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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, 907 908 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 909 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 910 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. 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PAMELA SOLTIS, reviewing editor Accepted January 31, 2001