Download Identification and Structure of the Rhizobium galegae Common

Identification and Structure of the Rhizobium galegae Common Nodulation
Genes: Evidence for Horizontal Gene Transfer
Leena Suominen,* Christophe Roos,† Gilles Lortet,* Lars Paulin,† and Kristina Lindström*‡
*Department of Applied Chemistry and Microbiology and †Institute of Biotechnology, University of Helsinki, Helsinki,
Finland; and ‡Department of Genetics, Harvard Medical School, and Department of Molecular Biology, Massachusetts
General Hospital, Boston, Massachusetts
Rhizobia are soil bacteria able to fix atmospheric nitrogen in symbiosis with leguminous plants. In response to a
signal cascade coded by genes of both symbiotic partners, a specific plant organ, the nodule, is formed. Rhizobial
nodulation (nod) genes trigger nodule formation through the synthesis of Nod factors, a family of chitolipooligosaccharides that are specifically recognized by the host plant at the first stages of the nodulation process. Here, we
present the organization and sequence of the common nod genes from Rhizobium galegae, a symbiotic member of
the Rhizobiaceae. This species has an intriguing phylogenetic position, being symbiotic among pathogenic agrobacteria, which induce tumors instead of nodules in plant shoots or roots. This apparent incongruence raises special
interest in the origin of the symbiotic apparatus of R. galegae. Our analysis of DNA sequence data indicated that
the organization of the common nod gene region of R. galegae was similar to that of Sinorhizobium meliloti and
Rhizobium leguminosarum, with nodIJ downstream of nodABC and the regulatory nodD gene closely linked to the
common nod operon. Moreover, phylogenetic analyses of the nod gene sequences showed a close relationship
especially between the common nodA sequences of R. galegae, S. meliloti, and R. leguminosarum biovars viciae
and trifolii. This relationship in structure and sequence contrasts with the phylogeny based on 16S rRNA, which
groups R. galegae close to agrobacteria and separate from most other rhizobia. The topology of the nodA tree was
similar to that of the corresponding host plant tree. Taken together, these observations indicate that lateral nod gene
transfer occurred from fast-growing rhizobia toward agrobacteria, after which the symbiotic apparatus evolved under
host plant constraint.
Introduction
Soil bacteria in the family Rhizobiaceae include
phytopathogenic agrobacteria that induce tumors in
plant roots and shoots and a divergent group of symbiotic rhizobia that are able to fix atmospheric nitrogen in
association with leguminous plants. At present, rhizobia
are divided into six main genera: Mesorhizobium, Sinorhizobium, Rhizobium, Allorhizobium, and the more
distantly related Bradyrhizobium and Azorhizobium
(Young and Haukka 1996; de Lajudie et al. 1998). Agrobacterium is intertwined with Rhizobium in the phylogenetic tree based on 16S rRNA sequences (Willems
and Collins 1993).
Rhizobial classification has traditionally been based
on a ‘‘cross-inoculation concept,’’ which means that the
early rhizobial taxonomy was based to a large degree
on host specificity (Fred, Baldwin, and McCoy 1932).
Thus, the symbiotic nodulation (nod) genes of rhizobia,
which are the determinants of the host range, had an
important role in rhizobial taxonomy. More recently,
phylogenetic analyses combined with molecular methods, such as DNA hybridization, DNA sequencing, and
typing methods (Vandamme et al. 1996), have provided
tools to generate more reliable classifications. At present, the sequencing of 16S rRNA genes is one of the
basic methods used for bacterial classification (Graham
et al. 1991; Young and Haukka 1996; Terefework et al.
Key words: legumes, Rhizobium, phylogeny, nodulation genes,
host constraint.
Address for correspondence and reprints: Leena Suominen, Department of Applied Chemistry and Microbiology, Division of Microbiology, P.O. Box 56, Biocenter 1, FIN-00014, University of Helsinki,
Finland. E-mail: [email protected].
Mol. Biol. Evol. 18(6):907–916. 2001
q 2001 by the Society for Molecular Biology and Evolution. ISSN: 0737-4038
1998). As a result, the taxonomy of rhizobia is undergoing rapid revision.
