same symbiovar may be present in distinct species as a consequence of the lateral transfer of symbiotic plasmids or islands.
symbiotic genes and other genes associated with niche adaptation may have evolutionary histories independent of the evolution of the chromosomal genes  <cit> .
two symbiovars are recognized in rhizobium etli: phaseoli  and mimosae .
symbiovar mimosae strains were isolated from mimosa affinis in morelos and have a broad host range, including plants of m. affinis, leucaena leucocephala, calliandra grandiflora, acaciella angustissima as well as p. vulgaris
<cit> .
symbiovar mimosae was originally distinguished from sv.
phaseoli by the sequence of a few symbiotic genes and by the organization of nif and common nod genes.
multiple copies of nifh genes and a noda gene separated from nodbc found in sv.
phaseoli and not in sv.
mimosae served as a molecular basis to distinguish these symbiovars  <cit> .
phylogenies indicated that symbiovar mimosae and phaseoli nifh genes were related  <cit>  and similar to the corresponding gene in sv.
gallicum  <cit> .
different origins of replication were found in sv.
phaseoli and sv.
mimosae symbiotic plasmids and both symbiotic plasmids were compatible  <cit> .
it has been proposed that sv.
mimosae is older than sv.
phaseoli and that the phaseoli symbiotic plasmid was selected by p. vulgaris
<cit> .
p. vulgaris is a recent species , while mimosas seem to be older.
mimosas are distributed worldwide with brazil and mexico as main diversification sites.
mimosas in south america are nodulated by β-proteobacteria like burkholderia or other β-proteobacteria  while mimosas in mexico are only exceptionally nodulated by burkholderia .
mimosas from mexico and brazil are phylogentically separated  <cit> .
additionally abiotic conditions like ph and soil nitrogen content may account for their differences in symbionts  <cit> .
native mimosas in india are nodulated by sinorhizobia  <cit> , that we have also found in some mimosa nodules in mexico .
based on multilocus enzyme electrophoresis analysis, m. affinis isolate mim <dig> was found to group closely to r. etli sv.
phaseoli cfn  <dig>  <cit> .
other m. affinis isolates such as mim <dig> were separated from cfn <dig>  and thus mim <dig> has been recently reassigned to rhizobium phaseoli
<cit> .
therefore we recognize now that symbiovar mimosae exists in r. phaseoli as well as in r. etli.
it is the aim of this work to define the genomic differences between two r. etli strains  representing the symbiovars phaseoli and mimosa, respectively.
methods
strains and growth conditions
rhizobium strains were grown overnight at 28ºc in py medium  <cit>  after recovery from glycerol stocks at -80ºc.
bacterial strains to be inoculated on plants were grown on solid py media and resuspended in water to an od <dig> of  <dig> .
for pcr or dna isolation, bacteria were grown in liquid py cultures  <cit> .
plasmid profiles
plasmid profiles were visualized on agarose gels according to the protocol described by hynes and mcgregor  <cit> .
plasmid patterns from r. etli cfn <dig> or r. leucaenae cfn <dig> were used as references.
plant nodulation assays
l. leucocephala seeds were treated with concentrated sulfuric acid for 15 min, rinsed with water and surface disinfected with sodium hypochlorite as described  <cit> ; the same procedure was used to disinfect p. vulgaris seeds.
seeds were germinated in water-agar plates in the dark and transferred to flasks after 3 days.
l. leucocephala plants were grown in vermiculite flasks with n free fahraeus nutrient solution for 40 days and p. vulgaris plants in agar flasks with the same nutrient solution for 14 days.
genome sequencing, assembly and annotation
genomic dna from r. etli mim <dig> was sheared to produce two paired-end libraries for  <dig> pyrosequencing, one with  <dig> kb inserts and the other with  <dig> kb inserts.
an additional  <dig> kb library was sequenced only at one end.
the total amount of reads were  <dig>  paired reads and  <dig>   <dig> single reads.
library construction and sequencing was done at mogene lc .
additionally two paired-end libraries were constructed, one with 200 bp inserts and the second with  <dig> kb inserts.
both libraries were sequenced by illumina at bgi .
a total amount of  <dig>   <dig>   <dig> short-paired readings  were assembled.
to improve the scaffolding a bac library was constructed in pindogo bac-5™ vector by bios&t  using fragments from a partial genomic dna hindiii digestion.
<dig> bac-ends were sequenced with abi3730xl sequencer by sanger method.
additionally, three bacs were completely sequenced with the same technology at the center for genomic sciences .
two of these bacs were selected by hybridizing with nifh and other psym probes; they embraced half of the psym plasmid sequence.
the third bac was from the chromosome.
final assembly of the symbiotic plasmid was obtained using sequence reads from the three sources of information: bac sequences, illumina, and  <dig> pyrosequencing.
different assembly strategies were used with the following programs: newbler  <dig> .
<dig> , velvet  <dig> .
<dig>  <cit> , sspace-basic  <dig>   <cit> , and consed v <dig>  <cit> .
orfs were predicted with glimmer  <dig>  <cit> , and annotations were done in artemis  <dig>  graphic display  <cit>  using previous annotations made for r. etli cfn <dig>  <cit>  and comparing with the non-redundant data base of the genbank  <cit> , interpro database  <cit> , and is database .
sequence analysis
average nucleotide identity  was calculated using the jspecies software  <cit> .
the dna conservation between two genomes or replicons was estimated by obtaining an alignment with nucmer  <cit> , run with default parameters, and dividing the summed lengths of all aligned regions by the length of the genome or replicon and expressing the value obtained as a percentage.
common and specific protein families between r. etli cfn <dig> and mim <dig> were detected using mcl as described  <cit> .
genomic islands
alien hunter v <dig>  software  <cit>  was used to analyze the chromosome sequence of r. etli sv.
mimosae mim <dig> and r. etli cfn <dig>  the minimum region length for ht detection was  <dig> kbs.
the score thresholds were  <dig>  and  <dig>  for mim <dig> and cfn <dig>  respectively.
phylogenetic analysis
alignments were performed with muscle  <cit>  and manually verified.
maximum likelihood trees were generated with phyml  <cit>  with tree node support evaluated by bootstrap analysis based on  <dig> pseudoreplicate datasets.
phylogenetic relationships were also assessed by bayesian inference using mrbayes  <dig>   <cit> .
analyses were initiated with random starting trees, run for  <dig> , <dig> generations and three separate analyses were executed.
markov chains were sampled every  <dig> generations.
we discarded 25% of trees as “burn in”.
genome accession numbers
sequences and annotations were deposited in the genbank database under accession numbers cp <dig> , cp <dig> , cp <dig> , cp <dig> , cp <dig> , cp <dig>  and cp <dig> .
