A common genomic framework for a diverse assembly of plasmids in the symbiotic nitrogen fixing bacteria.
ABSTRACT This work centres on the genomic comparisons of two closely-related nitrogen-fixing symbiotic bacteria, Rhizobium leguminosarum biovar viciae 3841 and Rhizobium etli CFN42. These strains maintain a stable genomic core that is also common to other rhizobia species plus a very variable and significant accessory component. The chromosomes are highly syntenic, whereas plasmids are related by fewer syntenic blocks and have mosaic structures. The pairs of plasmids p42f-pRL12, p42e-pRL11 and p42b-pRL9 as well large parts of p42c with pRL10 are shown to be similar, whereas the symbiotic plasmids (p42d and pRL10) are structurally unrelated and seem to follow distinct evolutionary paths. Even though purifying selection is acting on the whole genome, the accessory component is evolving more rapidly. This component is constituted largely for proteins for transport of diverse metabolites and elements of external origin. The present analysis allows us to conclude that a heterogeneous and quickly diversifying group of plasmids co-exists in a common genomic framework.
- SourceAvailable from: Jerzy Wielbo[Show abstract] [Hide abstract]
ABSTRACT: Rhizobium leguminosarum bv. trifolii TA1 (RtTA1) is a soil bacterium establishing a highly specific symbiotic relationship with clover, which is based on the exchange of molecular signals between the host plant and the microsymbiont. The RtTA1 genome is large and multipartite, composed of a chromosome and four plasmids, which comprise approximately 65 % and 35 % of the total genome, respectively. Extrachromosomal replicons were previously shown to confer significant metabolic versatility to bacteria, which is important for their adaptation in the soil and nodulation competitiveness. To investigate the contribution of individual RtTA1 plasmids to the overall cell phenotype, metabolic properties and symbiotic performance, a transposon-based elimination strategy was employed. RtTA1 derivatives cured of pRleTA1b or pRleTA1d and deleted in pRleTA1a were obtained. In contrast to the in silico predictions of pRleTA1b and pRleTA1d, which were described as chromid-like replicons, both appeared to be completely curable. On the other hand, for pRleTA1a (symbiotic plasmid) and pRleTA1c, which were proposed to be unessential for RtTA1 viability, it was not possible to eliminate them at all (pRleTA1c) or entirely (pRleTA1a). Analyses of the phenotypic traits of the RtTA1 derivatives obtained revealed the functional significance of individual plasmids and their indispensability for growth, certain metabolic pathways, production of surface polysaccharides, autoaggregation, biofilm formation, motility and symbiotic performance. Moreover, the results allow us to suggest broad functional cooperation among the plasmids in shaping the phenotypic properties and symbiotic capabilities of rhizobia.Journal of applied genetics 05/2014; · 1.85 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: A core component of the α-proteobacterial general stress response (GSR) is the extracytoplasmic function (ECF) sigma factor EcfG, exclusively present in this taxonomic class. Half of the completed α-proteobacterial genome sequences contain two or more copies of genes encoding σ(EcfG) -like sigma factors, with the primary copy typically located adjacent to genes coding for a cognate anti-sigma factor (NepR) and two-component response regulator (PhyR). So far, the widespread occurrence of additional, non-canonical σ(EcfG) copies has not satisfactorily been explained. This study explores the hierarchical relation between Rhizobium etli σ(EcfG1) and σ(EcfG2) , canonical and non-canonical σ(EcfG) proteins, respectively. Contrary to reports in other species, we find that σ(EcfG1) and σ(EcfG2) act in parallel, as nodes of a complex regulatory network, rather than in series, as elements of a linear regulatory cascade. We demonstrate that both sigma factors control unique yet also shared target genes, corroborating phenotypic evidence. σ(EcfG1) drives expression of rpoH2, explaining the increased heat sensitivity of an ecfG1 mutant, while katG is under control of σ(EcfG2) , accounting for reduced oxidative stress resistance of an ecfG2 mutant. We also identify non-coding RNA genes as novel σ(EcfG) targets. We propose a modified model for GSR regulation in R. etli, in which σ(EcfG1) and σ(EcfG2) function largely independently. Based on a phylogenetic analysis and considering the prevalence of α-proteobacterial genomes with multiple σ(EcfG) copies, this model may also be applicable to numerous other species.MicrobiologyOpen. 12/2013; 2(6):976-87.
