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Ó Springer-Verlag 1997 Mol Gen Genet (1997) 255:131±140 ORIGINAL PAPER B. SaÂnchez-AnduÂjar á C. Coronado S. Philip-Hollingsworth á F. B. Dazzo á A. J. Palomares Structure and role in symbiosis of the exoB gene of Rhizobium leguminosarum bv trifolii Received: 31 May 1996 / Accepted: 18 December 1996 Abstract The Rhizobium leguminosarum bv trifolii exoB gene has been isolated by heterologous complementation of an exoB mutant of R. meliloti. We have cloned a chromosomal DNA fragment from the R. leguminosarum bv trifolii genome that contains an open reading frame of 981 bp showing 80% identity at the amino acid level to the UDP-glucose 4-epimerase of R. meliloti. This enzyme produces UDP-galactose, the donor of galactosyl residues for the lipid-linked oligosaccharide repeat units of various heteropolysaccharides of rhizobia. An R. leguminosarum bv trifolii exoB disruption mutant diered from the wild type in the structure of both the acidic exopolysaccharide and the lipopolysaccharide. The acidic exopolysaccharide made by our wild-type strain is similar to the Type 2 exopolysaccharide made by other R. leguminosarum bv trifolii wild types. The exopolysaccharide made by the exoB mutant lacked the galactose residue and the substitutions attached to it. The exoB mutant induced the development of abnormal root nodules and was almost completely unable to invade plant cells. Our results stress the importance of exoB in the Rhizobium-plant interaction. Key words Rhizobium leguminosarum bv trifolii á Trifolium subterraneum á Trifolium repens á exoB gene á Exopolysaccharide Communicated by E. CerdaÂ-Olmedo B. SaÂnchez-AnduÂjar á C. Coronado á A. J. Palomares (&) Departamento de MicrobiologõÂ a, Facultad de Farmacia, Universidad de Sevilla, E-41012 Sevilla, Spain e-mail [email protected] S. Philip-Hollingsworth á F. B. Dazzo Department of Microbiology, Michigan State University, East Lansing, Michigan 48824, USA F. B. Dazzo Center for Microbial Ecology, Michigan State University, East Lansing, Michigan 48824, USA The nucleotide sequence data reported in this paper are available from the EMBL data bank under accession number X96507 Introduction The extracellular and surface polysaccharides produced by Rhizobium constitute the interface between the bacterial cell and its environment and are involved in the interactions leading to the establishment of an eective Rhizobium-legume symbiosis (Coronado et al. 1996). These polysaccharides include extracellular heteropolysaccharides (EPS), capsular polysaccharides, and lipopolysaccharides (LPS) of the outer membrane. Cyclic (1±2)-b-D glucan is found in the periplasm and culture medium of some strains. Rhizobium meliloti synthesizes two dierent heteropolysaccharides. EPSI is a succinoglycan necessary for the invasion of alfalfa root nodules; EPSI-de®cient mutants fail to form infection threads. EPSII is a galactoglucan with a completely dierent structure; EPSIIde®cient mutants are still able to form an eective symbiosis with alfalfa. Twenty-two exo genes for EPSI synthesis have been identi®ed in R. meliloti Rm2011; 19 of them reside in a contiguous 24-kb cluster on the second symbiotic megaplasmid (pSymb) (Leigh and Walker 1994). The R. meliloti exoB gene encodes a UDP-glucose 4epimerase (BuendõÂ a et al. 1991) that converts UDPglucose into UDP-galactose. Since galactose is a common component of both EPS and LPS (Diebold and Noel 1989; Bhagwat et al. 1991), exoB mutants should be pleiotropic and defective in the synthesis of both polysaccharides. The symbiotic behavior of exoB mutants depends on both the Rhizobium and the legume host species. An exoB mutant of R. meliloti induces empty, ineective nodules on alfalfa (Leigh and Lee 1988). An exoB mutant of Rhizobium sp. GRH2 induces a few empty nodules on Acacia and fails to nodulate most herbaceous legume hosts (Lopez-Lara et al. 1995). R. leguminosarum bv viciae exoB mutants produce only some root hair deformation and rare, abortive infection threads on Vicia villosa (Canter Cremers et al. 1990). In contrast, a Rhizobium sp.(Ollero et al. 1994) exoB mu- 132 tant induces eective, indeterminate nodules on the host, Hedysarum coronarium. Strains of R. leguminosarum bv trifolii may be classi®ed into two major groups and one minor group according to the stoichiometry and the linkage site of the non-carbohydrate substituents in EPS (Philip-Hollingsworth et al. 1989). The symbiotic behavior of some exo mutants of R. leguminosarum bv trifolii has been described, but they have not been characterized at the genetic level. Spontaneous and Tn5-induced exo mutants of dierent strains of R. leguminosarum bv trifolii induce small, white, and ineective nodules on clover roots (Rolfe et al. 1980; Chakravorty et al. 1982; Derylo et al. 1986). These nodules contain