RESULTS
genome of r. etlisymbiovar mimosae strain mim1
the final assembly of the r. etli sv.
mimosae mim <dig> genome rendered seven circular molecules: one chromosome and six plasmids at 150× coverage on average.
the chromosome was  <dig>  mb in size while the plasmids ranged in size from 181 kb to  <dig>  mb .
average nucleotide identity  and the percentage of conserved dna between mim <dig> and cfn <dig> were  <dig> % and  <dig> % respectively on a whole genome analysis, confirming that both strains belong to the same species.
lower ani  was found between mim <dig> and strains of other species such as r. phaseoli and r. leguminosarum.figure  <dig>
schematic representation of plasmid patterns of
rhizobium etli
symbiovars phaseoli and mimosae and
r. leguminosarum
.
equivalent replicons are indicated with the same color.
*indicates the symbiotic plasmid.
c indicates chromids.
there were more than twice as many unique genes in mim <dig> than in cfn <dig>  mainly in plasmids.
the respective chromosomes of each strain had around  <dig> unique genes.
in chromosomes,  <dig> genomic islands were identified only in mim <dig> and  <dig> only in cfn <dig>  ; genes found in mim <dig> genomic islands are shown in additional file 1: table s <dig>  examples of unique genes found in mim <dig> and not in cfn <dig> are those encoding cytochrome oxidases, some chaperonins, dipeptide transporters, lactate dehydrogenase, a phb depolymerase, a ferritin–like protein, exopolysaccharide biosynthesis genes, a type vi secretion system and menaquinone biosynthesis as well as many hypothetical genes.figure  <dig>
representation of
r. etli
mim <dig> chromosome.
circles from outermost to innermost indicate: genomic islands of mim <dig> , orfs in the leading strand, orfs in the lagging strand, blast matches against cfn <dig> chromosome, genomic islands of cfn <dig> , gc content, gc skew, coordinates.
the conserved genome in r. etli strains mim <dig> and cfn <dig> includes not only the chromosome but two extrachromosomal replicons  that have been designated as chromids in cfn <dig>  <cit>  and one plasmid  .
each of the chromids had less than  <dig> unique genes and the chromid pairs had an ani around 99% .
the small replicons precfn42a and pretmim1c were partially conserved and large genomic differences were found in the symbiotic plasmids .figure  <dig>
graphic comparison of equivalences among the
r. etli
cfn <dig> and
r. etli
mim <dig> genomes.
letters indicate the different extrachromosomal replicons found in both strains .
letters in bold indicate the symbiotic plasmids.
average nucleotide identity  and conservation in percent between
r. etli
sv.
mimosae mim <dig> and
r. etli
sv.
phaseoli cfn  <dig> or
r. leguminosarum
<dig> replicons
*average nucleotide identity was calculated using all portions of the replicons that could be aligned with the nucmer program.
these regions included both genic and intergenic regions.
§percentage of the mim <dig> replicon involved in the ani calculation is expressed here as the conservation value.
graphic representation of the alignments of cfn <dig> -mim <dig>  replicons.
a) pretmim1a-pretcfn42b, b) pretmim1b-pretcfn42c, c) pretmim1d-pretcfn42e, d) pretmim1e-pretcfn42d, e) pretmim1f-pretcfn42f.
orfs of each replicon are depicted with light blue arrows in their corresponding reading frame.
syntenic segments oriented in the same or opposite direction are joined by red and blue regions, respectively.
mim <dig> and cfn <dig> chromosomes were syntenic, as were the chromid pairs pretmim1a-pretcfn42b and pretmim1d-pretcfn42e, and plasmids pretmim1b-pretcfn42c.
in comparison to r. leguminosarum  <dig>  mim <dig> plasmid equivalences were similar to those reported previously for r. etli cfn <dig>  <cit> .
ani values were lower with r. leguminosarum  <dig> that among the r. etli strains .
all extrachromosomal replicons in mim <dig> belong to the repabc plasmid family  <cit> .
the protein products of the repabc operons of the replicon homologous pairs  were almost identical with a sequence identity greater than  <dig> %, strongly suggesting that members of each replicon pair belong to the same incompatibility group.
pretcfn42f-pretmim1f comparison
the largest extrachromosomal replicon in mim <dig>  was only partially conserved in the putative chromid pretcfn42f .
pretmim1f and pretcfn42f possess two repabc operons: repabc <dig> and repabc <dig>  the sequence identity between the two repabc operons in mim <dig> is low.
the degree of sequence identities between the corresponding repabc genes of pretcfn42f and pretmim1f is large enough to suggest that both plasmids share the same incompatibility group and evolutionary origin.
however only 51% of the pretmim1f replicon is conserved in cfn <dig> while 86% of pretcfn42f was found in pretmim1f .
type vi secretion system genes were only found in the megaplasmid pretmim1f and the conserved impb component is phylogenetically related to the corresponding genes in rhizobium sp.
kim <dig>  and r. leguminosarum strain  <dig>  mim <dig> pretmim1f has a duplicated citrate synthase gene as in sv.
tropici symbiotic plasmids.
the plasmid duplicated citrate synthase gene in sv.