- [Show abstract] [Hide abstract]
ABSTRACT: Rhizobium grahamii belongs to a new phylogenetic group of rhizobia together with Rhizobium mesoamericanum and other species. R. grahamii has a broad-host-range that includes Leucaena leucocephala and Phaseolus vulgaris, although it is a poor competitor for P. vulgaris nodulation in the presence of Rhizobium etli or Rhizobium phaseoli strains. This work analyzed the genome sequence and transfer properties of R. grahamii plasmids. Genome sequence was obtained from R. grahamii CCGE502 type strain isolated from Dalea leporina in Mexico. The CCGE502 genome comprises one chromosome and two extrachromosomal replicons (ERs), pRgrCCGE502a and pRgrCCGE502b. Additionally, a plasmid integrated in the CCGE502 chromosome was found. The genomic comparison of ERs from this group showed that gene content is more variable than average nucleotide identity (ANI). Well conserved nod and nif genes were found in R. grahamii and R. mesoamericanum with some differences. R. phaseoli Ch24-10 genes expressed in bacterial cells in roots were found to be conserved in pRgrCCGE502b. Regarding conjugative transfer we were unable to transfer the R. grahamii CCGE502 symbiotic plasmid and its megaplasmid to other rhizobial hosts but we could transfer the symbiotic plasmid to Agrobacterium tumefaciens with transfer dependent on homoserine lactones. Variable degrees of nucleotide identity and gene content conservation were found among the different R. grahamii CCGE502 replicons in comparison to R. mesoamericanum genomes. The extrachromosomal replicons from R. grahamii were more similar to those found in phylogenetically related Rhizobium species. However, limited similarities of R. grahamii CCGE502 symbiotic plasmid and megaplasmid were observed in other more distant Rhizobium species. Interestingly, the set of conserved genes in R. grahamii comprises some of those that are highly expressed in R. phaseoli on plant roots, suggesting that they play an important role in root colonization.BMC Microbiology 01/2014; 14(1):6. · 3.10 Impact Factor
A Common Genomic Framework for a Diverse Assembly
of Plasmids in the Symbiotic Nitrogen Fixing Bacteria
Lisa C. Crossman1*, Santiago Castillo-Ramı ´rez2, Craig McAnnula3, Luis Lozano2, Georgios S. Vernikos1,
Jose ´ L. Acosta2, Zara F. Ghazoui4, Ismael Herna ´ndez-Gonza ´lez2, Georgina Meakin5, Alan W. Walker1,
Michael F. Hynes6, J. Peter W. Young4, J. Allan Downie3, David Romero2, Andrew W. B. Johnston5,
Guillermo Da ´vila2, Julian Parkhill1, Vı ´ctor Gonza ´lez2*
1The Wellcome Trust Sanger Institute, Hinxton, Cambridge, United Kingdom, 2Universidad Nacional Auto ´noma de Me ´xico, Cuernavaca, Me ´xico, 3John Innes Centre,
Norwich, United Kingdom, 4Department of Biology, University of York, York, United Kingdom, 5School of Biological Sciences, University of East Anglia, Norwich, United
Kingdom, 6Department of Biological Sciences, University of Calgary, Calgary, Canada
This work centres on the genomic comparisons of two closely-related nitrogen-fixing symbiotic bacteria, Rhizobium
leguminosarum biovar viciae 3841 and Rhizobium etli CFN42. These strains maintain a stable genomic core that is also
common to other rhizobia species plus a very variable and significant accessory component. The chromosomes are highly
syntenic, whereas plasmids are related by fewer syntenic blocks and have mosaic structures. The pairs of plasmids p42f-
pRL12, p42e-pRL11 and p42b-pRL9 as well large parts of p42c with pRL10 are shown to be similar, whereas the symbiotic
plasmids (p42d and pRL10) are structurally unrelated and seem to follow distinct evolutionary paths. Even though purifying
selection is acting on the whole genome, the accessory component is evolving more rapidly. This component is constituted
largely for proteins for transport of diverse metabolites and elements of external origin. The present analysis allows us to
conclude that a heterogeneous and quickly diversifying group of plasmids co-exists in a common genomic framework.
Citation: Crossman LC, Castillo-Ramı ´rez S, McAnnula C, Lozano L, Vernikos GS, et al. (2008) A Common Genomic Framework for a Diverse Assembly of Plasmids in
the Symbiotic Nitrogen Fixing Bacteria. PLoS ONE 3(7): e2567. doi:10.1371/journal.pone.0002567
Editor: Richard R. Copley, Wellcome Trust Centre for Human Genetics, United Kingdom
Received February 19, 2008; Accepted May 6, 2008; Published July 2, 2008
Copyright: ? 2008 Crossman et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: CONACyT 46333-Q and PAPIIT-UNAM IN223005-5 grants supported work on Rhizobium etli. Work on Rhizobium leguminosarum was supported by the
Wellcome Trust and the BBSRC under grants 104/P16988, 208/BRE13665, and 208/PRS12210.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org (LCC); email@example.com (VC)
Rhizobium etli and Rhizobium leguminosarum bv viciae (henceforth
called R. leguminosarum) are closely related species which are able to
fix atmospheric nitrogen in symbiosis with specific leguminous
plants. The common bean is the natural host of R. etli whereas R.
leguminosarum interacts with peas, lentils, vetches and Lathyrus spp.
Recently, we reported the complete genome sequences of a strain
of R. etli and a strain of R. leguminosarum [1,2], but no
comprehensive genome comparison between these species had
been carried out. To date, several other complete genome
sequences of symbiotic nitrogen fixing bacteria have been
published: Mesorhizobium loti, Bradyrhizobium japonicum, B. spp.
ORS278, B. spp. BTAi1 and Sinorhizobium meliloti [3–6]). Our
comparisons of R. etli and R. leguminosarum show that: 1) Rhizobium
genomes are composed of ‘‘core’’ and ‘‘accessory’’ components; 2)
the chromosomes are markedly conserved in gene content (despite
differences in size) and amongst the closest species gene order is
also conserved; 3) the plasmids are heterogeneous in size and gene
content and in some cases no synteny can be seen even in
comparison with phylogenetic neighbours.
Rhizobium field isolates have the unusual feature of harbouring
several plasmids, ranging in size from 100 kb to .1,000 kb and
the plasmid profiles of a particular isolate can be used to type
strains reliably . Since R. etli CFN42 and R. leguminosarum 3841
are the most closely-related rhizobial species yet sequenced and
both strains have six large plasmids, a detailed genome
comparison between them may help us interpret the evolutionary
history of these prototypical accessory elements. Indeed, whole
genome comparisons allowed us to discern the distinctive
properties of the core genome, and also to highlight the genetic
differences between these species.
Main features of the compared species
Both R. etli CFN42 and R. leguminosarum 3841 have large
genomes composed of a circular chromosome and six large
plasmids [1,2]. The six CFN42 plasmids, pRetCFN42a-f, will be
referred to as p42a-f throughout this article, whilst the six 3841
plasmids (sometimes known as pRL7JI-pRL12JI) are termed
pRL7-12. The total size of the R. etli CFN42 genome is
1,221,081 bp shorter than that of R. leguminosarum 3841 (Table
S1). The two smaller plasmids of R. etli are substantially larger than
the two smallest plasmids of R. leguminosarum, whilst the opposite is
the case for the other four plasmids (Table S1). R. leguminosarum
plasmids comprise 34.8% of the total genome, whilst R. etli
plasmids comprise an equivalent 32.9%. The two smallest R.
leguminosarum plasmids are of lower than average GC content,
whilst in R. etli the major nitrogen fixation plasmid (pSym; p42d)
PLoS ONE | www.plosone.org1 July 2008 | Volume 3 | Issue 7 | e2567
and the smallest plasmid (p42a) are the only plasmids of
significantly lower GC content. The largest plasmids in both
genomes resemble their corresponding chromosomes both in GC
content and dinucleotide signatures. Symbiotic functions, specified
by the nod, nol, nif and fix genes, are mainly encoded by a single
plasmid (p42d in R. etli and pRL10 in R. leguminosarum), but other
symbiosis-related genes are located on other plasmids and in the
chromosome [1,8]. The R. etli plasmid p42a is transferable at high
frequencies and can help the mobilization of p42d [9–11] and
p42d is also self-transmissible by conjugation  although its
transfer ability is tightly repressed . In R. leguminosarum, pRL7
and pRL8 are transmissible by conjugation, although neither
carries a full set of tra genes .