The rhizobial genes involved in the construction of
the symbiotic organ in plant roots or stems, the nodule,
are designated as nodulation (nod) genes. The transcription of the nodulation genes is induced through the regulatory nodD genes, which mediate host specificity by
activating nod operons in response to various flavonoid
compounds derived from legume hosts (Horvath et al.
1987; Spaink et al. 1987; Györgypal, Iyer, and Kondorosi 1988; Honma, Asomaning, and Ausubel 1990). The
expression of nod genes results in the synthesis of extracellular bacterial compounds, the Nod factors (Carlson, Price, and Stacey 1995; van Rhijn and Vanderleyden 1995). These factors act as signals, which, in the
appropriate host plant, elicit the first symptoms of nodule formation, such as deformation of root hairs, formation of infection threads, and cell division in the root
cortex (Lerouge et al. 1990).
Common nodulation genes nodABC and nodIJ are
involved in the synthesis of the basic oligosaccharide
core of the Nod factors. The highly conserved nature of
the common nodulation genes among rhizobial species
is clearly indicated by sequence data available from various rhizobia (for references, see the accession numbers
in table 1). However, in spite of their high sequence
similarity, the common nod genes are not functionally
conserved among rhizobial species (Debelle et al. 1996).
The Nod factors are acetylated with fatty acids either
from the general lipid metabolism or from a specific
a,b-unsaturated fatty acid pool (Yang et al. 1999). The
nodA gene, which codes for an acyltransferase and determines the type of the N-acyl substitution transferred
into the oligosaccharide backbone of the Nod factor,
plays a critical role in making this distinction. Thereby,
907
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Suominen et al.
Table 1
Sequence Data of the Rhizobial Species and Strains Included in the Phylogenetic Analysis of nod Genes
Species, Strain, and Plasmid
Azorhizobium caulinodans, ORS571 . . . . . . . . . . . . . . . . . .
Bradyrhizobium elkanii, USDA94 . . . . . . . . . . . . . . . . . . . .
Bradyrhizobium japonicum, USDA110 . . . . . . . . . . . . . . . .
Bradyrhizobium sp. (Parasponia), ANU289 . . . . . . . . . . . .
Bradyrhizobium sp. (Arachis), NC92 . . . . . . . . . . . . . . . . .
Mesorhizobium loti, NZP2213 . . . . . . . . . . . . . . . . . . . . . . .
M. loti, NZP2037 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Rhizobium sp. Oxytropis arctobia, N33 . . . . . . . . . . . . . . .
Rhizobium sp., NGR234 . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Rhizobium etli, CE-3, plasmid p42d . . . . . . . . . . . . . . . . . .
Rhizobium galegae, HAMBI1174 . . . . . . . . . . . . . . . . . . . .
Rhizobium leguminosarum bv. phaseoli, p42d . . . . . . . . . .
R. leguminosarum, pRle1001a . . . . . . . . . . . . . . . . . . . . . . .
R. leguminosarum, bv. trifolii, ANU843. . . . . . . . . . . . . . .
R. leguminosarum bv. viciae, 248, pRL1JI. . . . . . . . . . . . .
Rhizobium tropici, BR816 . . . . . . . . . . . . . . . . . . . . . . . . . .
R. tropici, CFN299 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Sinorhizobium meliloti, Rm1021 . . . . . . . . . . . . . . . . . . . . .
S. meliloti, Rm41. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Sinorhizobium fredii, USDA257 . . . . . . . . . . . . . . . . . . . . .
EMBL
Accession No.
L18897
U04609
M16488
J03685
X03720
U33192
L06241
X55705
X52958
U53327
X73362
M58625
M58626
X87578
M58626
J03671
X03721
X51411
Y00548
U11928
X98514
M11268
X01649
M73699
the function of the NodA protein specifies the Nod factor structure and the host range (Ritsema et al. 1996).
Because the bacterial Nod factor is a key signal molecule in the initiation of the plant root nodule, it is expected to evolve under constraints imposed by interaction with the host plant. To date, evolution of the Nod
factor under such constraint has not been clearly
demonstrated.
The location of nod genes varies in different rhizobia, being on either chromosomal or plasmid DNA
(Mercado-Blanco and Toro 1996). The position of an
individual nodulation gene or the internal structure of a
nod operon may vary in rhizobial genomes (Lindström
et al. 1995). Interestingly, the phylogeny of rhizobia
based on nodulation (nod) genes is different from that
based on 16S rRNA (Györgypal, Kiss, and Kondorosi
1991; Dobert, Breil, and Triplett 1994), and the evolutionary history of nodulation genes has remained
unclear.