infection threads, but few of the plant cells contain intracellular bacteria. We have constructed an exoB disruption mutant of R. leguminosarum bv trifolii to determine the role of this gene in EPS structure and symbiotic behavior. Materials and methods Strains, plasmids, and media Strains and plasmids used in this study are listed in Table 1. R. meliloti strains were grown in rich medium YTA (Orosz et al. 1973) or LB medium (Ausubel et al. 1989) supplemented with 2.5 mM MgSO4 and 2.5 mM CaCl2 at 30° C. R. leguminosarum bv trifolii strains were grown in YT medium (Beringer 1974) or de®ned BIII agar medium (Dazzo 1982) at 30° C. For Congo red absorption, bacteria were streaked on plates of YEMCR agar (Zevenhuizen et al. 1986). Escherichia coli strains were grown in LB medium at 37° C. When appropriate, media were supplemented with 200 lg/ml neomycin, 10 lg/ml nalidixic acid, 50 lg/ml kanamycin, 100 lg/ml ampicillin, 20 lg/ml gentamicin, 100 lg/ml spectinomycin, 5 lg/ml tetracycline for Rhizobium strains and 15 lg/ml for E. coli strains or 200 lg/ml Calco¯uor white M2R (Fluorescent Brightener 28, Sigma, St. Louis, Mo., USA). An R. leguminosarum bv trifolii gene bank, previously constructed in E. coli HB101 (Rodriguez-QuinÄones et al. 1989), was used for the heterologous complementation of the exoB mutant, R. meliloti 7094. Cosmid pVK102 derivatives were transferred conjugatively in triparental matings as described by Ditta (1986). DNA manipulations and analysis E. coli plasmid DNA was isolated by alkaline lysis (Ausubel et al. 1989). Rhizobium plasmid DNA was isolated according to Ish-Horowicz and Burke (1981). Bacterial genomic DNA was isolated by selective precipitation with CTAB (hexadecyltrimethyl ammonium bromide) as described by Ausubel et al. (1989). Plasmid characterization was performed according to the procedure of Eckhardt (1978) as modi®ed by Rosenberg et al. (1982). DNA was digested, electrophoresed in 0.7% agarose gels, and blotted onto nylon membranes (Hybond-N, Amersham, Buckinghamshire, UK, 0.45 lm) according to the Southern blotting procedure. DNA probes were prepared using the Multiprime DNA labelling system (Amersham). Hybridizations were performed overnight at 37° C in 3 ´ SSC, 50% formamide, 25 mM NaH2PO4, 25 mM Na2HPO4, 5 ´ Denhardt's solution, containing 500 lg/ml of herring sperm DNA (Kondorosi et al. 1982). Tn5 insertions in plasmid pBSB43 were physically mapped as described by Ditta (1986). DNA sequences were obtained from overlapping nested deletion clones generated by exonuclease III digestion according to Heniko (1984). Sequencing reactions were performed using the Auto-Read Sequencing kit (Pharmacia Biotech, Uppsala, Sweden). Sequence data were obtained and processed using the A.L.F. DNA Sequencer (Pharmacia Biotech) according to the manufacturer's instructions. The nucleotide and amino acid sequences were analyzed using the BESTFIT program of the Genetics Computer Group (GCG) program package of the University of Wisconsin (Devereux et al. 1984). Plant nodulation Rhizobium strains were assayed for their symbiotic phenotypes on alfalfa (Medicago sativa cv Moapa) and clover plants (Trifolium repens cv Huia and T. subterraneum cv Seaton Park) grown in Leonard jars, and analyzed 4±5 weeks after inoculation, (Corbin et al. 1982). For each strain, at least six nodules from dierent plants were collected. Nodules were ®xed for 2 h at 4° C with glutaraldehyde (30 ml/l in 100 mM sodium cacodylate, pH 7.2). After two rinses in this buer, the nodules were post®xed for 2 h in OsO4 (10 g/l in 100 mM sodium cacodylate, pH 7.2). After three further rinses, the samples were dehydrated in a graded acetone series at 4° C. Samples were maintained for 2 h in uranyl acetate (10 g/l in 70% acetone) before complete dehydration. Finally, nodules were embedded in Spurr medium. Sections were examined by combined light and transmission electron microscopy following staining with (1 g/l) toluidine blue or lead citrate/uranyl acetate, respectively. Exopolysaccharide analysis Bacteria were grown in shaker ¯asks containing 500 ml of BIII medium at 30° C for 5 days to a density of 0.6 OD600. After removing the bacteria by centrifugation, two volumes of cold ethanol were added to the supernatant. The EPS precipitate was collected by centrifugation, lyophilized, redissolved in distilled water, and dialyzed (12 000±14 000 molecular weight cuto) against 2 l of distilled water at 4° C with ®ve to seven changes over a 3-day period to remove the majority of the b-1,2 glucans and other oligomers. Finally, the EPS solution was lyophilized. The EPS were analyzed by proton nuclear magnetic resonance (1H-NMR) spectroscopy and combined gas-liquid chromatography/mass spectrometry (GLC/MS). For 1H-NMR analysis, 3 mg of puri®ed EPS were dissolved in deuterium oxide (99.7%, 0.4 