Phylogenomic relatedness between R. etli and R.
R. leguminosarum and R. etli are closely related species, judged by
16S rRNA comparisons and other molecular criteria (Figure S1).
We first tested the consistency of these traditional phylogenies with
genome phylogenies obtained with all individual proteins included
in quartops (QUARtet of Orthologous Proteins). To do this, we
incorporated two other species of the Rhizobiaceae family, S.
meliloti and the non-nitrogen-fixing Agrobacterium tumefaciens, whose
complete genomes are also available.. A total of 33% and 39% of
R. leguminosarum and R. etli proteins, respectively, were present in
the Quartops; this equates to 2,392 predicted proteins representing
core genes that are common to these four organisms (Table 1).
Most of these predicted proteins are chromosomally encoded
(2,054) but 338 belong to plasmids pRL9, pRL11 and pRL12.
Three of the plasmids (pRL7, pRL8 and pRL10) do not have any
proteins in Quartops. A total of 2,241 (85% of all proteins included
in quartops) supports the phylogenetic relationship that proposes
R. leguminosarum and R. etli are the most closely related. However,
the high numbers of proteins absent from Quartops suggests that
gene losses and gains might significantly have driven the
diversification of the fast growing rhizobia. To investigate this
area, we clustered all the predicted proteins of R. etli, R.
leguminosarum, S. meliloti and A. tumefaciens into families by means
of the MCL algorithm . About 28% of the protein families
identified (1,965 out 6,827) are shared by the four species, whereas
about 10% (668) are only present in three species (Figure 1, bars
1–6). The rest of the protein families (13% or 908) occur in just
two species. Most of these families (443) belong to the R. etli-R.
leguminosarum pair, giving further support to the quartop phylogeny
and the recent divergence of these two species (Figure 1, bars 7–
11). Moreover, an appreciable number of families were particular
to individual genomes. They belong to known and hypothetical
Table 1. Quartops analysis with R. leguminosarum, R. etli, A.
tumefaciens and S. meliloti.
quartops Rl-Re Rl-At Rl-Sm
Chr4736 2054 43.419512523
Figure 1. Distribution of protein families in the genomes of S. meliloti (S), A. tumefaciens (A), R. leguminosarum (L) and R. etli (E). - Bar
number indicates the assignation of the protein families to the corresponding genome according to the following letters code: 1, SALE; 2, SAL; 3, ALE;
4, SAE; 5, SLE; 6. SA; 7, LE; 8, AL; 9, SE; 10, SL; 11, AE; 12, S; 13, A; 14, L; 15, E. Bars 12-15 show in red the proportion of orphan genes compared with
those which match with known or hypothetical proteins present in the nr database of Genbank (yellow).
PLoS ONE | www.plosone.org2 July 2008 | Volume 3 | Issue 7 | e2567
families already present in the Genbank or they are orphan genes
(Figure 1, bars 12–15). This confirms the previous findings that the
coding potential of the rhizobial species is very variable while
maintaining a stable common core.
To investigate whether the evolutionary relationship between R.
etli and R. leguminosarum is also maintained at the level of gene
order, the whole genomes were compared using ACT and
Nucmer softwares [15,16]. A clear syntenic pattern is distinguished
between both chromosomes but it is also noticeable for some pairs
of plasmids: (p42f-pRL12), (p42e-pRL11) and (p42b-pRL9) as well
as large parts of p42c woth pRL10, suggesting a common origin
(Figure 2). These observations are supported by the similarity of
the replication genes, repABC, of those pairs of plasmids, as well as
experimental demonstration of incompatibility between the
plasmid pairs (Clark, Mattson, Garcia and Hynes, in preparation).
Plasmids pRL7 and pRL8 appear to be unique to R. leguminosarum
whilst p42a is peculiar to R. etli (see below). A more accurate
measure of synteny between the genomes was obtained by
calculating the length and number of colineal blocks (CBs). To
do this, we employed a whole alignment obtained by Nucmer ,
then individual matches were clustered in CBs taking all the
continuous segments separated by gaps less than 1kb. In total,
4,557,466 bp (70%) of the R. etli genome is contained in CBs with
nucleotide identity about 85–95% (to R. leguminosarum). In the total
genome of R. leguminosarum, 4,931,491 bp (63%) are contained in
CBs. A total of 353 CBs .1 kb were recognized. The largest and
most abundant (221) CBs are located on the chromosome and the
rest on plasmids. Figure 3 shows that 81% and 74% of the
chromosomes of R. etli and R. leguminosarum respectively are
contained in CBs. Three of the R. etli plasmids have 44–58% of
their genetic information in CBs that also occur in R. leguminosarum.
