Host-range related structural features of the acidic extracellular polysaccharides of Rhizobium trifolii and Rhizobium leguminosarum

Proton nuclear magnetic resonance (1H NMR) and fast atom bombardment mass spectrometric analyses were performed on enzymatically derived oligosaccharides from the acidic excreted polysaccharides (EPS) from representative bacterial strains of the pea-nodulating symbiont, Rhizobium leguminosarum (128C53, 128C63, and 300) and the clover-nodulating symbiont, Rhizobium trifolii (NA-30, ANU843, 0403, TA-1, LPR5035, USDA20.102, and 4S). The results revealed structural similarities and differences between EPS of these two species. Octasaccharide units containing galactose, glucuronic acid, alpha-L-threo-hex-4-enopyranosyluronic acid, and glucose in a molar ratio of 1:1:1:5 were obtained from the EPS of the three R. leguminosarum strains and had the same primary glycosyl sequence and location of pyruvate, acetate, and 3-hydroxybutyrate substituents. About 80% of the galactose residues were acylated with 3-hydroxybutyrate, and there were two acetyl groups per repeating unit distributed between the 2 glucose residues of the main chain-derived sequence of the octasaccharides. In contrast, the R. trifolii strains had varied EPS structures, each of which differed from the common R. leguminosarum EPS structure. The EPS from one group of R. trifolii strains (0403 and LPR5035) most closely resembled the R. leguminosarum EPS but differed in that a lower number of galactose and glucose residues were substituted by 3-hydroxybutyryl and acetyl groups, respectively. The EPS from a second group of R. trifolii strains (ANU843, TA-1, and NA-30) was even more different than the R. leguminosarum EPS. These R. trifolii octasaccharides bore a single acetyl group on O-3 of the glucuronic acid residue. In addition, the level of acylation by 3-hydroxybutyryl groups was 50% of that present in the R. leguminosarum EPS. The remaining two strains of R. trifolii (USDA20.102 and 4S) had very different patterns of acylation to each other and to all of the other strains. The EPS from strain USDA20.102 practically lacked 3-hydroxybutyryl groups and had a unique degree and pattern of acetylation. The oligomers from the EPS of R. trifolii strain 4S completely lacked 3-hydroxybutyryl groups and galactose. The latter EPS contained only one O-1-carboxyethylidene group and had a different degree and pattern of acetylation. Interestingly, these two latter strains differ from the other R. trifolii strains in nodulation rates on rare clover species in the Trifolium cross-inoculation group. Thus, we define several groups of R. trifolii based upon their EPS structures and establish their similarities and distinct differences with the EPS of R. leguminosarum.(ABSTRACT TRUNCATED AT 400 WORDS)     

Heterogeneity of Rhizobium lipopolysaccharides.

The lipopolysaccharides ( LPSs ) from strains of Rhizobium leguminosarum, Rhizobium trifolii, and Rhizobium phaseoli were isolated and partially characterized by mild acid hydrolysis and by polyacrylamide gel electrophoresis. Mild acid hydrolysis results in a precipitate which can be removed by centrifugation or extraction with chloroform. The supernatant contains polysaccharides which, in general, are separated into two fractions ( LPS1 and LPS2 ) by Sephadex G-50 gel filtration chromatography. The higher-molecular-weight LPS1 fractions among the various Rhizobium strains are highly variable in composition and reflect the variability reported in the intact LPSs (R. W. Carlson and R. Lee, Plant Physiol. 71:223-228, 1983; Carlson et al., Plant Physiol. 62:912-917, 1978; Zevenhuizen et al., Arch. Microbiol. 125:1-8, 1980). The LPS1 fraction of R. leguminosarum 128C53 has a higher molecular weight than all other LPS1 fractions examined. All LPS2 fractions examined are oligosaccharides with a molecular weight of ca. 600. The major sugar component of all LPS2 oligosaccharides is uronic acid. The LPS2 compositions are similar for strains of R. leguminosarum and R. trifolii, but the LPS2 from R. phaseoli was different in that it contained glucose, a sugar not found in the other LPS2 fractions or found only in trace amounts. Polyacrylamide gel electrophoretic analysis shows that each LPS contains two banding regions, a higher-molecular-weight heterogeneous region often containing many bands and a lower-molecular-weight band. The lower-molecular-weight bands of all LPSs have the same electrophoretic mobility, which is greater than that of lysozyme. The banding pattern of the heterogeneous regions varies among the different Rhizobium strains. In the case of R. leguminosarum 128C53 LPS, the heterogeneous region of a higher molecular weight than is this region from all other Rhizobium strains examined and consists of many bands separated from one another by a small and apparently constant molecular weight interval. When the heterogeneous region of R. Leguminosarum 128C53 LPS was cut from the gel and analyzed, its composition was found to be that of the intact LPS, whereas the lower-molecular-weight band contains only sugars found in the LPS2 oligosaccharide. In the case of R. leguminosarum 128C63 and R. trifolii 0403 LPSs, the heterogeneous regions are similar and consist of several band s separated by a large-molecular-weight interval with a the major band of these heterogeneous regions having the lowest molecular weight with an electrophoretic mobility near that of beta-lactoglobulin. The heterogeneous region from R. phaseoli 127K14 consists of several bands with electrophoretic mobilities near that of beta-lactoglobulin, whereas this region from R. trifolii 162S7 shows a continuous staining region, indicating a great deal of heterogeneity. The results described in this paper are discussed with regard to the reported properties of Escherichia coli and Salmonella LPSs.     