ml), lyophilized, redissolved in deuterium oxide (99.99%, 0.4 ml), and sonicated for 1 min at room temperature. Spectra were recorded at 500 MHz and 70° C with presaturation of the solvent peak at d 4.25. For GLC/MS analysis of glycosyl compositions, EPS samples were acid-hydrolyzed and the released sugars converted to their corresponding alditol acetate derivatives as previously described (Hollingsworth et al. 1984). Uronic acids were identi®ed by carboxyl reduction with sodium borodeuteride followed by GLC/ MS analysis of the alditol acetate derivatives with selected ion monitoring for 217 a.m.u. and 219 a.m.u. as previously described (Hollingsworth et al. 1984; Philip-Hollingsworth et al. 1989). Electrophoresis of lipopolysaccharides For SDS-polyacrylamide gel electrophoresis, the LPS were solubilized from proteinase K-treated cells and electrophoresed in 16.5% polyacrylamide gels as described by KoÈplin et al. (1993). Gels were ®xed and silver-stained as described by Kittelberger and Hilbink (1993). LPS from the smooth strain Salmonella typhimurium and the rough strains S. typhimurium TV119 and SL1181, purchased from Sigma, were used as standards. Results A gene from R. leguminosarum bv trifolii homologous to the R. meliloti exoB gene Ten exconjugants of the exoB mutant, R. meliloti 7094, carrying a cosmid from the R. leguminosarum bv trifolii 133 Table 1 Strains and plasmids (rantibiotic resistance, Nal nalidixic acid, Sp spectinomycin, Sm streptomycin, Tc tetracycline, Km kanamycin, Rf rifampicin, Gm gentamicin) Strain or plasmid Reference or source Rhizobium strains R. leguminosarum bv trifolii RS800 RS12 Wild type, Nalr exoB mutant RS800 derivative, Spr Megias et al. (1988) This work R. meliloti Rm1021 Rm7094 Rm7031 Rm7055 Rm8457 Smr, SU47 derivative Rm1021 exoB::Tn5 Rm1021 exoA::Tn5 Rm1021 exoF::Tn5 Rm1021 exoM::Tn5 Meade et al. (1982) Leigh et al. (1985) Leigh et al. (1985) Leigh et al. (1985) Long et al. (1988) hsdS, hsdM, pro, leu, thi, gal, lacY, recA, Smr recA1, endA1, gyrA96, thi, hsdR17, supE44, relA1, D(lac-proAB) F¢ (traD36, proAB, lacI qZDM15) F-, recA, hsdR, RP4-2 (Tc::Mu), (Km::Tn7) polA1, rha, his, Nalr, Rf r Tn5 is present at an indeterminate chromosomal location Boyer and Roulland-Dussoix (1969) Escherichia coli strains HB101 JM109 S17-1 C2110 HB101::Tn5 Plasmids pRK2073 pVK102, gene bank pD2 pRK404 pUT miniTn5 Sp/Sm pJQ200SK pBSB4 pBSB43 pBSB42 pBSB43-3 pBSB43-4 pBSB43-6 pBSB43-8 pJQ2003 ``Helper'' plasmid, Spr R. leguminosarum bv trifolii genomic DNA HindIII fragments cloned in pVK102, Tcr pLAFR1 derivative containing the exoB gene of Rm1021 pRK290 derivative, Tcr pUT derivative containing the miniTn5 Sp/Sm element pACYC184 derivative, Gmr mob, sacB, lacZa Cosmid containing exoB gene of RS800 pRK404 derivative containing the 3.7-kb PstI fragment DNA of RS800 pRK404 derivative containing the 2.2-kb BamHI fragment DNA of RS800 pBSB43 derivative containing a Tn5 insertion pBSB43 derivative containing a Tn5 insertion pBSB43 derivative containing a Tn5 insertion pBSB43 derivative containing a Tn5 insertion pJQ200SK derivative containing the RS800 exoB gene disrupted by the Sp/Sm interposon gene bank, were examined for their capacity to form ¯uorescent colonies on Calco¯uor LB media. Physical studies showed that all ten Rhizobium clones analyzed were identical. The cosmid, pBSB4, which restored colony ¯uorescence to R. meliloti 7094, was chosen. This cosmid did not complement R. meliloti exoA, exoF or exoM mutants. Hybridization with an internal fragment of the R. meliloti exoB gene contained in cosmid pD2 revealed Yanisch-Perron et al. (1985) Simon et al. (1989) Ditta et al. (1980) Ditta et al. (1980) Ditta et al. (1980) Rodriguez-QuinÄones et al. (1989) Leigh et al. (1985) Ditta et al. (1985) De Lorenzo et al. (1990) Quandt and Hynes (1993) This work This work This work This work This work This work This work This work that the cosmid pBSB4 contained a 3.7-kb PstI fragment and a 2.2-kb BamHI fragment that hybridized to the probe. These fragments were subcloned in pRK404 to obtain plasmids pBSB43 and pBSB42, respectively. Plasmid pBSB43 complemented R. meliloti 7094, allowing formation of ¯uorescent colonies on Calco¯uor LB agar and eective nodulation of alfalfa, but plasmid pBSB42 did not complement any of the mutant phenotypes. These results of hybridization and complementa- 134 tion studies suggested that an R. leguminosarum bv trifolii exoB homolog, which contains a BamHI restriction site, is located on the 3.7-kb PstI fragment. R. leguminosarum bv trifolii RS800 harbors three plasmids with molecular weights of about 190, 280, and 470 MDa (Rodriguez-QuinÄones et al. 1989). In order to locate the exoB gene in the genome of RS800, a blotted Eckhardt gel (Rosenberg et al. 1982) was hybridized using the 3.7-kb PstI fragment as a probe. This fragment hybridized with