Plasmids with fewer CBs are p42a, p42d, pRL7 and pRL8. Some
of the plasmid pairs can be functionally identified by the presence
of specific genes. For example, p42f and pRL12 carry some genes
for flagellar biosynthesis (flgLKE) and for oxidative stress protection
(oxyR and katG); p42e and pRL11 harbor cell division genes
(minCDE), as well as thiamin, cobalamin, NAD biosynthetic genes
(thiMED, cobFGHIJKLM, nadABC), and an isolated flagellin (fla)
gene, as well as a rhamnose catabolism operon. In some cases,
e.g. thiMED, these genes are functionally interchangeable between
these species . A duplication of the fixNOQP operon in p42f
, in R. leguminosarum is located in pRL9, a plasmid with
Figure 2. ACT View of Chromosome and Plasmids. The chromosomal and plasmid DNAs have been laid end-to-end and analysed using the
Artemis comparison tool (ACT) . Red bars represent close matches, whilst blue bars represent inverted close matches. The R. leguminosarum
genome is at the top of the figure with replicons in the order Chromosome, pRL7, pRL8, pRL9, pRL10, pRL11, pRL12 whilst the R. etli genome is shown
at the bottom of the figure in order Chromosome, p42a, p42b, p42c, p42d, p42e, p42f.
PLoS ONE | www.plosone.org3 July 2008 | Volume 3 | Issue 7 | e2567
homologous segments to p42b. Conjugative plasmids pRL8, pRL7
and p42a, which are otherwise unrelated to each other, have
homologous tra-trb systems.
Core genome composition and evolution
R. etli and R. leguminosarum share 5,470 genes with approximately
89–100% similarity (see methods). A significant fraction of these
common genes (3,359 or 62%) is solely present in both
chromosomes (Chromosomal Only, CHR-O). The rest are
situated either in the chromosome or plasmids or exclusively in
the plasmids (Non-Chromosomal, N-CHR). Using the Riley
classification scheme , CHR-O genes are overrepresented in
the categories corresponding to small and macromolecule
metabolism, structural elements, regulators and hypothetical
conserved genes. In contrast, the N-CHR group tends to contain
genes implicated in processes like chemotaxis, chaperones,
transport, and elements of external origin (Figure 4a). A detailed
classification using COGs  reveals other differences between
CHR-O and N-CHR groups. Some of the COGs that are
overrepresented in N-CHR are COG K (replication, recombina-
tion and repair) and the COGs related with predicted transport
and metabolism of carbohydrates, amino acids, lipids and
inorganic ions (COG G, E, I, and P) (Figure 4b), but not COGs
related to information storage and processing.
Differences between the CHR-O and the N-CHR gene
compartments were also detected in regard to rates of evolution.
To do this, we calculated the rates of nucleotide substitution per
synonymous (Ks) and non-synonymous sites (Ka), for a subset of
2,917 single copy homologues (see methods; Figure 5). It is clear
that both CHR-O and N-CHR homologous groups are under
negative selection. Nevertheless, as seen by the slopes of the
regression lines, the CHR-O group seems to be under stronger
negative selection than the N-CHR group. However, many genes
of the N-CHR group show higher Ka (.0.19) and Ks (.2.0)
values than those of the CHR-O group. Therefore, negative
selection is acting on the whole genome, but overall, the N-CHR
gene compartment is less constrained.
Despite the high level of genome conservation, it is reasonable
to expect that some degree of intra-genomic recombination has
occurred since these two strains R. etli and R. leguminosarum had a
common ancestor. This was substantiated by comparing the
locations of the N-CHR group of genes in the different replicons of
both genomes. Approximately 7% of the chromosomal genes of R.
leguminosarum are represented in the plasmids of R. etli, and 10% of
the chromosomal genes of R. etli are located in the R. leguminosarum
plasmids. As shown before, some pairs of plasmids are likely
equivalent in terms of their global similarity, but they are mosaic
replicons that contain genes from the other replicons. For instance,
pRL12 has significant similarity with p42f, but also possesses genes
that in R. etli are chromosomal or on another plasmid (Figure 6). A
similar pattern is observed in the other replicons (Figure 6). Such
heterogeneous composition of the plasmids has precluded any
attempt to make a reliable plasmid phylogeny. One way to assess
the phylogenetic relatedness among plasmids is to compare their
RepABC proteins that are essential components for plasmid
replication .. However, we observed here that only the p42c-
pRL10, p42d-pRL11 and p42f-pRL12 pairs carry closely related
replication systems. They share nucleotide identities greater than
82% in the three proteins, whereas the RepABC proteins of the
other plasmids are poorly related. Therefore, the replication genes
might have been shuffled several times among the distinct
plasmids, perhaps to allow a number of plasmids to coexist in
the same cell.
A potential symbiosis cassette
A comparison of the major symbiotic plasmids (pSyms) pRL10
and p42d shows that the nif-nod region in pRL10 is compacted into
60 kb, whereas in p42d it encompasses 125 kb. As many as 20
Figure 3. The proportion of synteny in the R.etli genome as compared to the R. leguminosarum genome. - The proportion of synteny is
expressed as the percentage of the total DNA in CBs (Y axis) considering the total length of the pairs of replicons (X axis). Blue color R. etli CFN42;
magenta, R. leguminosarum 3841.
PLoS ONE | www.plosone.org4 July 2008 | Volume 3 | Issue 7 | e2567
common nod and nif genes have been identified in comparisons
among complete sequences of pSyms and symbiotic islands of
different rhizobia . The plasmid pRL10 contains 18 of these
genes and has a particularly enhanced set of nodulation genes,
including genes that lack homologs in R. etli, such as nodTNMLEF
and rhiABCR. In contrast, pRL10 has a restricted set of genes for
nitrogenase maturation, lacking nifS, nifW, nifZ, nifX, iscN, and
nifU, which are present in R. etli and in other rhizobia. Besides the
common nodulation genes, the R. etli pSym possesses nolT, nolL,
nolR, noeI, noeJ, and a Type III secretion system.