Rhizobial polysaccharide-degrading enzymes from roots of legumes

The surface structure and chemistry of symbiotic bacteria from the genus Rhizobium are probably important for the outcome of the infection of legume hosts. Exopolysaccharide, capsular polysaccharide, lipopolysaccharide and a low-molecular-weight polysaccharide were isolated from R. trifolii UTC 110-1 and R. leguminosarum UTC 114-5 and partially characterized. No or only minor differences in sugar composition could be found for the corresponding fractions from the two organisms. A general method to measure low activities of polymer-degrading enzymes was developed, and used to determine enzyme activities in root extracts of Trifolium repens L. cv. Lena and Pisum xativiini L. cv. Little Marvel against the isolated rhizobial polysaccharides. An enzyme preparation from T. repens partially degraded all polysaccharides isolated from its symbiont R. trifolii while polysaccharides from R. leguminosarum , symbiont of P. sativum , were degraded to a much lesser extent. Correspondingly, an enzyme preparation from P. sativum degraded all polysaccharides isolated from both its symbiont R. leguminosarum and its non-symbiont R. trifolii. The amount of symbiont polysaccharides degraded was larger than the amount of polysaccharides degraded from the non-symbiont R. trifolii.     

Cloning and characterization of ce/8A gene from Rhizobium leguminosarum bv. trifolii 1536

AIMS: To isolate the cellulase gene from Rhizobium leguminosarum bv. trifolii 1536. METHODS AND RESULTS: By the shot-gun method a clone (cel8A) harbouring 3.1 kb genomic DNA fragment from R. leguminosarum bv. trifolii 1536 was obtained. The cel8A gene coded 348 amino acids and it belongs to the glycosyl hydrolase family 8. The molecular mass of Cel8A protein induced from Escherichia coli DH5alpha, appeared to be 35 kDa. The optimum pH and optimum temperature was 7.0, and about 30 degrees C for its enzymatic activity respectively. CONCLUSIONS: R. leguminosarum bv. trifolii 1536 had cel8A gene having an open reading frame of 1047 bp coded for the activity of hydrolyzation of carboxymethyl cellulose. SIGNIFICANCE AND IMPACT OF THE STUDY: The production of celluloytic enzyme by R. leguminosarum bv. trifolii was confirmed, which would play specific roles in rhizobia. Future study should focus on its role in the infection and nodulation phenomena.     

Genetic analysis and cellular localization of the Rhizobium host specificity-determining NodE protein.