The major questions about the evolution of nodulation concern the origin of rhizobial nod genes, the relationship between the phylogeny of the nod genes and
that derived from genomic sequences, and the influence
of the host plant on nod gene evolution (Young 1993,
1994; Dobert, Breil, and Triplett 1994; Young and Haukka 1996; Doyle 1998).
In this work, we describe the cloning, sequencing,
and structure of the common nod region of Rhizobium
galegae, a rhizobial species with a peculiar position in
the Agrobacterium clade based on 16S rRNA sequence
analyses (Lindström 1989; Willems and Collins 1993;
Young and Haukka 1996). We used phylogenetic analysis to examine the evolutionary relationships of the
nodulation genes to the bacterial and the host plant
genomes.
Database Entry Feature Table
nodA, nodB, nodC, nodI, nodJ
nodA, nodB, nodC (partial)
nodA (partial)
nodC (partial), nodI, nodJ
nodA, nodB, nodC (partial)
nodA
nodA (partial), C-terminus partial, nodC
nodI
nodC
nodA, nodB, nodC, nodI, nodJ
nodA, nodB, nodC
nodA
nodB, nodC
nodA, nodB, nodC, nodI, nodJ
nodB
nodA (partial)
nodA, nodB, nodC (partial)
nodI (partial), nodJ
nodA, nodB, nodC, nodI, nodJ
nodA (partial)
nodA, nodB, nodC
nodA, nodB, nodC, nodI (partial)
nodA, nodB, nodC
nodA, nodB, nodC
Materials and Methods
Growth Media, Strains, Plasmids, and Phages
Bacterial strains, plasmids, and phages used in this
work are listed in Table 2. Rhizobium strains were
grown at 288C on tryptone yeast (TY) complete medium
(Beringer 1974) or on yeast extract mannitol (YEM) medium (Lindström et al. 1985). Selections after the conjugations were performed on def8 medium with 1,000
mg/ml streptomycin and 10 mg/ml tetracycline (Lindström and Lehtomäki 1988). Escherichia coli strains
were grown at 378C in Luria broth (Maniatis, Fritsch,
and Sambrook 1982) or in TY; in the cosmid selections,
the media were supplemented with 10 mg/ml tetracycline. Test plants Medicago sativa L. (cv. Iroquois) and
Galega orientalis Lam. were grown on Jensen agar
slants (Vincent 1970) in a growth chamber with a 16-h
light period at 258C and an 8-h dark period at 188C using
a 400-W Na-lamp (Airam) as a light source.
DNA Isolation and Manipulation Procedures
Subcloning and screening of DNA were performed
as described by Maniatis, Fritsch, and Sambrook (1982).
Total DNA, probe DNA, and plasmid isolations were
performed according to Meade et al. (1982) and by standard procedures described by Maniatis, Fritsch, and
Sambrook (1982). The 25-bp synthetic nod-box core oligonucleotide was synthesized following the data of
Rostas et al. (1986). Probe DNAs were labeled according to standard methods (Hames and Higgins 1985).
Southern hybridizations were performed as described by
Kaijalainen and Lindström (1989).
Nodulation Gene Evolution
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Table 2
Bacterial Strains and Plasmids Used for the Cloning and Identification of the Common nod Region
Designation
Characteristics
Sources and References
Bacterial strains
Rhizobium galegae
HAMBI 1174 . . . . . . . .
HAMBI 1587 . . . . . . . .
Smr, Spcr
1174::Tn5 in nodABC, Smr, Spcr, Kmr
Lindström et al. (1985)
This work
Sinorhizobium meliloti
Rm1126 . . . . . . . . . . . . .
Rm1027 . . . . . . . . . . . . .