The symbiotic genes of R. leguminosarum may have been acquired
by horizontal gene transfer, since an in silico analysis of pRL10 with
the Alien Hunter program  reveals that its symbiotic gene
cluster, which includes the nif, nod, rhi and fix genes, is located in a
short potentially mobile region of DNA (,63.5 kb). Internal to this
region are the nifNEKDH genes that are found bounded by two
identical IS element repeat regions. The rhi and nod gene cluster,
together with fixABCX, lie adjacent on this potential genomic
island and are potentially bounded by 20 bp repeats, whilst the
fixNOPQ and fixGHIS genes lie immediately downstream on a
separate putative genomic island of approximately 11,000 bp,
potentially bounded by 18 bp repeats (Figure 7). It is possible that
the fixNOPQ, fixGHIS island represents a second acquisition of
DNA as an independent event. These adjacent symbiotic nitrogen
Figure 4. Functional bias in CHR-O (chromosomal only) and N-CHR (non-chromosomal) classes of homologues. - Figure 4a) Rileys
categories: 1. small molecule metabolism. 2. Macromolecule metabolism. 3. Structural elements. 4. Cell process. 5. External origin. 6. Miscellaneous.
4b) COGs functional classification. Bars indicate the relative frequency for each COG J, Translation, ribosomal structure and biogenesis; K,
Transcription; L, Replication, recombination and repair; B, chromatin structure and dynamics; D, Cell cycle control; V, Defense mechanisms; T, Signal
transduction mechanisms; M, Cell wall, membrane envelope biogenesis; N, Cell motility; U, Intracellular trafficking and secretion; 0, Postranslational
modification and chaperones; C, Energy production and conversion; G, Carhohydrate transport and metabolism; E, Amino acid transport and
metabolism; F, Nucleotide transport and metabolism; H, Coenzyme transport and metabolism; I, Inorganic ion transport and metabolism; P, inorganic
ion transport and metabolism; Q, Secondary metabolites biosynthesis, transport and catabolism; R, General function prediction; S, function unknown;
X, No COG.
PLoS ONE | www.plosone.org5July 2008 | Volume 3 | Issue 7 | e2567
fixation gene clusters are located in one particular region of the
plasmid with six other short potentially horizontally transferred
areas. The remainder of the pRL10 plasmid is highly similar to the
p42c plasmid of Rhizobium etli. By contrast, the symbiotic nitrogen
fixation genes are scattered throughout 125 kb of the p42d
plasmid of R. etli. However, this region is surrounded by insertion
sequences, which prompted the idea that it might be transposable
. When plasmid p42d was analysed by the Alien Hunter
program 16 regions were detected as atypical. These regions
contain the Type III transport system genes, nod genes, genes for
virulence and conjugation (vir and tra), as well as cytochrome and
chemotaxis genes (Figure S2). They are bordered by repeated
sequences that might represent potential composite transposons
when the repeats are homologous insertion sequences. Alterna-
tively, the chimeric structure of p42d might have been the result of
multiple gene exchanges and rearrangements.
The consequences of the evolutionary process of gain and losses
are reflected in some physiological differences. For example, no
in the R. etli or R. leguminosarum genomes, however, the nirK gene for
the respiratory nitrite reductase is present on R. etli p42f
(RE1PF0000526). This gene appears to participate in nitrite
detoxification . Nitric oxide (NO) removal is encoded by R. etli
as a predicted norECBD operon on the p42f plasmid located in
proximity to the nirKV and probable regulators. These genes are
absent in R. leguminosarum, although there are possible alternative NO
consumption systems. One of such pathways encoded chromosom-
ally by both R. etli and R. leguminosarum is via the assimilatory nitrite
reductase. Another difference is the presence of erythritol catabolic
Figure 5. Rates of synonymous (Ks) and non-synonymous substitutions (Ka) in orthologous genes of R. etli and R. leguminosarum. -
Neutrality line (Ka=Ks) is indicated in yellow. Linear regressions for CO class (blue color line and diamonds) and NC class (rose color line and
diamonds) are indicated. As neutrality assumes equal nucleotide substitutions rates per synonymous and non-synonymous sites, points under the
neutrality line indicate negative selection. Strong selective constraints are acting on genes of the CHR-O class (R2 = 0.6124; P%0.001) but are slightly
less intense for some genes of the N-CHR class (R2= 0.5094), as can be seen by the dispersion of the rose color diamonds.
Figure 6. Composition of the R. leguminosarum and R.etli
genomes according to N-CHR homologues. - The composition
of the R. leguminosarum genome compared to the replicons of R. etli is
shown. The replicon name is given at the base of the figure and color
key to the right of the figure. Genes on the R. etli chromosome may be
elsewhere on the R. leguminosarum genome (as shown in pale blue),
genes from R. etli p42a (burgundy), p42b (cream), p42c (cyan), p42d
(purple), p42e (salmon) and p42f are shown in royal blue.
PLoS ONE | www.plosone.org6 July 2008 | Volume 3 | Issue 7 | e2567
genes, possibly originating from a horizontal transfer event, on
pRL12 . This gene cluster is absent from the CFN42 genome.
Since the lifestyles of R. etli and R. leguminosarum bv. viciae are
similar, they may have similar responses to environmental stimuli.
Thus, they may respond similarly with respect to environmental
stimuli. For instance, population-density-dependent gene induc-
tion by N-acyl homoserine lactones (AHLs) influence symbiotic
functions such as nodulation, nitrogen fixation, and surface
polysaccharide production as well as several aspects of growth
including plasmid transfer and stationary phase adaptation [8,26]
for reviews). Comparative analysis with known AHL regulators
shows that there are 11 LuxR-type regulators in R. etli and 9 in R.
leguminosarum. Some of them are known AHL regulators (CinR,
TraR and RhiR) with associated AHL synthases (CinI, TraI, and
RhiI) but there are also three other regulators, ExpR, AvhR and
AsaR, for which there are no matching AHL synthases. In
addition, we identified three LuxR-like sequences in R. legumino-
sarum (RL0606, RL0607 and RL3528) that matched the LuxR
family over their entire length, but they could not be identified
using protein domain searches. Two of these (RL0606 and
RL0607) are highly conserved in R. etli, and in each case, they are
located within a cluster of genes associated with bacterial motility,
chemotaxis and flagella biosynthesis. Two related genes, visN and
visR from S. meliloti strain RU10/406 act as global regulators of
flagellar motility and chemotaxis, their products probably
functioning as a heterodimer . Although the third regulator
(RL3258) also appears to be conserved in R. etli (CH03080) it has
no known function. Remarkably, RhiR regulates the rhiABC
operon that plays an undefined role in legume infection in R.
leguminosarum, although this regulator is not present in R. etli, .