The nucleotide sequence of the nodE gene of Rhizobium trifolii strain ANU843 was determined. Like the nodE gene of R. leguminosarum strain 248 it encodes a protein with a predicted mol. wt of 42.0 kd. The predicted NodE proteins of R.trifolii and R.leguminosarum have a homology of 78%. Using antibodies raised against the NodE protein of R.trifolii it was shown that the NodE products of R.leguminosarum and R.trifolii are localized in the cytoplasmic membrane. Furthermore, these NodE proteins are predicted to contain at least two non-polar transbilayer alpha-helices. The nodE genes of R.trifolii and R.leguminosarum were separated from the nodF genes that precede them in the respective nodFE operons. Using the resulting clones, in which NodE was produced under control of the flavonoid-inducible nodABCIJ promoter of R.leguminosarum, it was shown that the NodE product is the main factor that distinguishes the host range of nodulation of R.trifolii and R.leguminosarum. Hybrid nodE genes, which consist of a 5' part of the R.leguminosarum nodE gene and a 3' part of the R.trifolii gene, were constructed. From the properties of these hybrid genes it was concluded that a central region of 185 amino acids at the most, containing only 44 non-identical amino acids, determines the difference in the host-specific characteristics of the two NodE proteins.     

Distribution of O-acetyl groups in the exopolysaccharide synthesized by Rhizobium leguminosarum strains is not determined by the Sym plasmid

The patterns of O-acetylation of the exopolysaccharide (EPS) from the Sym plasmid-cured derivatives of Rhizobium leguminosarum bv. trifolii strain LPR5, R. leguminosarum bv. trifolii strain ANU843 and R. leguminosarum bv. viciae strain 248 were determined by 1H and 13C NMR spectroscopy. Beside a site indicative of the chromosomal background, these strains have one site of O-acetylation in common, namely residue b of the repeating unit. The O-acetyl esterification pattern of EPS of the Sym plasmid-cured derivatives of strains LPR5, ANU843, and 248 was not altered by the introduction of a R. leguminosarum bv. viciae Sym plasmid or a R. leguminosarum bv. trifolii Sym plasmid. The induction of nod gene expression by growth of the bacteria in the presence of Vicia sativa plants or by the presence of the flavonoid naringenin, produced no significant changes in either amount or sites of O-acetyl substitution. Furthermore, no such changes were found in the EPS from a Rhizobium strain in which the nod genes are constitutively expressed. The substitution pattern of the exopolysaccharide from R. leguminosarum is, therefore, determined by the bacterial genome and is not influenced by genes present on the Sym plasmid. This conclusion is inconsistent with the suggestion of Philip-Hollingsworth et al. (Philip-Hollingsworth, S., Hollingsworth, R. I., Dazzo, F. B., Djordjevic, M. A., and Rolfe, B. G. (1989) J. Biol. Chem. 264, 5710-5714) that nod genes of R. leguminosarum bv. trifolii, by influencing the acetylation pattern of EPS, determine the host specificity of nodulation.     

AUTOMATED 35S-LABELLED PROTEIN ELECTROPHORESIS FOR IDENTIFICATION OF STRAINS OF RHIZOBIUM LEGUMINOSARUM bv. TRIFOLII

A technique for strain-level identification within a Rhizobium biovar is described, based on automated sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of ³⁵S-labelled proteins, offering substantial improvements on existing SDS-PAGE methods particularly in the areas of standardization of electrophoresis conditions and rapidity of positive identification. Gels were analysed with a β-scanner, the beta particle emission data being directly relayed to an IBM PC/AT computer for subsequent manipulation. Analysis of the total protein profiles obtained by this method revealed regions of variability between strains of R. leguminosarum bv. trifolii. Automated comparison of these regions enabled identification of strains. The method was successfully used for identifying root nodule isolates obtained from competition studies between known pairs of R. leguminosarum bv. trifolii strains inoculated onto Trifolium repens.     

Exopolysaccharides of Rhizobium leguminosarum biovar trifolii harbouring cloned exo region.

Rhizobium leguminosarum bv. trifolii produces an acidic exopolysaccharide (EPS) which plays an important role in the development of nitrogen-fixing nodules. Tn5 mutant of R. trifolii 93 defective in EPS production (Exo-) forms ineffective (Fix-) nodules on red clover. This Exo- mutation is complemented by the pARF1368 and pARF25 cosmids isolated from gene bank of Rhizobium trifolii TA1, but the complementation is not correlated with restoration of Fix+ phenotype. Furthermore, these cosmids introduced to wild-type of R. trifolii 24 repress its ability to form nitrogen-fixing nodules. These results might suggest that bacteria with cosmids carrying the exo region form EPS of altered structure. It has been shown by 1H-n.m.r. that exopolysaccharides produced by R. trifolii 93pARF-1368 and 93pARF25 contain less non-carbohydrate residues (acetyl, pyruvyl and 3-hydroxybutanoyl) than the wild type EPS. These data suggest that the biological activity of the exopolysaccharide of R. trifolii depends on the contents of the non-carbohydrate substitutions.     