tropici is required for eliciting a normal number of nodules and is regulated differently than the copy in the chromosome  <cit> .
a plasmid borne citrate synthase was not found in cfn <dig>  phylogenetic analysis of citrate synthase genes showed that the mim <dig> chromosomal gene product is identical to that of cfn <dig>  while the gene in pretmim1f has a novel sequence only distantly related to a plasmid copy of r. gallicum r <dig> .
genes such as raii and rair were found in the conserved region of pretcfn42f and pretmim1f.
raii produces homoserine lactones and rair is the transcriptional regulator.
the rai system in r. phaseoli sv.
phaseoli cnpaf  <dig> affects nodule number but not nitrogen fixation in p. vulgaris
<cit> .
this system also controls growth in r. phaseoli
<cit>  and if this is the case in r. etli, this would explain its conservation in both symbiovars.
a transcriptomic study compared the genome expression of r. phaseoli ch24- <dig> in maize and p. vulgaris rhizospheres  <cit> .
over 50% of the extrachromosomal genes highly expressed in p. vulgaris but not in maize roots were found in a ch24- <dig> replicon equivalent to pretcfn42f.
it seems that genes in this replicon are involved in plant specific interactions.
symbiosis plasmid gene comparison
large differences were observed between the symbiotic plasmids of cfn <dig> and mim <dig> .
around 10% and 15% of the symbiotic plasmids of mim <dig> and cfn <dig> had conserved syntenic regions with an average nucleotide identity of  <dig> % .
differences in symbiosis genes in cfn <dig> and mim <dig> genomes are shown in table  <dig>  the nod factor from r. etli sv.
phaseoli strain cfn <dig> is a pentamer of n-acetylglucosamine with an acetyl fucose at the reducing end and methyl and carbamoyl groups at the non reducing end  <cit> .
the heterologous expression in azorhizobium caulinodans of nod modification genes showed that fucosylated nod factors were the most suitable to induce p. vulgaris nodulation  <cit> .
in symbiovar mimosae no genes related to nod factor fucosylation  were observed , in their place, nodhpq genes that modify the nod factor with sulfate, were found.
such genes are present in sv.
tropici strains that are also leucaena symbionts  <cit> .
like r. etli strain cfn <dig>  mim <dig> may produce nodulation factors bearing carbamoyl groups at their non reducing end residues but the position of these decorations must differ because their psyms encoded distinct carbamoyl transferases, nolo in cfn <dig> and nodu in mim <dig>  carbamoylation at the c- <dig> position introduced by nodu maybe promotes leucaena nodulation  <cit> .
both mim <dig> and cfn <dig> symbiotic plasmids carry nods involved in methylation at the non reducing end, a decoration that is essential for bean and leucaena nodulation in r. tropici ciat  <dig>  <cit> .
a nodo homologue, 70% identical to that of rhizobium sp.
br <dig>  was found only in the sv.
mimosae psym.
it has been shown that heterologous expression of nodo can improve nodulation of l. leucocephala by different rhizobia and can even extend the host range  <cit> .table  <dig>
relevant symbiotic plasmid differences between
r. etli
sv.
mimosae mim <dig> and
r. etli
sv.
phaseoli cfn 42
nodhpq
nodz
noll
nodo
nolo
nodu
fixkl
fxkr
acds
teu
hup-hyp
*functions of each gene are explained in the text.
mim <dig> nod gene phylogenies are congruent, resembling the corresponding genes from sv.
giardinii  while mim <dig> nifh genes resemble those from sv.
phaseoli.
different nodds in phaseoli and mimosae symbiovars may reflect their affinities for the different flavonoids exuded by the different host plants.
mim <dig> nodh gene  resembles the corresponding gene in rhizobium sp.
ie <dig> that represents a novel genomic lineage related to r. etli and r. phaseoli
<cit> .
acds gene coding acc-deaminase was found in the symbiotic plasmid of mim <dig> but not in cfn <dig>  acc-deaminase decreases the amount of acc that is a precursor of ethylene that may diminish nodule number.
a heterologous acc-deaminase in rhizobium sp tal  <dig> enhanced nodulation in leucaena
<cit> .
in cfn <dig>  the fixghis-fixnoqp genes required for biosynthesis of the symbiotic terminal oxidase are present in the psym and also in pretcfn42f  <cit> .
the regulatory genes fixk and fixl are adjacent to this reiteration in pretcfn42f while a fixk pseudogene is found in the psym  <cit> .
in mim <dig>  we found that the symbiotic terminal oxidase genes are also reiterated in pretmim1f but, in contrast to cfn <dig>  the sv mimosae psym carried complete fixk and fixl genes.
the fixghis-fixnoqp-fixkl region shared by psym and pretmim1f plasmids in mim <dig> is 95% identical while the reiterated regions in cfn <dig> are only 87% identical.
the recently described fxkr gene in pretcfn42f  <cit> , coding for a response regulator that acts in conjunction with fixl and fixk, is present in the sv.
mimosae psym.
there is a reiteration of this gene in pretmim1f as well.
genes involved in p. vulgaris exudate uptake  are found in the symbiotic plasmids in symbiovar phaseoli strains r. etli cfn <dig> and r. phaseoli ciat <dig> but they were not found in the mim <dig> genome.
rhizobium mutants in teu genes had reduced nodulation competitiveness in p. vulgaris
<cit> .
the symbiosis plasmid of mim <dig> has genes for a type iii secretion system  that are more closely related to those found in mesorhizobium and sinorhizobium strains than to those encoded in the cfn <dig> psym .
this difference may contribute to the disparate host ranges displayed by sv.
phaseoli and mimosae strains considering the function of rhizobial t3ss in specificity  <cit> .
a cluster of hup-hyp genes encoding the components of an uptake hydrogenase  was found in the psym of mim <dig> but not in cfn <dig>  the mim <dig> products showed high identities  to their counterparts coded in r. leguminosarum and r. tropici
<cit> .