Rm1021::Tn5, Smr, Nmr, Nod2, Hac2
Rm1021::Tn5, Smr, Nmr, Nod2, Hac2
Meade et al. (1982)
Meade et al. (1982)
IncP, repRK2, Tcr
IncP, repRK2, Tcr, cos
repcolE1, Nmr
pBR325 carrying common nod BamHI-HindIII fragment of
pRmJ1
8.7-kb common nod fragment from pRmSL26 in pBR325, Apr,
Tcr
pLAFR1 carrying 26-kb common nod clone of HAMBI 1174,
Tcr
6-kb common nod fragment of pRg30 subcloned in pWB5a, Tcr
Ditta et al. (1980)
Friedman et al. (1982)
Ditta et al. (1980)
Egelhoff et al. (1985)
Plasmids
pRK290 . . . . . . . . . . . . . . .
pLAFR1. . . . . . . . . . . . . . .
pRK2013 . . . . . . . . . . . . . .
pRmSL42. . . . . . . . . . . . . .
pRmJ1 . . . . . . . . . . . . . . . .
pRg30 . . . . . . . . . . . . . . . .
pRg33 . . . . . . . . . . . . . . . .
Jacobs, Egelhoff, and Long (1985)
This work
This work
NOTE.—Smr, Spcr, Kmr, Nmr, Tcr, and Apr indicate resistance to streptomycin, spectinomycin, kanamycin, neomycin, tetracycline, and ampicillin, respectively;
Nod2 indicates the inability to form nodules on the hosts and Hac2 indicates the inability to curl the root hairs of the hosts.
Cloning and Identification of the Common nod Region
Sequencing of the Common nod Region
The construction of the R. galegae HAMBI 1174
clone library has been described previously (Suominen
et al. 1999). To clone the common nodulation genes, the
cosmid library was conjugated into two mutants of Sinorhizobium meliloti, Rm1126 and Rm1027, both carrying a mutated nodC gene (Buikema et al. 1983). Conjugations were conducted according to protocols described by Ruvkun and Ausubel (1981), with pRK2013
as a helper plasmid. Transconjugants were used in en
masse infection of alfalfa as described by Marvel et al.
(1985). After 30 days, bacteria from the nodules showing activity in acetylene reduction measurements (Lindström 1984) were isolated, and their plasmid content
was examined by EcoRI digestion. A 28-kb plasmid,
pRg30, which carried a 9.2-kb EcoRI fragment dominating in restriction profiles, gave a positive reaction in
a hybridization assay with the common nod probe
pRmJ1 from S. meliloti and was chosen for further
study. A restriction map of pRg30 was constructed. The
location of the nodulation genes in pRg30 was investigated by hybridizations using internal fragments of S.
meliloti nodABCD and nodD genes from plasmid pRmSL42 and a synthetic nod promoter sequence, nodbox, as probes.
To verify the functionality of the R. galegae common nodulation genes, site-directed Tn5 mutagenesis
was performed in the nod gene region by the method of
de Bruijn and Lupski (1984), and the insertion sites were
mapped by restriction endonucleases. For marker exchange, pRg33, a 6-kb subclone of pRg30 (see fig. 1),
carrying a Tn5 in nodABC, was conjugated into R. galegae HAMBI 1174. Selection for strains in which Tn5
had recombined with the R. galegae genome was carried
out following Ruvkun and Ausubel (1981). The correct
location of the Tn5 insertion was verified by DNA
hybridization.
The 6-kb common nod fragment, pRg33, was sequenced using the AutoRead Kit (Pharmacia, Uppsala,
Sweden), and the sequencing reactions were run on an
Automated DNA Sequencer A.L.F. (Pharmacia). A nested set of deletions of the larger fragments to be sequenced was obtained using the Nested Deletion Kit
(Pharmacia). Sequences were assembled using the program Xdap (Dear and Staden 1991). The common nod
of R. galegae appears in the EMBL/GenBank/DDBJ database under accession number X87578.
Phylogenetic Analysis of nod Genes
Bacterial nod DNA sequences used for the phylogenetic studies are listed in table 1, and the accession
numbers for 16S rRNA and legume internal transcribed
sequence (ITS) sequences are shown in table 3. For the
nod DNA sequences, only the coding regions were used.