Rhizobium genomes consist of single circular chromosomes and
several large plasmids. It is not understood why these genomes are
so large and divided. Young et al. (2006) proposed that microbial
life in the soil, a very heterogeneous environment, selects for a
versatile genomes that encode multiple capabilities . Therefore,
genome comparisons between closely related Rhizobium species
may indicate how variable these capabilities could be, as well as
establishing whether they are distributed throughout the genome
or in particular replicons. The comparative analysis presented here
allows us to conclude that most of the differences between R. etli
and R. leguminosarum tend to be in the plasmids. Previous genomic
comparisons of S. meliloti, A. tumefaciens, and R. etli have shown that
chromosomes are well conserved both in gene content and gene
order, whereas plasmids have few common regions (nif-nod, tra-trb,
vir, and others) and a lack of synteny . These comparisons
indicate that the plasmids in those three species are not closely
related phylogenetically or that they have undergone many
recombination events. Our analysis reveals many syntenic blocks
exist between some pairs of plasmids of R. etli and R. leguminosarum
(p42f-pRL12, p42e-pRL11 and p42b-pRL9 as well large parts of
p42c with pRL10) suggesting a common origin. Plasmids of R. etli
are smaller than those of R. leguminosarum, and 44–58% of their
length is contained in CBs common to R. leguminosarum.
Nonetheless, the phylogenetic relationships among the plasmids
A particular case of the mosaic structure of Rhizobium plasmids is
shown by comparison of the symbiotic plasmids. In R. legumino-
sarum the pSyms are variable in size and also differ in repC group
. It has been noted that pRL10 and pRL1 (a pSym of 200 kb in
R. leguminosarum) have a virtually identical nod-nif region, but the
remainder of these plasmids appear to be dissimilar .
Speculatively, the entire symbiotic region may be a mobile
element in R. leguminosarum, as has been proposed for the symbiotic
region of p42d . Although direct evidence for this scenario is
still lacking, it is plausible given the observed recombinational
plasticity displayed by rhizobial plasmids (reviewed by [29,30].
Nevertheless, the overall structure of pRL10 more closely
resembles p42c than p42d of R. etli (Figure 2). Extensive syntenic
regions are common between pRL10 and p42c, accounting for
Figure 7. Diagram of the R. leguminosarum major nitrogen fixation gene cluster. - This cluster represents a potentially laterally transferred
region of DNA. Major nitrogen fixation genes are represented as blocks and are as shown in the color key.
PLoS ONE | www.plosone.org7July 2008 | Volume 3 | Issue 7 | e2567
59% of the length of p42c (Figure 2). Thus, either pRL10 has
gained a large insertion carrying the symbiotic nitrogen fixation
functions, or p42c has suffered a large deletion of these genes. We
show here that the former possibility could be plausible since the
nif-nod region is a potential symbiotic cassette surrounded by
repeated sequences. Furthermore, the structural differences
between the pSyms of R. etli and R. leguminosarum, prompt us to
suggest that they have evolved differently. In R. leguminosarum the
Sym region resembles an specific ‘‘cassette’’, whereas in R. etli the
partial nucleotide sequence of different pSyms suggests that their
diversification is driven by general recombination .
Some authors have proposed that bacterial genomes consist of
‘‘core’’ and ‘‘accessory’’ components [2,32]. The ‘‘Core’’ compo-
nent, exemplified by the chromosome, is more stable and changes
more slowly over time than the ‘‘accessory’’ component. Plasmids
are prototypical accessory elements composed of genes from
different genomic contexts and evolutionary origin. As shown
here, R. etli and R. leguminosarum are good models to study the
evolution of plasmid (‘‘accessory’’) versus chromosome (‘‘core’’)
evolution. Their chromosomes are nearly identical and harbor a
distinct collection of plasmids that have evolved at different rates to
the chromosome. It is tantalizing to speculate that these organisms
can recruit plasmids from a pool in their soil environment .
Plasmids p42a, p42d, pRL7 and pRL8, in particular, seem to be
the outliers. Other plasmids share many common regions and
might have been part of the ancestral chromosome. Shuffling of
the repABC genes might be a strategy to allow many plasmids to
coexist in the same bacterium, and might explain the amazing
plasmid diversity of Rhizobium. A more comprehensive picture of
the evolution of the partitioned genomes can only be reached by
comparing the respective plasmid pool of additional strains of R.
etli and R. leguminosarum to describe how they are able to function in
a common genomic framework.
The 16S rRNA sequences were downloaded from EMBL for R.
C58, Sinorhizobium meliloti 2011, Mesorhizobium loti MAFF303099,
Bradyrhizobium japonicum USDA 110 and Escherichia coli T10. We first
likelihood tree using the PHYLIP package .
The complete nucleotide sequences of the R. etli CFN42 and R.
leguminosarum Rlv3841 were obtained from Genbank (Accession
numbers: R. etli, NC_007761-NC_007766, and NC_004041; R.
leguminosarum NC_008378-NC008384). The sequences of the
replicons for each genome were concatenated and used in a
global comparison using ACT  and the Nucmer application of
the Mummer package , with the default settings. To calculate
the CBs, we took the nucmer.delta output and then parsed it with
the show-coords utility. Syntenic segments .1 kb and separated
by .1 kb were curated with ad hoc perl scripts and manual editing.
Clustering of protein families. First we did BLAST-P compari-
S.melilotiand A.tumefaciens.Clusteringwasachievedwith MCLusing
an e-value of 1027and an inflation parameter of 1.5 .