r. tropici ciat  <dig> lacks several hup genes and displays a hup minus phenotype.
in mim <dig>  all genes except hupe are present.
hupe is an uptake transporter for nickel  <cit> , a metal required for hup function.
since another nickel transporter is encoded elsewhere in mim <dig> symbiotic plasmid, the hup system could be functional.
mim <dig> symbiotic plasmid has a repabc origin of replication as well as a repc gene that are not phylogenetically related to the r. etli cfn <dig> corresponding genes.
pretmim1e repc resembles those from r. gallicum and rhizobium sp.
sv.
giardinii ie <dig> .
mim <dig> repabc from the psym resembles the corresponding genes in r. endophyticum ccge <dig> but an extensive plasmid conservation was not observed.
ccge <dig> is nod- and does not have a symbiotic plasmid  <cit> .
the analysis of insertion sequences in r. etli cfn <dig> suggested that the symbiotic plasmid did not significantly share is sequences with the rest of the genome  <cit> .
this was interpreted as evidence that the psym was a recent acquisition in this bacterium.
the analysis of is sequences in mim <dig> indicated that the symbiotic plasmid had the largest number of is sequences, some of them shared with the chromosome, pretmim1f and pretmim1b, this may perhaps indicate that this symbiotic plasmid has an older history with the r. etli genomic background than the phaseoli plasmid.
mim <dig> has a large number of is <dig> that are common in rhizobia.
genome similarities were found among the different mim <dig> replicons.
pretmim1e  has similar sequences to pretmim1c and the same is observed among pretmim1e  and pretmim1f .
the similarities of mim <dig> symbiotic plasmid and the putative chromid pretmim1f could support that symbiovar mimosae symbiotic plasmid is ancestral in r. etli.
in contrast the phaseoli symbiotic plasmid was found dissimilar to the rest of the genomic background in r. etli, except to precfn42a  <cit> .figure  <dig>
graphic comparison of the mim <dig> symbiotic plasmid pretmim1e compared to other rhizobial replicons.
in r. etli cfn <dig>  the symbiotic plasmid and pretcfn42a have common sequences such as tra and vir genes and repeated sequences that mediate the natural cointegration of both plasmids for the conjugative transfer of cfn <dig> psym  <cit> .
in contrast to precfn42a, pretmim1c from mim <dig> does not seem to participate in the transfer of the mim <dig> symbiotic plasmid, which we have been unable to transfer to other bacterial hosts .
plant-interaction genes not in the symbiosis plasmid
some genes involved in plant interactions were found conserved in both symbiovars, such as those encoding rmr extrusion pumps that may be involved in eliminating plant produced phytoalexins.
r. etli mutants in these genes had reduced nodulation  <cit> .
those genes are encoded in chromid pretcfn42b in cfn <dig> and in the corresponding replicon pretmim1a.
rmra gene had 97% identity and rmrb and rmrr genes 98% in cfn <dig> and mim <dig> genomes.
homologous genes were found being expressed in different plant rhizospheres  <cit> .
even though sv.
mimosae strains are capable of forming nodules in leucaena, we did not find genes resembling mid or pyd genes involved in the catabolism of the toxic amino acid derivative mimosine found in leucaena plants.
such genes were described from a rhizobium sp.
strain tal  <dig>  that was isolated from leucaena plants  <cit> .
mimosa pigra has a much lower level of mimosine that leucaena plants  <cit>  and data for other mimosa species is not available.
symbiovar phaseoli is prevalent in different phaseolus vulgarisnodule bacteria
r. etli sv.
mimosae strains have a broader host range than symbiovar phaseoli strains.
in particular, l. leucocephala plants served as a host to distinguish symbiovar mimosae strains.
thirty-six p. vulgaris nodulating bacteria  obtained from the rain forest of los tuxtlas in mexico corresponded to symbiovar phaseoli on the basis of noda gene organization  <cit>  and for being unable to nodulate leucaena plants.
we found that some strains that were previously classified as r. etli such as ciat <dig>  ch24- <dig>  cnpaf <dig>  8c- <dig> and brasil <dig> now assigned to r. phaseoli
<cit> , as well as others like kim <dig>  gr <dig> and ciat  <dig> now assigned to recently named novel lineages  <cit>  corresponded to sv.
phaseoli when we analyzed their genomes.
symbiotic plasmids are highly conserved in symbiovar phaseoli  <cit>  perhaps from being recently evolved  <cit> .
considering that the majority of p. vulgaris nodule isolates tested corresponded to sv.
phaseoli we may conclude that this symbiovar is better adapted to its host, thus phaseoli seems to be a specialist symbiovar having a narrow range not including mimosa plants.
the phaseoli symbiovar is found in several rhizobium species or lineages .
the widespread of this symbiovar may be in relation to its host historic worldwide distribution and to the transferability of the symbiotic plasmid, seemingly an epidemic plasmid.
besides having the phaseoli symbiovar, the rhizobium species mentioned above have additional generalist symbiovars: gallicum or giardinii or mimosae having alternative symbiovars with different host ranges may be advantageous in rhizobia, as it expands their legume niches and allows them to avoid the specialist-generalist dilemma.
CONCLUSIONS
the term symbiovar is validated with genomic analyses that show that a common genomic background may harbor different symbiotic plasmids determining host specificity.
however, besides differences in the symbiotic plasmids there were differences in other ers and in the chromosomes in the two strains analyzed, cfn <dig> and mim <dig>  in mim <dig>  nod factors with sulfate modifications, secretion systems or acc-deaminase may help explain the extended host range of symbiovar mimosae.
in cfn <dig>  teu genes that participate in exudate uptake  <cit>  and genes involved in nod factor fucosylation  may contribute to p. vulgaris host specialization.
the discussion that mimosae is older than phaseoli may apply to gallicum and giardinii, thus we propose that gallicum and giardinii are older than phaseoli.
electronic supplementary material
additional file 1: table s1: genes found in different genomic islands exclusive of mim <dig>
competing interests
the authors declare that they have no competing interests.
authors’ contributions
emr initiated and designed the study.
mar performed experiments.
vg, dr, mac contributed reagents and analyzed data.
eoo, pb, ris, jmr, jcm, ll performed data analysis.
emr, eoo, mar wrote the paper.
all authors read and approved the manuscript.