DNA sequences were aligned with the program PILEUP
(Wisconsin Package, version 10.0; Genetics Computer
Group, Madison, Wis.) or with CLUSTAL W, version
1.7 (Thompson, Higgins, and Gibson 1994), using an
identity matrix, a gap weight of 8, and a gap length
weight of 0.1. Amino acid sequences were aligned with
the same programs using a Blosum82 protein weight
matrix, a gap weight of 12, and a gap length weight of
0.5. The nod DNA alignments were checked by eye and
corrected to avoid alignments with disrupted reading
frames. Trees were constructed from the data by maximum parsimony and neighbor joining (NJ) using programs from the PHYLIP (Felsenstein 1993) and Treecon
(Van de Peer and De Wachter 1994) packages and the
GCG implementation of PAUP* (Wisconsin package).
Heuristic searches were utilized in parsimony analyses
due to the large number of taxa examined. Branch swapping was done by tree bisection-reconnection. For NJ
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Suominen et al.
FIG. 1.—Restriction map of the common nod gene region of Rhizobium galegae HAMBI 1174. The cosmid clone pRg30 carries the six
open reading frames homologous to nodDABCIJ genes subcloned in pRg33. m indicates the site of the Tn5 insertion in pRg33. The black
square indicates the nod-box sequence. Restriction enzymes used were as follows: E 5 EcoRI; B 5 BamHI; C 5 ClaI; H 5 HindIII; P 5 PstI;
S 5 SalI.
Table 3
Accession Numbers of the Rhizobial 16S rRNA Gene
Sequences and Legume ITS-2 Sequences Used in the
Phylogenetic Analysis
Rhizobial Species
Accession No.
16S rRNA gene sequences
Azorhizobium caulinodans . . . . . . . . . . . . . .
X67221
Bradyrhizobium
B. elkanii. . . . . . . . . . . . . . . . . . . . . . . . . . . . .
B. japonicum USDA6 . . . . . . . . . . . . . . . . . .
B. japonicum USDA94 . . . . . . . . . . . . . . . . .
U35000
U69638
D13429
Mesorhizobium
M. huakuii. . . . . . . . . . . . . . . . . . . . . . . . . . . .
M. loti . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
D13431
X67229
Rhizobium
R. etli. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
R. galegae. . . . . . . . . . . . . . . . . . . . . . . . . . . .
R. leguminosarum bv. viciae. . . . . . . . . . . . .
R. leguminosarum bv. trifolii . . . . . . . . . . . .
R. tropici . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
U28916
X67226
U31074
U29386
X67234
Sinorhizobium
S. fredii . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
S. meliloti LMG6133 . . . . . . . . . . . . . . . . . . .
S. meliloti LMG8890 . . . . . . . . . . . . . . . . . . .
D14516
X67222
X77121
Legume ITS-2 sequences
Arachis major . . . . . . . . . . . . . . . . . . . . . . . . . .
Astragalus asterias . . . . . . . . . . . . . . . . . . . . . .
Cytisus arboreus . . . . . . . . . . . . . . . . . . . . . . . .
Galega orientalis . . . . . . . . . . . . . . . . . . . . . . . .
Genista januensis. . . . . . . . . . . . . . . . . . . . . . . .
Glycine max . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Lupinus luteus . . . . . . . . . . . . . . . . . . . . . . . . . .
Medicago sativa. . . . . . . . . . . . . . . . . . . . . . . . .
Oxytropis campestris . . . . . . . . . . . . . . . . . . . . .
Pisum sativum . . . . . . . . . . . . . . . . . . . . . . . . . .
Phaseolus vulgaris. . . . . . . . . . . . . . . . . . . . . . .
Trifolium longipes . . . . . . . . . . . . . . . . . . . . . . .
AF203552
L10765
Z72241
U56016
Z72269
U60551
Z72207
AF053142
L10803
U50862
AF074398
U56018
analyses, distance measures were employed using a Kimura two-parameter correction for multiple hits and a
transition/transversion ratio of 2. Bootstrap analyses
with 1,000 replicates were performed to examine the
relative support for relationships in the resultant topologies. Rhizobium trees were rooted with respect to Azorhizobium caulinodans, which, according to polyphasic
taxonomic studies (Vandamme et al. 1996), is the most
distant species among the rhizobia included in the analyses (Dreyfus, Garcia, and Gillis 1988). A test of phylogenetic congruence among phylogenies inferred for
the nod genes, the 16S rRNA genes, and the host plants
was conducted using TreeMap (Page 2000). TreeView
(Page 1996) was used to prepare illustrations of the
trees.