the second symbiotic megaplasmid of R. meliloti 1021 and with chromosomal DNA of RS800, but not with plasmid DNA of RS800. This con®rmed the plasmid location of gene exoB in R. meliloti and showed the chromosomal location of the homologous gene of R. leguminosarum bv trifolii. The 2-kb EcoRV-PstI fragment internal to the 3.7-kb PstI fragment was sequenced. It contained an open reading frame from the only ATG codon at position 210 to the TGA codon at position 1191 (Fig. 1). A potential ribosome-binding site (AGGG) was identi®ed just 6 bp upstream of the putative start codon. The deduced polypeptide product was 327 amino acids in length and had a molecular weight of 35 926 Da. This open reading frame was identi®ed as gene exoB from its ability to complement the exoB mutation of R. meliloti, and from sequence comparison with exoB genes of other organisms. The exoB gene coded for a predicted protein that showed 80.0% identity and 89.2% similarity to the deduced amino acid sequence of the UDP-glucose 4epimerase of R. meliloti. The exoB gene of R. leguminosarum bv trifolii also showed signi®cant homology to gene sequences coding for UDP-glucose 4-epimerase in Azospirillum brasilense (49.5% identity, 64.3% similarity) (De Troch et al. 1994) and E. coli (41.7% identity, 58.5% similarity) (Lemaire and MuÈller-Hill 1986). nies on Calco¯uor LB agar and to nodulate alfalfa, but the other two had lost both phenotypes. Chromosomal exoB Tn5 insertions could not be obtained by following the plasmid incompatibility method (Ditta 1986). A disruption mutant of the chromosomal exoB gene was obtained by using gene sacB (Quandt and Hynes 1993), which renders cells unable to grow in the presence of sucrose. The Tn5 sequence in plasmid pBSB43-4 was excised by HpaI digestion and replaced by a 2-kb SmaI fragment of plasmid pUTminiTn5 Sp/Sm that contains a spectinomycin resistance gene. The 5.7-kb PstI fragment of the modi®ed plasmid was subcloned into the PstI site of plasmid pJQ200SK that contains gene sacB. The resulting plasmid, pJQ2003, was transferred from E. coli S17-1 to R. leguminosarum bv trifolii RS800 by selecting for resistance to spectinomycin and sucrose. The resulting colonies had undergone double recombination events that were con®rmed by hybridization. The result was the replacement of the chromosomal exoB gene by the disrupted homolog from the plasmid. One of these strains was designated RS12. Mutant RS12 grew more slowly than its parental strain, tended to ¯occulate, and formed deep red colonies on Congo red plates, whereas wild-type colonies were pale pink. A lack of EPS seems to increase the uptake of Congo red in other exo mutants (Breedveld et al. 1993). The LPS pro®le of the wild-type strain (Fig. 2, lane 4) consisted of two major LPS species, a slow-migrating ladder-like structure (LPSI) which could represent the O-antigen-containing LPS, and the faster migrating band (LPSII) that consists of lipid A and core only (Carlson 1984). Our exoB mutant lacked the higher molecular weight form of the LPS, and contained only LPSII, which had a somewhat higher electrophoretic mobility than LPSII of wild type (Fig. 2, lane 5). The An exoB disruption mutant of R. leguminosarum bv trifolii Four Tn5 insertion mutants of plasmid pBSB43 were analyzed (Fig. 1). Two of them retained the ability to complement R. meliloti 7094 to form ¯uorescent colo- Fig. 1 Restriction map of the 3.7-kb PstI fragment of the RS800 chromosomal DNA. Bars with open circles indicate Tn5 insertions that give rise to ¯uorescent colonies on Calco¯uor media; insertions indicated by closed circles do not result in ¯uorescence. The numbers are the last digit in the name of the plasmid that bears the insertion (Table 1). The box indicates the exoB open reading frame and the arrow the direction of transcription. (P, PstI, Ss SspI, Pv PvuI, B BamHI, R RsaI, Sp SphI, EV EcoRV, A AvaI, Sal I) Fig. 2 SDS-polyacrylamide gel electrophoresis of lipopolysaccharides (LPS) from the wild-type RS800 (lane 4), the exoB mutant strain, RS12 (lane 5), and the same mutant with plasmid pBSB43 (lane 6). LPS from smooth Salmonella typhimurium (lane 1), and rough S. typhimurium TV119 (lane 2), and S. typhimurium SL1181 (lane 3) were used as standards 135 LPS pro®le of RS12 was restored to the wild-type LPS pro®le by complementation with plasmid pBSB43 that contains the complete exoB gene of R. leguminosarum bv trifolii RS800 (Fig. 2, lane 6). Analysis of RS800 and RS12 EPS The 1H-NMR spectrum of the EPS from the parent strain RS800 (Fig. 3A) was consistent with a polysaccharide bearing several dierent non-carbohydrate substitutions. The proton resonances between d 3.0±5.0 were assigned to glycosyl components, and the resonances at d 1.2, 1.4, and 2.1 were assigned to methyl protons of 3-hydroxybutyryl, pyruvate, and acetate group