 bacterial nitrogen fixation in legume nodules contributes to plant nutrition and allows plants to grow in nitrogen deficient soils. genes for plant nodulation and nitrogen fixation are plasmid-borne in rhizobium spp. and symbiovars define host specificity. there are over twenty different symbiovars reported not only in rhizobium but also in bradyrhizobium and in other genera of nodule forming bacteria. the term symbiovar was proposed as a counterpart to the term pathovar in pathogenic bacteria. a theoretical model proposes that a single species may exhibit alternative symbiovars depending on the presence of symbiotic plasmids or symbiotic islands. the same symbiovar may be present in distinct species as a consequence of the lateral transfer of symbiotic plasmids or islands. symbiotic genes and other genes associated with niche adaptation may have evolutionary histories independent of the evolution of the chromosomal genes. two symbiovars are recognized in rhizobium etli: phaseoli and mimosae. symbiovar mimosae strains were isolated from mimosa affinis in morelos and have a broad host range, including plants of m. affinis, leucaena leucocephala, calliandra grandiflora, acaciella angustissima as well as p. vulgaris. symbiovar mimosae was originally distinguished from sv. phaseoli by the sequence of a few symbiotic genes and by the organization of nif and common nod genes. multiple copies of nifh genes and a noda gene separated from nodbc found in sv. phaseoli and not in sv. mimosae served as a molecular basis to distinguish these symbiovars. phylogenies indicated that symbiovar mimosae and phaseoli nifh genes were related and similar to the corresponding gene in sv. gallicum. different origins of replication were found in sv. phaseoli and sv. mimosae symbiotic plasmids and both symbiotic plasmids were compatible. it has been proposed that sv. mimosae is older than sv. phaseoli and that the phaseoli symbiotic plasmid was selected by p. vulgaris. p. vulgaris is a recent species, while mimosas seem to be older. mimosas are distributed worldwide with brazil and mexico as main diversification sites. mimosas in south america are nodulated by β-proteobacteria like burkholderia or other β-proteobacteria while mimosas in mexico are only exceptionally nodulated by burkholderia. mimosas from mexico and brazil are phylogentically separated. additionally abiotic conditions like ph and soil nitrogen content may account for their differences in symbionts. native mimosas in india are nodulated by sinorhizobia, that we have also found in some mimosa nodules in mexico. based on multilocus enzyme electrophoresis analysis, m. affinis isolate mim was found to group closely to r. etli sv. phaseoli cfn. other m. affinis isolates such as mim were separated from cfn and thus mim has been recently reassigned to rhizobium phaseoli. therefore we recognize now that symbiovar mimosae exists in r. phaseoli as well as in r. etli. it is the aim of this work to define the genomic differences between two r. etli strains representing the symbiovars phaseoli and mimosa, respectively. methods strains and growth conditions rhizobium strains were grown overnight at 28ºc in py medium after recovery from glycerol stocks at -80ºc. bacterial strains to be inoculated on plants were grown on solid py media and resuspended in water to an od of. for pcr or dna isolation, bacteria were grown in liquid py cultures. plasmid profiles plasmid profiles were visualized on agarose gels according to the protocol described by hynes and mcgregor. plasmid patterns from r. etli cfn or r. leucaenae cfn were used as references. plant nodulation assays l. leucocephala seeds were treated with concentrated sulfuric acid for 15 min, rinsed with water and surface disinfected with sodium hypochlorite as described; the same procedure was used to disinfect p. vulgaris seeds. seeds were germinated in water-agar plates in the dark and transferred to flasks after 3 days. l. leucocephala plants were grown in vermiculite flasks with n free fahraeus nutrient solution for 40 days and p. vulgaris plants in agar flasks with the same nutrient solution for 14 days. genome sequencing, assembly and annotation genomic dna from r. etli mim was sheared to produce two paired-end libraries for pyrosequencing, one with kb inserts and the other with kb inserts. an additional kb library was sequenced only at one end. the total amount of reads were paired reads and single reads. library construction and sequencing was done at mogene lc. additionally two paired-end libraries were constructed, one with 200 bp inserts and the second with kb inserts. both libraries were sequenced by illumina at bgi. a total amount of short-paired readings were assembled. to improve the scaffolding a bac library was constructed in pindogo bac-5™ vector by bios&t using fragments from a partial genomic dna hindiii digestion. bac-ends were sequenced with abi3730xl sequencer by sanger method. additionally, three bacs were completely sequenced with the same technology at the center for genomic sciences. two of these bacs were selected by hybridizing with nifh and other psym probes; they embraced half of the psym plasmid sequence. the third bac was from the chromosome. final assembly of the symbiotic plasmid was obtained using sequence reads from the three sources of information: bac sequences, illumina, and pyrosequencing. different assembly strategies were used with the following programs: newbler., velvet., sspace-basic, and consed v. orfs were predicted with glimmer, and annotations were done in artemis graphic display using previous annotations made for r. etli cfn and comparing with the non-redundant data base of the genbank, interpro database, and is database. sequence analysis average nucleotide identity was calculated using the jspecies software. the dna conservation between two genomes or replicons was estimated by obtaining an alignment with nucmer, run with default parameters, and dividing the summed lengths of all aligned regions by the length of the genome or replicon and expressing the value obtained as a percentage. common and specific protein families between r. etli cfn and mim were detected using mcl as described. genomic islands alien hunter v software was used to analyze the chromosome sequence of r. etli sv. mimosae mim and r. etli cfn the minimum region length for ht detection was kbs. the score thresholds were and for mim and cfn respectively. phylogenetic analysis alignments were performed with muscle and manually verified. maximum likelihood trees were generated with phyml with tree node support evaluated by bootstrap