Results
Identification of the Common Nodulation Gene Region
in R. galegae
The cosmid pRg30 was able to complement the nod
gene mutations of S. meliloti and showed sequence similarity to common nod genes. The physical map (fig. 1)
shows the EcoRI, SalI, BamHI, ClaI, and HindIII sites
of pRg30. The hybridization patterns obtained with the
common nod and the internal nodD fragments from
pRmSL42 and nod-box as probes indicated that the
common nod gene region was located within a 6-kb
ClaI-ClaI fragment of pRg30, designated pRg33. The
functionality of the common nodulation genes of R. galegae was further verified by homologous exchange of
wild type and a Tn5 insertion in the nodABC gene region. The mutant strain designated HAMBI 1587 had
lost its ability to form nodules on G. orientalis. Nodulation was restored to 100% in HAMBI 1587 by the
conjugation of pRg33 into the strain.
Sequencing of the Common Nodulation Region
The sequence data verified the restriction sites previously detected by restriction analysis and revealed the
Nodulation Gene Evolution
existence of six major open reading frames in the pRg33
region (fig. 1). The organization of the common nod
gene region of R. galegae was similar to that of S. meliloti and Rhizobium leguminosarum, with nodIJ downstream of nodABC and the regulatory nodD gene closely
linked to the common nod operon. These genes code for
the NodD, NodA, NodB, NodC, NodI, and NodJ proteins. The highest protein sequence identities with
known rhizobial nodulation proteins were 74% for
NodA with R. leguminosarum biovar trifolii, 65% for
NodB with R. leguminosarum biovar viciae, 70% for
NodC with Rhizobium sp. NGR234, 74% for NodI and
NodJ with R. leguminosarum biovar viciae, and 79% for
NodD with both S. meliloti and R. leguminosarum biovar viciae. The percentages were measured over the total
length of the protein. The organization of the common
nod gene region was similar to that of R. leguminosarum
and S. meliloti, with nodIJ downstream of nodABC
(Rossen, Johnston, and Downie 1984; Egelhoff et al.
1985; Surin et al. 1990). The regulatory nodD gene was
located adjacent to the common nod operon and translated from the opposite strand of a stretch of DNA lying
248 bp upstream of nodA (fig. 1).
Phylogenetic Analysis of the Sequences
Phylogenetic analyses of the DNA sequences were
conducted using the R. galegae nod genes sequenced in
this study and corresponding sequences published in the
EMBL database (table 1). Phylogenetic trees obtained
from maximum-likelihood, parsimony, and neighborjoining analyses were identical with respect to the clustering of the main groups. The inferred phylogeny of
complete nodA gene sequences at both the DNA (fig.
2A) and the protein (fig. 2B) levels grouped R. galegae
HAMBI 1174 with S. meliloti and R. leguminosarum, in
contradiction to what would be expected from previous
work based on 16S rRNA genes (Willems and Collins
1993), which grouped R. galegae with Agrobacterium.
The bootstrap support for this group was 87% in analyses based on amino acid sequences (fig. 2B). The observed grouping of R. galegae nodA genes with S. meliloti and R. leguminosarum nodA genes seemed to follow previously published hypotheses of host plant phylogeny (Polhill 1981; Doyle, Lavin, and Bruneau 1992;
Doyle et al. 1997).
To further study the observed concordance between
the phylogenies of nod genes and hosts, we performed
a congruence analysis following Page (1994). For this
purpose, we compared the nodA gene tree with established bacterial 16S rRNA phylogenies on the one hand
and with host plant phylogenies on the other. We chose
the nodA gene for our study because it was in a cardinal
position in the specificity recognition event. The 16S
rRNA trees were calculated on complete sequence data
available in the public databases. The phylogenetic trees
of the relevant members of the host plant family were
based on ITS-1 and ITS-2 sequences; trees of ITS-1 and
ITS-2 showed similar grouping (data not shown). The
16S rRNA and ITS-2 reference trees were concordant
with previously proposed phylogenies (Polhill 1981;
911
Doyle, Lavin, and Bruneau 1992; Willems and Collins
1993; Doyle et al. 1997).