substitutions, respectively. GLC/MS analyses indicated that the EPS from this wild-type strain was an acidic heteropolysaccharide containing glucose, glucuronic acid, and galactose residues in an approximate glycosyl molar ratio of 4.5 : 1.2 : 1 (Fig. 4A). These 1HNMR and GLC/MS data were similar to the Type 2 EPS previously shown to be made by R. leguminosarum bv trifolii wild-type strains ANU843, NA-30, and TA-1 (Hollingsworth et al. 1984; Philip-Hollingsworth et al. 1989). The oligosaccharide repeat unit of the Type 2 acidic EPS structure consists of an octasaccharide with two glucuronic acid residues and two glucose residues Fig. 3 1H-NMR spectra of isolated acidic exopolysaccharide from Rhizobium leguminosarum bv trifolii strains RS800 (A, wild type) and RS12 (B, exoB mutant). Resonances for pyruvate (Py), acetate (Ac), and 3-hydroxybutyrate (3-Hb) are indicated that constitute the tetrasaccharide main chain and a tetrasaccharide branch that contains three internal glucose residues and a galactose residue at the non-reducing terminus (Fig. 5). The multiplicity and splitting pattern of the non-carbohydrate resonances in the 1H-NMR spectrum of the EPS from strain RS800 (Fig. 3A) were consistent with their location in the Type 2 structure presented in Fig. 5. The single acetate group was primarily attached to O-3 (with minor migration to O-2) of the glucuronic acid residue adjacent to glucose in the main chain. The two pyruvate groups were attached to the 4 and 6 positions of the terminal galactose and adjacent glucose residues of the side chain, while the 3hydroxybutyryl group was esteri®ed on the O-3 position of the galactose residue. From its 1H-NMR spectrum, the EPS from the exoB mutant (Fig. 3B) was similar to that of the RS800 parent, but lacked the 3-hydroxybutyrate moiety and one of the pyruvate groups (d 1.2, 1.4). The other pyruvate group and the acetate group were still present in the exoB mutant EPS. GLC/MS analysis of the EPS from the exoB mutant (Fig. 4B) indicated the presence of glucose and glucuronic acid, in a ratio of 4.8 : 1.3, and a total absence of galactose. Nodulation of clover by RS800 and RS12 Nodules induced by the RS800 wild-type strain were typical of an eective symbiosis. They were pink, cylindrical, normal in size (3±4 mm in T. repens, 5±6 mm in T. subterraneum), and developed mainly on primary roots. In contrast, nodules induced by the RS12 mutant strain were indicative of an ineective symbiosis. They lacked the cylindrical shape and they were white, smaller, and more numerous per plant than those of the wild type, and frequently located at the site of secondary root emergence. The distribution of nodule development on roots was aected by the mutation of the bacterial exoB gene. In contrast to the nodules induced by the RS800 wildtype strain in T. subterraneum (Fig. 6A,B), those induced by RS12 mutant strain were uninvaded and lacked infection threads and bacteroids. Host cells in the central region of these nodules were smaller and contained more starch grains than did host cells in nodules induced by wild-type RS800. Few nodules on plants inoculated with the RS12 mutant strain contained bacterial cells. In these rare cases, host cells that contained bacteria were located near the periphery of the central region (Fig. 6C,D). Deeper sections of the same nodules (Fig. 6E,F) revealed only uninfected host cells. These results indicated a signi®cant restriction in the ability of the exoB mutant to invade host cells within clover nodules. Transmission electron microscopy shows club-shaped bacteroids of wild-type RS800 enclosed within symbiosomes of infected nodule cells (Fig. 7A). Most host cells in the nodule central region were uninfected by the exoB mutant, RS12, and contained an abundance of starch granules (Fig. 7B). In the rare host cells that are invaded 136 Fig. 4 Total ion chromatogram of alditol acetate derivatives of glycosyl components from exopolysaccharide of wild type, RS800 (A) and the exoB mutant, RS12 (B). Peak 2 is the galactosyl derivative, whereas peak 3 contains both the glucosyl and glucuronosyl derivatives, respectively. The latter two components are distinguished by selected ion monitoring mass spectrometry. Peak 1 represents a trace of the decarboxylated product of uronic acid by the bacterial mutant, bacteria were released from infection threads but were not maintained in symbiosomes (Fig. 7C,D). Instead, the released bacteria were in direct contact with host cytosol, which itself appeared to undergo degradation and to accumulate numerous membraneous vesicles. The degeneration of the bacterial and host cell cytosol and the lack of intact peribacteroid membranes indicated an impaired root-nodule symbiosis. Discussion The present work is the ®rst study of the exoB gene of R. leguminosarum bv trifolii, involved in polysaccharide