Rhizobium lipopolysaccharide modulates infection thread development in white clover root hairs.

The interaction between Rhizobium lipopolysaccharide (LPS) and white clover roots was examined. The Limulus lysate assay indicated that Rhizobium leguminosarum bv. trifolii (hereafter called R. trifolii) released LPS into the external root environment of slide cultures. Immunofluorescence and immunoelectron microscopy showed that purified LPS from R. trifolii 0403 bound rapidly to root hair tips and infiltrated across the root hair wall. Infection thread formation in root hairs was promoted by preinoculation treatment of roots with R. trifolii LPS at a low dose (up to 5 micrograms per plant) but inhibited at a higher dose. This biological activity of LPS was restricted to the region of the root present at the time of exposure to LPS, higher with LPS from cells in the early stationary phase than in the mid-exponential phase, incubation time dependent, incapable of reversing inhibition of infection by NO3- or NH4+, and conserved among serologically distinct LPSs from several wild-type R. trifolii strains (0403, 2S-2, and ANU843). In contrast, infections were not increased by preinoculation treatment of roots with LPSs from R. leguminosarum bv. viciae strain 300, R. meliloti 102F28, or members of the family Enterobacteriaceae. Most infection threads developed successfully in root hairs pretreated with R. trifolii LPS, whereas many infections aborted near their origins and accumulated brown deposits if pretreated with LPS from R. meliloti 102F28. LPS from R. leguminosarum 300 also caused most infection threads to abort. Other specific responses of root hairs to infection-stimulating LPS from R. trifolii included acceleration of cytoplasmic streaming and production of novel proteins. Combined gas chromatography-mass spectroscopy and proton nuclear magnetic resonance analyses indicated that biologically active LPS from R. trifolii 0403 in the early stationary phase had less fucose but more 2-O-methylfucose, quinovosamine, 3,6-dideoxy-3-(methylamino)galactose, and noncarbohydrate substituents (O-methyl, N-methyl, and acetyl groups) on glycosyl components than did inactive LPS in the mid-exponential phase. We conclude that LPS-root hair interactions trigger metabolic events that have a significant impact on successful development of infection threads in this Rhizobium-legume symbiosis.     

Identification of a Rhizobium trifolii plasmid coding for nitrogen fixation and nodulation genes and its interaction with pJB5JI, a Rhizobium leguminosarum plasmid

Rhizobium trifolii T37 contains at least three plasmids with sizes of greater than 250 megadaltons. Southern blots of agarose gels of these plasmids probed with Rhizobium meliloti nif DNA indicated that the smallest plasmid, pRtT37a, contains the nif genes. Transfer of the Rhizobium leguminosarum plasmid pJB5JI, which codes for pea nodulation and the nif genes and is genetically marked with Tn5, into R. trifolii T37 generated transconjugants containing a variety of plasmid profiles. The plasmid profiles and symbiotic properties of all of the transconjugants were stably maintained even after reisolation from nodules. The transconjugant strains were placed into three groups based on their plasmid profiles and symbiotic properties. The first group harbored a plasmid similar in size to pJB5JI (130 megadaltons) and lacked a plasmid corresponding to pRtT37a. These strains formed effective nodules on peas but were unable to nodulate clover and lacked the R. trifolii nif genes. This suggests that genes essential for clover nodulation as well as the R. trifolii nif genes are located on pRtT37a and have been deleted. The second group harbored hybrid plasmids formed from pRtT37a and pJB5JI which ranged in size from 140 to ca. 250 megadaltons. These transconjugants had lost the R. leguminosarum nif genes but retained the R. trifolii nif genes. Strains in this group nodulated both peas and clover but formed effective nodules only on clover. The third group of transconjugants contained a hybrid plasmid similar in size to pRtT37b. These strains contained the R. trifolii and R. leguminosarum nif genes and formed N2-fixing nodules on both peas and clover.     