The comparison of the bacterial nodA gene/host
plant phylogeny to the bacterial nodA gene/bacterial 16S
rRNA phylogeny performed with TreeMap was indicative of a high congruence between the nodA gene and
host plant ITS-based phylogeny but not the 16S rRNA
phylogeny. Also, visual inspection of both tree pairs, or
‘‘tanglegrams’’ (fig. 3), unveils a major difference in the
position of the R. galegae grouping. In the host tanglegram, R. galegae clearly groups with S. meliloti and R.
leguminosarum (fig. 3B), whereas this is not the case in
the 16S rRNA tanglegram (fig. 3A). This difference is
taken as evidence of lateral transfer of the nodA gene
into R. galegae from a common ancestor of S. meliloti
and R. leguminosarum.
In analyses of nodB and nodC sequences, the observed clustering was less obvious. The nodB and nodC
genes of R. galegae were closely related to those of R.
leguminosarum based on the inferred phylogeny of amino acid sequences. Because of the small number of nodI
and nodJ sequences in the database, inferences of the
relationship of the R. galegae genes are less reliable.
Our results nevertheless indicate that even the nodI and
nodJ of R. galegae cluster closer to those of R. leguminosarum than to other rhizobial sequences included (fig.
2).
Discussion
The phylogeny of rhizobia and the origin of symbiotic nodulation (nod) genes have been the subject of
several studies (Provorov 1994; Ueda et al. 1995; Martinez-Romero and Caballero-Mellado 1996; Young and
Haukka 1996; Wernegreen and Riley 1999). To address
the question of evolution of nodulation, we chose to
study R. galegae. Rhizobium galegae belongs to the
agrobacterial clade of Rhizobiaceae, as shown by phylogenetic analysis of 16S rRNA genes (for references
see Wang and Martinez-Romero 2000). Agrobacteria
form a group of phytopathogenic soil bacteria that upon
infection induce tumors in shoots and roots of angiosperms. Rhizobium galegae has an exceptional position
among agrobacteria because its relation to the host plant
is symbiotic, not pathogenic. Symbiotic members of
Rhizobiaceae consist of three main groups: stem-nodulating Azorhizobium, the slow-growing genus Bradyrhizobium, and the fast-growing rhizobial genera Allorhizobium, Mesorhizobium, Rhizobium, and Sinorhizobium (de Lajudie et al. 1998).
In this work, we present the organization of the
symbiotic nod genes in R. galegae and confirm that the
phylogeny of the nod genes is clearly different from the
phylogeny based on the 16S rRNA genes. This difference, together with the fact that many nonsymbiotic bacteria are intermingled with rhizobia in the phylogenetic
tree of the alpha subdivision of the Proteobacteria, implies that all rhizobia did not inherit their nod genes
directly from their ancestor (Young and Haukka 1996;
Terefework et al. 1999).
FIG. 2.—Dendrograms obtained by the neighbor-joining method, showing the phylogeny of rhizobial nodA, nodB, nodC, nodI, and nodJ (A) DNA and (B) amino acid sequences based on coding
regions. Bootstrap values, based on 1,000 replicates, are indicated at the branching points. For the accession numbers and the sequences used, see table 1.
912
Suominen et al.
Nodulation Gene Evolution
913
FIG. 3.—Phylogenetic congruence (A) between the nodA and the 16S rRNA gene phylogenies and (B) between the nodA and host plant
phylogenies. The thin lines indicate the host-bacteria associations. The shaded areas emphasize the species for which differences in congruence
were observed. The accession numbers of the nodA sequences used are shown in table 1. The nodA sequence of Mesorhizobium huakuii was
kindly provided by Xue-Xian Zhang, Aachen University of Technology, Germany. Accession numbers of the sequences used for 16S rRNA
and ITS1 phylogenies are listed in table 3
.
The common nodulation genes that we identified
from R. galegae have a structure and orientation similar
to those of R. leguminosarum and S. meliloti (Rossen,
Johnston, and Downie 1984; Egelhoff et al. 1985; Surin
et al. 1990). In these rhizobia, the nodDABCIJ genes
form a cassette located on a plasmid, whereas in other
rhizobia the nod genes are scattered throughout the genome, and some additional nodulation genes may be inserted in the common nod region (Lindström 1989; Selenska-Trajkova et al. 1990; Lindström et al. 1995; Freiberg et al. 1997). The similarity of organization of the
nod genes of R. galegae, R. leguminosarum, and S. meliloti suggests that these genes originate from the same
evolutionary source. However, as R. galegae is rather
distant from R. leguminosarum and S. meliloti in the 16S
rRNA–based phylogeny, only lateral transfer of nodulation genes could account for this high similarity. The
concept of lateral transfer is given further support by
our phylogenetic analyses.