synthesis. This gene is chromosomally located, and the original cosmid that contained the gene complemented none of the R. meliloti exoA, exoF or exoM mutants. In R. meliloti 1021, exoB maps in a cluster of exo genes that includes exoA, exoF, and exoM, on the second symbiotic megaplasmid (Leigh and Walker 1994). Therefore, the genomic distribution of the exo loci diers in these two Rhizobium species. As in R. leguminosarum bv trifolii, exoB maps on the chromosome of other Rhizobiaceae species including one that nodulates H. coronarium (Ollero et al. 1994), and one that nodulates Acacia (Lopez-Lara et al. 1995) and Agrobacterium tumefaciens (Cangelosi et al. 1987). Despite the high divergence in genomic organization of the exo loci between R. meliloti and R. leguminosarum bv trifolii, DNA sequence similarity and functional interchangeability indicated parallel evolution of the exoB genes in both species. The EPS produced by R. leguminosarum bv trifolii wild-type strain corresponded to the Type 2 EPS synthesized by one of the major groups of R. leguminosarum bv trifolii strains. This EPS bears a single acetyl group on O-3 of one of the glucuronic acid residues in the backbone and contains only half as much 3-hydroxybutyryl groups as R. leguminosarum bv viciae (PhilipHollingsworth et al. 1989). The 1H-NMR and GLC/MS analyses of the acidic EPS produced by RS800 and RS12 exoB mutant strains were fully consistent with the assumed function of ExoB. Since the exoB mutant c Fig. 5 Structure of R. leguminosarum bv trifolii RS800 exopolysaccharide (EPS). Diagram showing the octasaccharide repeated unit of RS800 EPS. (Glc Glucose, Gal galactose, GlcA glucuronic acid, Pyr pyruvyl group, OAc acyl group, 3HB 3-hydroxybutyryl group) Fig. 6 Light micrographs of longitudinal nodule sections of Trifolium subterraneum inoculated with R. leguminosarum bv trifolii wild type, RS800 (A, B) or the exoB mutant, RS12 (C±F). B, D, and F are higher magni®cations of the center of nodule sections in A, C, and E, respectively. (Bar scales 20 lm (B, D, F) and 100 lm (A, C, E) 137 138 Fig. 7 Transmission electron micrographs of nodule sections of T. subterraneum inoculated with R. leguminosarum bv trifolii wild type, RS800 (A), or the exoB mutant, RS12 (B±D). Bar scales are 1 lm in A, C, and D; 2 lm in B cannot make the precursor for galactose incorporation, the EPS repeat unit that it makes lacks the terminal galactose in the branch, thus eliminating the site for attachment of the 3-hydroxybutyrate substitution and one of the pyruvate substitutions. This explains how mutation of a gene responsible for interconversion of a glycosyl component of EPS aects not only the incorporation of that carbohydrate residue but also of noncarbohydrate substitutions attached to it in the synthesis of the EPS repeat unit structure. Although a chemical analysis of LPS in the exoB mutant was not performed, a possible model to account for its altered electrophoretic pro®le is that the exoB mutation blocked the synthesis of the galactose-containing tetrasaccharide core (Hollingsworth et al. 1989) to which the O-antigen is normally attached. Because ExoB is required for the biosynthesis of precursors of EPS and LPS, a pleiotropic eect on both polymers has also been described for exoB mutants of R. leguminosarum bv viciae (Canter Cremers et al. 1990), a Rhizobium species that nodulates H. coronarium (Ollero et al. 1994), and one that nodulates Acacia (Lopez-Lara et al. 1995). Since EPS and LPS of RS12 were aected by the exoB mutation, the symbiotic defect of this mutant could not be attributed to alterations in any one of these two surface glycoconjugates. R. leguminosarum bv viciae exoB mutants fail to induce nodules or nodule-like structures on host plants (Canter Cremers et al. 1990). In contrast, the R. leguminosarum bv trifolii exoB mutant induced nodule 139 formation on clovers, but their dierentiation and distribution on the root diered considerably from nodules induced by the wild-type parent. Thus, the symbiotic phenotypes of exoB mutants of these closely related rhizobia are not identical. This implies that the requirement(s) for galactosyl-containing glycoconjugates of rhizobia may be more stringent in certain symbioses than others. Our results are consistent with the data of Derylo et al. (1986) which showed that exopolysaccharide de®cient mutants derived from R. leguminosarum bv trifolii 24AR5 were able to induce early steps of nodulation, although only a small number of bacteria were released from infection threads and some of them appeared to be lysed. Moreover, our results on the majority of RS12-induced nodules are in accordance with the analysis of R. meliloti 1021 exo mutant-induced nodules (Yang et al. 1992). These nodules also develop more frequently on secondary roots than on the primary root. The exo mutant-induced infection threads abort within the peripheral cells, and the nodules lack a wellde®ned apical meristem. In summary, our studies show that mutation of the exoB gene in R. leguminosarum bv trifolii leads to important alterations in EPS and LPS structures, and to symbiotic defects in nodule development, invasion of nodule host cells, and sustained maintenance of an intracellular endosymbiotic state. Acknowledgements G. C. Walker is gratefully acknowledged for providing strains and plasmids, and for receiving B.S.