analysis based on pseudoreplicate datasets. phylogenetic relationships were also assessed by bayesian inference using mrbayes. analyses were initiated with random starting trees, run for, generations and three separate analyses were executed. markov chains were sampled every generations. we discarded 25% of trees as “burn in”. genome accession numbers sequences and annotations were deposited in the genbank database under accession numbers cp, cp, cp, cp, cp, cp and cp.  genome of r. etlisymbiovar mimosae strain mim1 the final assembly of the r. etli sv. mimosae mim genome rendered seven circular molecules: one chromosome and six plasmids at 150× coverage on average. the chromosome was  mb in size while the plasmids ranged in size from 181 kb to  mb. average nucleotide identity and the percentage of conserved dna between mim and cfn were % and % respectively on a whole genome analysis, confirming that both strains belong to the same species. lower ani was found between mim and strains of other species such as r. phaseoli and r. leguminosarum.figure schematic representation of plasmid patterns of rhizobium etli symbiovars phaseoli and mimosae and r. leguminosarum. equivalent replicons are indicated with the same color. *indicates the symbiotic plasmid. c indicates chromids. there were more than twice as many unique genes in mim than in cfn mainly in plasmids. the respective chromosomes of each strain had around unique genes. in chromosomes, genomic islands were identified only in mim and only in cfn; genes found in mim genomic islands are shown in additional file 1: table s examples of unique genes found in mim and not in cfn are those encoding cytochrome oxidases, some chaperonins, dipeptide transporters, lactate dehydrogenase, a phb depolymerase, a ferritin–like protein, exopolysaccharide biosynthesis genes, a type vi secretion system and menaquinone biosynthesis as well as many hypothetical genes.figure representation of r. etli mim chromosome. circles from outermost to innermost indicate: genomic islands of mim, orfs in the leading strand, orfs in the lagging strand, blast matches against cfn chromosome, genomic islands of cfn, gc content, gc skew, coordinates. the conserved genome in r. etli strains mim and cfn includes not only the chromosome but two extrachromosomal replicons that have been designated as chromids in cfn and one plasmid. each of the chromids had less than unique genes and the chromid pairs had an ani around 99%. the small replicons precfn42a and pretmim1c were partially conserved and large genomic differences were found in the symbiotic plasmids.figure graphic comparison of equivalences among the r. etli cfn and r. etli mim genomes. letters indicate the different extrachromosomal replicons found in both strains. letters in bold indicate the symbiotic plasmids. average nucleotide identity and conservation in percent between r. etli sv. mimosae mim and r. etli sv. phaseoli cfn or r. leguminosarum replicons *average nucleotide identity was calculated using all portions of the replicons that could be aligned with the nucmer program. these regions included both genic and intergenic regions. §percentage of the mim replicon involved in the ani calculation is expressed here as the conservation value. graphic representation of the alignments of cfn -mim replicons. a) pretmim1a-pretcfn42b, b) pretmim1b-pretcfn42c, c) pretmim1d-pretcfn42e, d) pretmim1e-pretcfn42d, e) pretmim1f-pretcfn42f. orfs of each replicon are depicted with light blue arrows in their corresponding reading frame. syntenic segments oriented in the same or opposite direction are joined by red and blue regions, respectively. mim and cfn chromosomes were syntenic, as were the chromid pairs pretmim1a-pretcfn42b and pretmim1d-pretcfn42e, and plasmids pretmim1b-pretcfn42c. in comparison to r. leguminosarum mim plasmid equivalences were similar to those reported previously for r. etli cfn. ani values were lower with r. leguminosarum that among the r. etli strains. all extrachromosomal replicons in mim belong to the repabc plasmid family. the protein products of the repabc operons of the replicon homologous pairs were almost identical with a sequence identity greater than %, strongly suggesting that members of each replicon pair belong to the same incompatibility group. pretcfn42f-pretmim1f comparison the largest extrachromosomal replicon in mim was only partially conserved in the putative chromid pretcfn42f. pretmim1f and pretcfn42f possess two repabc operons: repabc and repabc the sequence identity between the two repabc operons in mim is low. the degree of sequence identities between the corresponding repabc genes of pretcfn42f and pretmim1f is large enough to suggest that both plasmids share the same incompatibility group and evolutionary origin. however only 51% of the pretmim1f replicon is conserved in cfn while 86% of pretcfn42f was found in pretmim1f. type vi secretion system genes were only found in the megaplasmid pretmim1f and the conserved impb component is phylogenetically related to the corresponding genes in rhizobium sp. kim and r. leguminosarum strain mim pretmim1f has a duplicated citrate synthase gene as in sv. tropici symbiotic plasmids. the plasmid duplicated citrate synthase gene in sv. tropici is required for eliciting a normal number of nodules and is regulated differently than the copy in the chromosome. a plasmid borne citrate synthase was not found in cfn phylogenetic analysis of citrate synthase genes showed that the mim chromosomal gene product is identical to that of cfn while the gene in pretmim1f has a novel sequence only distantly related to a plasmid copy of r. gallicum r. genes such as raii and rair were found in the conserved region of pretcfn42f and pretmim1f. raii produces homoserine lactones and rair is the transcriptional regulator. the rai system in r. phaseoli sv. phaseoli cnpaf affects nodule number but not nitrogen fixation in p. vulgaris. this system also controls growth in r. phaseoli and if this is the case in r. etli, this would explain its conservation in both symbiovars. a transcriptomic study compared the genome expression of r. phaseoli ch24- in maize and p. vulgaris rhizospheres. over 50% of the extrachromosomal genes highly expressed in p. vulgaris but not in maize roots were found in a ch24- replicon equivalent to pretcfn42f. it seems that genes in this replicon are involved in plant specific interactions. symbiosis plasmid gene comparison large differences were observed between the symbiotic plasmids of cfn and mim. around 10% and 15% of the symbiotic plasmids of mim and cfn had conserved syntenic regions