Homolog grouping and analysis of evolutionary rates
The most probable set of homologous proteins shared by R. etli
and R. leguminosarum was identified using a reciprocal best-hit
criterion. To that end, all R. etli predicted proteins were searched
against the R. leguminosarum predicted proteome and vice versa using
BLAST with cutoff e value of 10212and employing the Blosum-80
matrix . In addition to this criterion, to be included in a
homolog group the difference in length between the subject
protein and query protein had to be ,10%, the alignment region
had to be at least of 80%, and there had to be a at least 50%
similarity of both query and target sizes. We identified 5,470
homolog groups. The whole set was divided into two subdivisions.
The first subdivision contains all the homolog groups in which
there was only one protein per genome (unique bidirectional hits
or possible orthologs, 2,917). The second subdivision contains
homolog groups in which there is more than one protein in at least
one genome, that is, possible paralogs (2,533). Further classifica-
tion of the homolog groups was based on their localization. The
‘‘chromosomal-only’’ group (CHR-O) of homologs is present only
in the chromosomes of both genomes, whereas the non-
chromosomal group (N-CHR) was located either in chromosome
or in plasmids, or exclusively in plasmids. Exclusive genes were
recorded as those with no hits in the genomes at e-value of ,1026.
The number of nucleotide substitutions per synonymous site ‘‘Ks’’
and the number of nucleotide substitutions per non-synonymous
site ‘‘Ka’’ were determined with yn00 from PAML13.14 
Identification of genes involved in quorum sensing
We identified LuxI homologues using homology searches and
independently determined proteins matching InterPro family
IPR001690 (Autoinducer synthase). Both methods gave identical
results. LuxR homologues were identified using homology searches
as a guide, but were not by themselves used to identify likely LuxR
proteins since the C-terminal DNA-binding domain in LuxR is also
present at the C-terminus of a number of other proteins. Proteins
containing the InterPro domain IPR005143 were identified, which
corresponds to the N-terminal autoinducer-binding domain.
Identification of horizontally acquired regions
Potentially horizontally acquired areas of DNA were identified
with the Alien Hunter program, available from http://www.
R.leguminosarum. A comparison of the main features of the genomes
of Rhizobium leguminosarum and Rhizobium etli. Each replicon is
described in terms of length in base pairs, %G+C content and
number of coding sequences (CDS).
Found at: doi:10.1371/journal.pone.0002567.s001 (0.04 MB
General features of the Genomes of R.etli and
tree showing bacteria related to R.etli and R.leguminosarum
Found at: doi:10.1371/journal.pone.0002567.s002 (0.08 MB TIF)
Phylogenetic tree. Maximum likelihood phylogenetic
show (outermost to innermost): 1. Atypical regions as bars of
degraded colour (red to pale rose) according to the scores obtained
from Alien Hunter (red, highest score 73 over a threshold of 32). 2.
The 125 kb nif-nod region. 3, CDS of p42d according to the
following colour code: blue, nodulation genes; yellow, nif genes;
red, energy transfer genes (fix genes); green, insertion sequences;
pink, transfer and replication genes; brown, hypotheticals; grey,
transport (vir and tssIII genes); sky blue, regulators. 4. Insertion
sequences 5. Repeats from 100 to 300 identical nucleotides (black
lines); repeats higher than 300 nucleotides (red lines).
Found at: doi:10.1371/journal.pone.0002567.s003 (0.54 MB EPS)
Chimeric structure of R.etli plasmid p42d. The circles
PLoS ONE | www.plosone.org8July 2008 | Volume 3 | Issue 7 | e2567
The UNAM group wishes to thank the assistance of Patricia Bustos, Rosa I.
Santamarı ´a and Jose ´ L. Ferna ´ndez. Critical reading of the manuscript by
Xianwu Guo is also appreciated.
Analyzed the data: GV LC JY AW GM JD CM JA ZG IH DR VG SC.
Contributed reagents/materials/analysis tools: JP LC ZG VG. Wrote the
paper: JP AJ LC GM JD CM MH GD VG.
1. Gonza ´lez V, Santamarı ´a RI, Bustos P, Herna ´ndez-Gonza ´lez I, Medrano-Soto A,
et al. (2006) The partitioned Rhizobium etli genome: genetic and metabolic
redundancy in seven interacting replicons. Proc Natl Acad Sci U S A 103:
2. Young JP, Crossman LC, Johnston AW, Thomson NR, Ghazoui ZF, et al.
(2006) The genome of Rhizobium leguminosarum has recognizable core and
accessory components. Genome Biol 7: R34.
3. Kaneko T, Nakamura Y, Sato S, Asamizu E, Kato T, et al. (2000) Complete
genome structure of the nitrogen-fixing symbiotic bacterium Mesorhizobium loti.
DNA Res 7: 331–338.
4. Kaneko T, Nakamura Y, Sato S, Minamisawa K, Uchiumi T, et al. (2002)
Complete genomic sequence of nitrogen-fixing symbiotic bacterium Bradyrhizo-
bium japonicum USDA110 (supplement). DNA Res 9: 225–256.
5. Galibert F, Finan TM, Long SR, Pu ¨hler A, Abola P, et al. (2001) The composite
genome of the legume symbiont Sinorhizobium meliloti. Science 293: 668–672.
6. Giraud E, Moulin L, Vallenet D, Barbe V, Cytryn E, et al. (2007) Legumes
symbioses: absence of Nod genes in photosynthetic bradyrhizobia. Science 316:
7. Jumas-Bilak E, Michaux-Charachon S, Bourg G, Ramuz M, Allardet-Servent A
(1998) Unconventional genomic organization in the alpha subgroup of the
Proteobacteria. J Bacteriol 180: 2749–2755.
8. Gonza ´lez V, Bustos P, Ramı ´rez-Romero MA, Medrano-Soto A, Salgado H, et
al. (2003) The mosaic structure of the symbiotic plasmid of Rhizobium etli CFN42
and its relation to other symbiotic genome compartments. Genome Biol 4: R36.
9. Brom S, Garcı ´a-de los Santos A, Cervantes L, Palacios R, Romero D (2000) In
Rhizobium etli symbiotic plasmid transfer, nodulation competitivity and cellular
growth require interaction among different replicons. Plasmid 44: 34–43.