-A. for a short stay in his laboratory. We thank Ana BuendõÂ a for her collaboration in LPS analysis and AsuncioÂn Fernandez from the Servicio de MicroscopõÂ a ElectroÂnica-Universidad de Sevilla for preparation of samples for microscopical study. B.S.-A. and C.C. were supported by the Ministerio de EducacioÂn y Ciencia. This work was funded by grants BIO90-0521 and BIO93-0427 from the ComisioÂn EspanÄola Interministerial de Ciencia y TecnologõÂ a. Portions of this work were supported by the Michigan Agricultural Experiment Station, USDA-NRI grant 94±03537, and NSF grant BIR 9120006 to the MSU Center for Microbial Ecology. References Ausubel FM, Brent R, Kingston RE, Moore DD, Seichnan JG, Smith JA, Struhl F (1989) Current protocols in molecular biology. Greene and Wiley Interscience, New York Beringer JE (1974) R-factor transfer in Rhizobium leguminosarum. J Gen Microbiol 84:188±198 Bhagwat AA, Tully RE, Kesister DL (1991) Isolation and characterization of a competition-defective Bradyrhizobium japonicum mutant. Appl Environ Microbiol 57:3496±3501 Boyer HW, Roulland-Dussoix D (1969) A complementation analysis of restriction and modi®cation in Escherichia coli. J Mol Biol 41:459±472 Breedveld MW, Canter Cremers HCJ, Batley M, Posthumus MA, Zevenhuizen LPTM, Wijelman CA, Zehnder AJB (1993) Polysaccharide synthesis in relation to nodulation behavior of Rhizobium leguminosarum. J Bacteriol 175:750±757 BuendõÂ a AM, Enenkel B, KoÈplin R, Niehaus K, Arnold W, PuÈhler A (1991) The Rhizobium meliloti exoZ/exoB fragment of megaplasmid 2: ExoB functions as a UDP-glucose 4-epimerase and ExoZ shows homology to NodX of Rhizobium leguminosarum biovar viciae strain TOM. Mol Microbiol 5:1519± 1530 Cangelosi GA, Hung L, Puvanesarajah V, Stacey G, Ozga DA, Leigh JA, Nester EW (1987) Common loci for Agrobacterium tumefaciens and Rhizobium meliloti exopolysaccharide synthesis and their roles in plant interactions. J Bacteriol 169:2086±2091 Canter Cremers HCJ, Batley M, Redmond JW, Eydems L, Breedveld MW, Zevenhuizen LPTM, Pees E, Wijelman CA, Lugtenberg BJJ (1990) Rhizobium leguminosarum exoB mutants are de®cient in the synthesis of UDP-glucose 4¢ epimerase. J Biol Chem 265:21122±21127 Carlson RW (1984) The heterogeneity of Rhizobium lipopolysaccharides. J Bacteriol 158:1012±1017 Chakravorty A, Zurkowski W, Shine J, Rolfe BG (1982) Symbiotic nitrogen ®xation: molecular cloning of Rhizobium genes involved in exopolysaccharide synthesis and eective nodulation. J Mol Appl Genet 1:585±596 Corbin D, Ditta GS, Helinski DR (1982) Clustering of nitrogen ®xation (nif) genes in Rhizobium meliloti. J Bacteriol 149:221±228 Coronado C, Sanchez-Andujar B, Palomares AJ (1996) Rhizobium extracellular structures in symbiosis. World Microbiol Biotechnol 12:127±136 Dazzo FB (1982) Leguminous root nodules. In: Burns RG, Slater JH (eds) Experimental microbial ecology. Blackwell Scienti®c Publications, Oxford, pp 431±446 De Lorenzo V, Herrero M, Jakubzik U, Timmis UK (1990) MiniTn5 transposon derivatives for insertion mutagenesis, promoter probing, and chromosomal insertion of cloned DNA in gramnegative eubacteria. J Bacteriol 172:6568±6572 Derylo M, Skorupska A, Bednara J, Lorkiewicz Z (1986) Rhizobium trifolii mutants de®cient in exopolysaccharide production. Physiol Plant 66:699±704 De Troch P, Keijers V, Vanderleyden J (1994) Sequence analysis of the Azospirillum brasilense exoB gene, encoding UDP-glucose 4¢-epimerase. Gene 144:143±144 Devereux J, Haeberli P, Smithies O (1984) A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res 12: 387±395 Diebold R, Noel KD (1989) Rhizobium leguminosarum exopolysaccharide mutants: biochemical and genetic analyses and symbiotic behaviour on three hosts. J Bacteriol 171:4821±4830 Ditta G (1986). Tn5 mapping of Rhizobium nitrogen ®xation genes. Methods Enzymol 118:519±528 Ditta G, Stan®eld S, Corbin D, Helinski DR (1980). Broad hostrange DNA cloning system for gram-negative bacteria: construction of a gene bank of Rhizobium meliloti. Proc Natl Acad Sci USA 77:7347±7351 Ditta G, Schmidhauser T, Yakobson E, Lu P, Liang XW, Finlay DR, Guiney D, Helinski DR (1985) Plasmid related to the broad host-range vector, pRK290, useful for gene cloning and for monitoring gene expression. Plasmid 13:149±153 Eckhardt T (1978) A rapid method for the identi®cation of plasmid