Our phylogenetic study, which was based on parsimony and neighbor-joining analyses of complete,
aligned nodA, nodB, and nodC nucleotide and amino
acid sequences, provided a well-supported estimate of
nod gene phylogeny. The R. galegae Nod protein sequences were most closely related to those of R. leguminosarum and S. meliloti biovars viciae and trifolii in
spite of the distant taxonomic position of R. galegae
relative to other Rhizobium species. Rhizobium leguminosarum and S. meliloti are fast-growing rhizobia with
a fairly narrow host range. They infect the temperate
legume tribes Vicieae and Trifolieae, with a similar infection type and an indeterminate nodule structure. The
host tribe of R. galegae, Galegeae, is closely related to
Vicieae and Trifolieae (Doyle 1998). Thus, it seems
914
Suominen et al.
probable that these legumes share a similar receptor system for Nod factors, the symbiotic signals from rhizobia.
Therefore, it is likely that the host constrains the evolution of the nod region by selecting against changes
that prevent host infection.
Several authors have discussed the role of the host
plant in nod gene evolution. While some think that there
is little correlation between rhizobial and plant phylogenies (Young and Johnston 1989; Doyle 1994, 1998),
others suggest that the rhizobial nod gene phylogeny
might be congruent with the phylogeny of the host
plants while the bacterial phylogeny as a whole would
not (Dobert, Breil, and Triplett 1994). Indeed, Kaijalainen and Lindström (1989) studied several strains of R.
galegae by analysis of restriction fragment length polymorphism and showed that the symbiotic (common nod
and nifHDK) probes grouped the bacteria according to
the host plant Galega officinalis or G. orientalis, whereas the constitutive (hemA, glnA, ntrC, and recA) probes
did not. Our study shows that common nod genes from
R. galegae group according to host plant. However, differences in function of the nod genes are expected to be
reflected in how strictly they follow the host plant phylogeny rather than the 16S rRNA–based bacterial
phylogeny.
The Nod factors are acetylated with fatty acids either from the general metabolism or, in a subset of rhizobial strains, from a polyunsaturated fatty acid pool
(Yang et al. 1999). The nodA gene determines the type
of the N-acyl substitution of the Nod factor and therefore plays a crucial role in host plant recognition (Ritsema et al. 1996; Yang et al. 1999). The Nod factors
produced by R. leguminosarum and S. meliloti both have
polyunsaturated fatty acid chains (Lerouge et al. 1990;
Spaink et al. 1991; Spaink, Wijfjes, and Lugtenberg
1995). Rhizobium galegae has been shown to have the
same type of fatty acid substitution (Yang et al. 1999).
Moreover, we found that nodA was the nod gene that
showed the most strict hostlike phylogeny. Taken together, our results clearly demonstrate that the Nod factor in R. galegae has evolved under strong constraint
imposed by the host plant interaction following a historically more ancient lateral gene transfer event.
Several questions remain unanswered. Our studies
were based on the finding that the R. galegae nodDABCIJ genes form a plasmid-borne cassette and are
related to similar cassettes on nonrelated bacterial species that infect related host plants. This incongruence,
most apparent for the nodA gene, implies that the common nod genes have evolved through lateral transfer
between major chromosomal subdivisions. However,
further studies are required to identify the primary unit
of evolution: the cassette as a whole or, for example, the
nodA gene, with the other nod genes showing similar,
albeit less obvious, evolution simply due to some kind
of ‘‘hitchhiking’’ effect. Moreover, further study will be
needed to identify additional putative gene transfer
events that may have led to the present state of nod gene
organization in symbiotic rhizobia.
Acknowledgments
This work was initiated in the laboratory of Fred Ausubel. We express our warm thanks for his genuine interest and support. The work was funded by the Academy of Finland and by grants from EMBO, the Finnish
Culture Foundation, and the University of Helsinki.
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PAMELA SOLTIS, reviewing editor
Accepted January 31, 2001