with an average nucleotide identity of %. differences in symbiosis genes in cfn and mim genomes are shown in table  the nod factor from r. etli sv. phaseoli strain cfn is a pentamer of n-acetylglucosamine with an acetyl fucose at the reducing end and methyl and carbamoyl groups at the non reducing end. the heterologous expression in azorhizobium caulinodans of nod modification genes showed that fucosylated nod factors were the most suitable to induce p. vulgaris nodulation. in symbiovar mimosae no genes related to nod factor fucosylation were observed, in their place, nodhpq genes that modify the nod factor with sulfate, were found. such genes are present in sv. tropici strains that are also leucaena symbionts. like r. etli strain cfn mim may produce nodulation factors bearing carbamoyl groups at their non reducing end residues but the position of these decorations must differ because their psyms encoded distinct carbamoyl transferases, nolo in cfn and nodu in mim carbamoylation at the c- position introduced by nodu maybe promotes leucaena nodulation. both mim and cfn symbiotic plasmids carry nods involved in methylation at the non reducing end, a decoration that is essential for bean and leucaena nodulation in r. tropici ciat. a nodo homologue, 70% identical to that of rhizobium sp. br was found only in the sv. mimosae psym. it has been shown that heterologous expression of nodo can improve nodulation of l. leucocephala by different rhizobia and can even extend the host range.table relevant symbiotic plasmid differences between r. etli sv. mimosae mim and r. etli sv. phaseoli cfn 42 nodhpq nodz noll nodo nolo nodu fixkl fxkr acds teu hup-hyp *functions of each gene are explained in the text. mim nod gene phylogenies are congruent, resembling the corresponding genes from sv. giardinii while mim nifh genes resemble those from sv. phaseoli. different nodds in phaseoli and mimosae symbiovars may reflect their affinities for the different flavonoids exuded by the different host plants. mim nodh gene resembles the corresponding gene in rhizobium sp. ie that represents a novel genomic lineage related to r. etli and r. phaseoli. acds gene coding acc-deaminase was found in the symbiotic plasmid of mim but not in cfn acc-deaminase decreases the amount of acc that is a precursor of ethylene that may diminish nodule number. a heterologous acc-deaminase in rhizobium sp tal enhanced nodulation in leucaena. in cfn the fixghis-fixnoqp genes required for biosynthesis of the symbiotic terminal oxidase are present in the psym and also in pretcfn42f. the regulatory genes fixk and fixl are adjacent to this reiteration in pretcfn42f while a fixk pseudogene is found in the psym. in mim we found that the symbiotic terminal oxidase genes are also reiterated in pretmim1f but, in contrast to cfn the sv mimosae psym carried complete fixk and fixl genes. the fixghis-fixnoqp-fixkl region shared by psym and pretmim1f plasmids in mim is 95% identical while the reiterated regions in cfn are only 87% identical. the recently described fxkr gene in pretcfn42f, coding for a response regulator that acts in conjunction with fixl and fixk, is present in the sv. mimosae psym. there is a reiteration of this gene in pretmim1f as well. genes involved in p. vulgaris exudate uptake are found in the symbiotic plasmids in symbiovar phaseoli strains r. etli cfn and r. phaseoli ciat but they were not found in the mim genome. rhizobium mutants in teu genes had reduced nodulation competitiveness in p. vulgaris. the symbiosis plasmid of mim has genes for a type iii secretion system that are more closely related to those found in mesorhizobium and sinorhizobium strains than to those encoded in the cfn psym. this difference may contribute to the disparate host ranges displayed by sv. phaseoli and mimosae strains considering the function of rhizobial t3ss in specificity. a cluster of hup-hyp genes encoding the components of an uptake hydrogenase was found in the psym of mim but not in cfn the mim products showed high identities to their counterparts coded in r. leguminosarum and r. tropici. r. tropici ciat lacks several hup genes and displays a hup minus phenotype. in mim all genes except hupe are present. hupe is an uptake transporter for nickel, a metal required for hup function. since another nickel transporter is encoded elsewhere in mim symbiotic plasmid, the hup system could be functional. mim symbiotic plasmid has a repabc origin of replication as well as a repc gene that are not phylogenetically related to the r. etli cfn corresponding genes. pretmim1e repc resembles those from r. gallicum and rhizobium sp. sv. giardinii ie. mim repabc from the psym resembles the corresponding genes in r. endophyticum ccge but an extensive plasmid conservation was not observed. ccge is nod- and does not have a symbiotic plasmid. the analysis of insertion sequences in r. etli cfn suggested that the symbiotic plasmid did not significantly share is sequences with the rest of the genome. this was interpreted as evidence that the psym was a recent acquisition in this bacterium. the analysis of is sequences in mim indicated that the symbiotic plasmid had the largest number of is sequences, some of them shared with the chromosome, pretmim1f and pretmim1b, this may perhaps indicate that this symbiotic plasmid has an older history with the r. etli genomic background than the phaseoli plasmid. mim has a large number of is that are common in rhizobia. genome similarities were found among the different mim replicons. pretmim1e has similar sequences to pretmim1c and the same is observed among pretmim1e and pretmim1f. the similarities of mim symbiotic plasmid and the putative chromid pretmim1f could support that symbiovar mimosae symbiotic plasmid is ancestral in r. etli. in contrast the phaseoli symbiotic plasmid was found dissimilar to the rest of the genomic background in r. etli, except to precfn42a.figure graphic comparison of the mim symbiotic plasmid pretmim1e compared to other rhizobial replicons. in r. etli cfn the symbiotic plasmid and pretcfn42a have common sequences such as tra and vir genes and repeated sequences that mediate the natural cointegration of both plasmids for the conjugative transfer of cfn psym. in contrast to precfn42a, pretmim1c from mim does not seem to participate in the transfer of the mim symbiotic plasmid, which we have been unable to transfer to other bacterial hosts. plant-interaction genes not in the symbiosis plasmid some genes involved in plant interactions were found conserved in both symbiovars, such as those encoding rmr extrusion pumps that may be involved in