deoxyribonucleic acid in bacteria. Plasmid 1:584±588 Heniko S (1984) Unidirectional digestions with exonuclease III creates targeted breakpoints for DNA sequencing. Gene 28:351±359 Hollingsworth R, Abe M, Sherwood J, Dazzo FB (1984) Bacteriophage-induced acidic heteropolysaccharide lyases that convert the acidic heteropolysaccharides of Rhizobium trifolii into oligosaccharide units. J Bacteriol 160:510±516 Hollingsworth R, Carlson RW, Garcia F, Gage DA (1989) A new core tetrasaccharide component from the lipopolysaccharide from Rhizobium trifolii ANU843. J Biol Chem 264:9300±9304 Ish-Horowicz D, Burke JF (1981) Rapid and ecient cosmid cloning. Nucleic Acids Res 9:2989±2998 Kittelberger R, Hilbink F (1993) Sensitive silver-staining detection of bacterial lipopolysaccharides in polyacrylamide gels. J Biochem Biophys Methods 26:81±86 Kondorosi A, Kondorosi E, Pankhurst CE, Broughton W, Banfalvi S (1982) Mobilization of a Rhizobium meliloti megaplasmid carrying nodulation and nitrogen ®xation genes into other rhizobia and Agrobacterium. Mol Gen Genet 188:433±439 KoÈplin R, Wang G, HoÈtte B, Priefer UB, PuÈhler A (1993) A 3.9 kb DNA region of Xanthomonas campestris pv campestris that is 140 necessary for lipopolysaccharide production encodes a set of enzymes involved in the synthesis of dTDP-rhamnose. J Bacteriol 175:7786±7792 Leigh JA, Lee CC (1988) Characterization of polysaccharides of Rhizobium meliloti exo mutants that form ineective nodules. J Bacteriol 170:3327±3332 Leigh JA, Walker GC (1994) Exopolysaccharides of Rhizobium: synthesis, regulation and symbiotic function. Trends Genet 10: 63±67 Leigh JA, Signer ER, Walker GC (1985) Exopolysaccharide-de®cient mutants of Rhizobium meliloti that form ineective nodules. Proc Natl Acad Sci USA 82:6231±6235 Lemaire HG, MuÈller-Hill B (1986) Nucleotide sequences of the galE gene and the galT gene of E. coli. Nucleic Acids Res 14:7705±7711 Long S, Reed JW, Himawan J, Walker GC (1988) Genetic analysis of a cluster of genes required for synthesis of the Calco¯uorbinding exopolysaccharides of Rhizobium meliloti. J Bacteriol 170:4239±4248 Lopez-Lara IM, Orgambide G, Dazzo FB, Olivares J, Toro N (1995) Surface polysaccharide mutants of Rhizobium sp. (Acacia) strain GRH2: major requirement of lipopolysaccharide for successful invasion of Acacia nodules and host range determination. Microbiology 141:573±581 Meade HM, Long SR, Ruvkun GB, Brown SE, Ausubel FM (1982) Physical and genetic characterization of symbiotic and auxotrophic mutants of Rhizobium meliloti induced by transposon Tn5 mutagenesis. J Bacteriol 149:114±122 Megias M, Caviedes MA, Andres M, Sousa C, Ruiz-Berraquero F, Palomares AJ (1988) Localization of his genes on the Rhizobium trifolii RS800 linkage map. Mol Gen Genet 211:369±372 Ollero FJ, Valverde MA, SaÂnchez-PalazoÂn L, Villalobo E, Espuny MR, BellogõÂ n R (1994) An exoB mutant of Rhizobium sp. is eective in indeterminate nodules of Hedysarum coronarium. Microbiology 140:1389±1394 Orosz L, Svab Z, Kondorosi A, Silk T (1973) Genetic studies on Rhizobiophage 16-3. I. Genes and functions on the chromosome. Mol Gen Genet 125:341±350 Philip-Hollingsworth S, Hollingsworth R, Dazzo FB (1989) Hostrange related structural features of the acidic extracellular polysaccharides of Rhizobium trifolii and Rhizobium leguminosarum. J Biol Chem 264:5710±5714 Quandt J, Hynes MF (1993) Versatile suicide vectors which allow direct selection for gene replacement in gram-negative bacteria. Gene 127:15±21 Rodriguez-QuinÄones F, Fernandez-Burriel M, Banfalvi Z, Megias M, Kondorosi A (1989) Identi®cation of a conserved, reiterated DNA region that in¯uences the eciency of nodulation in strain RS1051 of Rhizobium leguminosarum bv trifolii. Mol. Plant-Microbe Interact 2:75±83 Rolfe BG, Gressho PM, Shine J, Vincent JW (1980) Interaction between a non-nodulating and an ineective mutant of Rhizobium trifolii resulting in eective (nitrogen ®xing) nodulation. Appl Environ Microbiol 39:449±452 Rosenberg C, Casse-Delbart F, Dusha I, David M, Boucher C (1982) Megaplasmid in the plant-associated bacteria Rhizobium meliloti and Pseudomonas solanacearum. J Bacteriol 150:402±406 Simon R, Quandt J, Klipp W (1989) New derivatives of transposon Tn5 suitable for mobilization of replicons, generation of operon fusions and induction of genes in gram-negative bacteria. Gene 80:161±169 Yang C, Signer ER, Hirsch AM (1992) Nodules initiated by Rhizobium meliloti exopolysaccharide mutants lack a discrete, persistent nodule meristem. Plant Physiol 98:143±151 Yanisch-Perron C, Vieira J, Messing J (1985) Improved M13 phage cloning vectors and host strains: nucleotide sequence of the M13mp18 and pUC19 vectors. Gene 33:103±119 Zevenhuizen LPTM, Bertocchi C, Van Neerven ARW (1986) Congo red absorption and cellulose synthesis by Rhizobiaceae. Antonie Van Leeuwenhoek 52:381±386