10. Tun-Garrido C, Bustos P, Gonza ´lez V, Brom S (2003) Conjugative transfer of
p42a from Rhizobium etli CFN42, which is required for mobilization of the
symbiotic plasmid, is regulated by quorum sensing. J Bacteriol 185: 1681–1692.
11. Brom S, Girard L, Tun-Garrido C, Garcı ´a-de los Santos A, Bustos P, et al.
(2004) Transfer of the symbiotic plasmid of Rhizobium etli CFN42 requires
cointegration with p42a, which may be mediated by site-specific recombination.
J Bacteriol 186: 7538–7548.
12. Pe ´rez-Mendoza D, Domı ´nguez-Ferreras A, Mun ˜oz S, Soto MJ, Olivares J, et al.
(2004) Identification of functional mob regions in Rhizobium etli: evidence for self-
transmissibility of the symbiotic plasmid pRetCFN42d. J Bacteriol 186:
13. Pe ´rez-Mendoza D, Sepu ´lveda E, Pando V, Mun ˜oz S, Nogales J, et al. (2005)
Identification of the rctA gene, which is required for repression of conjugative
transfer of rhizobial symbiotic megaplasmids. J Bacteriol 187: 7341–7350.
14. Enright AJ, Van Dongen S, Ouzounis CA (2002) An efficient algorithm for
large-scale detection of protein families. Nucleic Acids Res 30: 1575–1584.
15. Carver TJ, Rutherford KM, Berriman M, Rajandream MA, Barrell BG, et al.
(2005) ACT: the Artemis Comparison Tool. Bioinformatics 21: 3422–3423.
16. Delcher AL, Phillippy A, Carlton J, Salzberg SL (2002) Fast algorithms for large-
scale genome alignment and comparison. Nucleic Acids Res 30: 2478–2483.
17. Richardson JS, Hynes MF, Oresnik IJ (2004) A genetic locus necessary for
rhamnose uptake and catabolism in Rhizobium leguminosarum bv. trifolii. J Bacteriol
18. Karunakaran R, Ebert K, Harvey S, Leonard ME, Ramachandran V, et al.
(2006) Thiamine is synthesized by a salvage pathway in Rhizobium leguminosarum
bv. viciae strain 3841. J Bacteriol 188: 6661–6668.
19. Girard L, Brom S, Davalos A, Lo ´pez O, Sobero ´n M, et al. (2000) Differential
regulation of fixN-reiterated genes in Rhizobium etli by a novel fixL-fixK cascade.
Mol Plant Microbe Interact 13: 1283–1292.
20. Riley M (1993) Functions of the gene products of Escherichia coli. Microbiol Rev
21. Tatusov RL, Koonin EV, Lipman DJ (1997) A genomic perspective on protein
families. Science 278: 631–637.
22. Cevallos MA, Porta H, Izquierdo J, Tun-Garrido C, Garcı ´a-de-los-Santos A, et
al. (2002) Rhizobium etli CFN42 contains at least three plasmids of the repABC
family: a structural and evolutionary analysis. Plasmid 48: 104–116.
23. Vernikos GS, Parkhill J (2006) Interpolated variable order motifs for
identification of horizontally acquired DNA: revisiting the Salmonella
pathogenicity islands. Bioinformatics 22: 2196–2203.
24. Bueno E, Go ´mez-Herna ´ndez N, Girard L, Bedmar EJ, Delgado MJ (2005)
Function of the Rhizobium etli CFN42 nirK gene in nitrite metabolism. Biochem
Soc Trans 33: 162–163.
25. Yost CK, Rath AM, Noel TC, Hynes MF (2006) Characterization of genes
involved in erythritol catabolism in Rhizobium leguminosarum bv. viciae. Microbi-
ology 152: 2061–2074.
26. Sa ´nchez-Contreras M, Bauer WD, Gao M, Robinson JB, Allan Downie J (2007)
Quorum-sensing regulation in rhizobia and its role in symbiotic interactions with
legumes. Philos Trans R Soc Lond B Biol Sci 362: 1149–1163.
27. Sourjik V, Muschler P, Scharf B, Schmitt R (2000) VisN and VisR are global
regulators of chemotaxis, flagellar, and motility genes in Sinorhizobium (Rhizobium)
meliloti. J Bacteriol 182: 782–788.
28. Rosemeyer V, Michiels J, Verreth C, Vanderleyden J (1998) luxI- and luxR-
homologous genes of Rhizobium etli CNPAF512 contribute to synthesis of
autoinducer molecules and nodulation of Phaseolus vulgaris. J Bacteriol 180:
29. Palacios R, Flores M (2005) Genome dynamics in rhizobial organisms. In:
Newton WE, Palacios R, eds. In Genomes and Genomics of Nitrogen-fixing
Organisms Springer. pp 183–200.
30. Romero D, Brom S (2004) The symbiotic plasmids of the Rhizobiaceae. In:
(Chapter 12), pp 271–290. In: Phillips G, Funnell BE, eds. Plasmid Biology:
American Society for Microbiology.
31. Flores M, Morales L, Avila A, Gonza ´lez V, Bustos P, et al. (2005) Diversification
of DNA sequences in the symbiotic genome of Rhizobium etli. J Bacteriol 187:
32. Reanney D (1976) Extrachromosomal elements as possible agents of adaptation
and development. Bacteriol Rev 40: 552–590.
33. Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG (1997) The
CLUSTAL_X windows interface: flexible strategies for multiple sequence
alignment aided by quality analysis tools. Nucleic Acids Res 25: 4876–4882.
34. Felsenstein J (2005) ‘‘Phylip (Phylogeny Inference Package) version 3.6.’’
Distributed by the author, Department of Genome Sciences, University of
35. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local
alignment search tool. J Mol Biol 215: 403–410.
36. Yang Z (1997) PAML: a program package for phylogenetic analysis by
maximum likelihood. Comput Appl Biosci 13: 555–556.
PLoS ONE | www.plosone.org9 July 2008 | Volume 3 | Issue 7 | e2567