eliminating plant produced phytoalexins. r. etli mutants in these genes had reduced nodulation. those genes are encoded in chromid pretcfn42b in cfn and in the corresponding replicon pretmim1a. rmra gene had 97% identity and rmrb and rmrr genes 98% in cfn and mim genomes. homologous genes were found being expressed in different plant rhizospheres. even though sv. mimosae strains are capable of forming nodules in leucaena, we did not find genes resembling mid or pyd genes involved in the catabolism of the toxic amino acid derivative mimosine found in leucaena plants. such genes were described from a rhizobium sp. strain tal that was isolated from leucaena plants. mimosa pigra has a much lower level of mimosine that leucaena plants and data for other mimosa species is not available. symbiovar phaseoli is prevalent in different phaseolus vulgarisnodule bacteria r. etli sv. mimosae strains have a broader host range than symbiovar phaseoli strains. in particular, l. leucocephala plants served as a host to distinguish symbiovar mimosae strains. thirty-six p. vulgaris nodulating bacteria obtained from the rain forest of los tuxtlas in mexico corresponded to symbiovar phaseoli on the basis of noda gene organization and for being unable to nodulate leucaena plants. we found that some strains that were previously classified as r. etli such as ciat ch24- cnpaf 8c- and brasil now assigned to r. phaseoli, as well as others like kim gr and ciat now assigned to recently named novel lineages corresponded to sv. phaseoli when we analyzed their genomes. symbiotic plasmids are highly conserved in symbiovar phaseoli perhaps from being recently evolved. considering that the majority of p. vulgaris nodule isolates tested corresponded to sv. phaseoli we may conclude that this symbiovar is better adapted to its host, thus phaseoli seems to be a specialist symbiovar having a narrow range not including mimosa plants. the phaseoli symbiovar is found in several rhizobium species or lineages. the widespread of this symbiovar may be in relation to its host historic worldwide distribution and to the transferability of the symbiotic plasmid, seemingly an epidemic plasmid. besides having the phaseoli symbiovar, the rhizobium species mentioned above have additional generalist symbiovars: gallicum or giardinii or mimosae having alternative symbiovars with different host ranges may be advantageous in rhizobia, as it expands their legume niches and allows them to avoid the specialist-generalist dilemma.  the term symbiovar is validated with genomic analyses that show that a common genomic background may harbor different symbiotic plasmids determining host specificity. however, besides differences in the symbiotic plasmids there were differences in other ers and in the chromosomes in the two strains analyzed, cfn and mim in mim nod factors with sulfate modifications, secretion systems or acc-deaminase may help explain the extended host range of symbiovar mimosae. in cfn teu genes that participate in exudate uptake and genes involved in nod factor fucosylation may contribute to p. vulgaris host specialization. the discussion that mimosae is older than phaseoli may apply to gallicum and giardinii, thus we propose that gallicum and giardinii are older than phaseoli. electronic supplementary material additional file 1: table s1: genes found in different genomic islands exclusive of mim competing interests the authors declare that they have no competing interests. authors’ contributions emr initiated and designed the study. mar performed experiments. vg, dr, mac contributed reagents and analyzed data. eoo, pb, ris, jmr, jcm, ll performed data analysis. emr, eoo, mar wrote the paper. all authors read and approved the manuscript. 
differences in nod factors deduced from the presence of nod genes, in secretion systems or acc-deaminase could help explain the distinct host specificity. symbiosis genes involved in nodulation and nitrogen fixation in legumes are plasmid-borne in rhizobium. with the genomic analysis presented, the term symbiovar is validated. genes involved in p. vulgaris exudate uptake were not found in symbiovar mimosae but hup genes were found. in rhizobium etli or in rhizobium phaseoli, symbiovar phaseoli strains have the capacity to form nodules in phaseolus vulgaris while symbiovar mimosae confers a broad host range including different mimosa trees. we report on the genome of r. etli symbiovar mimosae strain mim and its comparison to that from r. etli symbiovar phaseoli strain cfn differences were found in plasmids especially in the symbiosis plasmid, not only in nod gene sequences but in nod gene content. furthermore, our data support that the generalist symbiovar mimosae may be older than the specialist symbiovar phaseoli. rhizobial symbiotic variants with distinct host specificity would depend on the type of symbiosis plasmid. the genomic differences between the two symbiovars, mimosae and phaseoli may explain different host specificity. legume nodulationbacterial symbiosisnitrogen fixationhost specificityissue-copyright-statement© the author 2014
 symbiosis genes involved in nodulation and nitrogen fixation in legumes are plasmid-borne in rhizobium. rhizobial symbiotic variants with distinct host specificity would depend on the type of symbiosis plasmid. in rhizobium etli or in rhizobium phaseoli, symbiovar phaseoli strains have the capacity to form nodules in phaseolus vulgaris while symbiovar mimosae confers a broad host range including different mimosa trees.  we report on the genome of r. etli symbiovar mimosae strain mim and its comparison to that from r. etli symbiovar phaseoli strain cfn differences were found in plasmids especially in the symbiosis plasmid, not only in nod gene sequences but in nod gene content. differences in nod factors deduced from the presence of nod genes, in secretion systems or acc-deaminase could help explain the distinct host specificity. genes involved in p. vulgaris exudate uptake were not found in symbiovar mimosae but hup genes were found. plasmid pretcfn42a was partially contained in mim and a plasmid was found only in mim chromids were well conserved.  the genomic differences between the two symbiovars, mimosae and phaseoli may explain different host specificity. with the genomic analysis presented, the term symbiovar is validated. furthermore, our data support that the generalist symbiovar mimosae may be older than the specialist symbiovar phaseoli. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords legume nodulationbacterial symbiosisnitrogen fixationhost specificityissue-copyright-statement© the author 2014 