The degree of esterification and points of substitution by O-acetyl and O-(3-hydroxybutanoyl) groups in the acidic extracellular polysaccharides secreted by Rhizobium leguminosarum biovars viciae, trifolii, and phaseoli are not related to host range

Rhizobium leguminosarum biovars viciae, trifolii, and phaseoli have been grown in the presence and absence of 4',5,7-trihydroxyflavonone (naringenin) or 4',5,7-trihydroxyflavone (apigenin), which induce the expression of nodulation genes of the bacteria. The acidic polysaccharides secreted by the Rhizobium were isolated from the culture media and purified. The polysaccharides were cleaved with a bacteriophage enzyme and the octasaccharide repeating units formed were isolated. The glycosyl sequence and type of nonglycosyl substituents of the repeating units derived from a number of these Rhizobium biovars were shown by fast atom bombardment-mass spectroscopy and proton nuclear magnetic resonance spectroscopy (1H NMR) to be identical. Minor variations in the degree of esterification of the repeating units by O-acetyl and O-(3-hydroxybutanoyl) substituents were observed when the Rhizobium were grown in the presence or absence of the flavonoids. The variation in content and the points of attachment of the O-acetyl and O-(3-hydroxybutanoyl) substituents were as great within each Rhizobium biovar as between different Rhizobium biovars and, contrary to two recent reports (Philip-Hollingsworth, S., Hollingsworth, R. I., and Dazzo, F. B. (1989) J. Biol. Chem. 264, 1461-1466; Philip-Hollingsworth, S., Hollingsworth, R. I., Dazzo, F. B., Djordjevic, M. A., and Rolfe, B. G. (1989) J. Biol. Chem. 264, 5710-5714), the O-acylation patterns were not correlated with the host specificity of the bacteria.     

Studies on the Characterization of Root Morphology of White Clover Infected by Transconjugant of Rhizobium leguminosarum

White clover plants were inoculated with transconjugant strain' 290 which was obtained from introduction of host specific nodulation genes of wild-type Rhizobium trifolii strain ANU 843 to Rhizobium leguminosarum strain 300. The characterization of root morphology of white clover induced by the transconjugant was observed and compared to the plants induced by the parent strains. White clover started tO form a typical root hair curling inoculated with transconjugant strain 290 24h after inoculation, at 48h a part of cell wall of root hair was degradated, infection thread was observed in the infected root hair cell, cortical cell divisions occurred extensively. All these characterizations were similar to that infected by strain ANU 843. Plant inoculation test indicated that no nodule was formed when inoculated by R. leguminosarum strain 300, while plants nodulated when inoculated with transconjugant strain 290 as well as R. trifolii ANU 843. This suggests that introduction of host specific nodulation genes of R. trifolii results in conferring the nodulation ability of R. leguminosarum on white clover.     

Large plasmids of fast-growing rhizobia: homology studies and location of structural nitrogen fixation (nif) genes.

A single large plasmid was isolated from multiplasmid-harboring strains Rhizobium leguminosarum 1001 and R. trifolii 5. These single plasmids, as well as the largest plasmid detectable in R. phaseoli 3622, hybridized with part of the nif structural genes of Klebsiella pneumoniae. In contrast, the plasmids of R. meliloti strains V7 and L5-30 did not show hybridization with the nif genes of K. pneumoniae, indicating that these genes might be located either on the chromosome or on a much larger plasmid which as yet has not been isolated. Studies of the homology between plasmids of fast-growing Rhizobium species showed that a specific deoxyribonucleic acid sequence, which carries the structural genes for nitrogenase, is highly conserved on a plasmid in R. leguminosarum, R. trifolii, and R. phaseoli. Furthermore, it was found that this type of plasmid in the different species shares extensive deoxyribonucleic acid homology, suggesting that strains in the R. leguminosarum cluster have preserved a nif plasmid.     

The Rhizobium meliloti host range nodQ gene encodes a protein which shares homology with translation elongation and initiation factors.

The Rhizobium meliloti nod region IIb is involved in host-range determination: (i) the presence of region IIb is necessary for transfer of alfalfa root hair curling ability to Rhizobium leguminosarum biovar trifolii; (ii) a mutation in region IIb extends the R. meliloti infection host range to Vicia sativa nigra; (iii) dominance of R. meliloti nod genes over R. leguminosarum biovar viciae nod genes is abolished by mutations in region IIb. The nucleotide sequence of this region has been determined. Genes corresponding to the two open reading frames identified are designated nodP and nodQ. The predicted amino acid sequence of the NodQ protein shows homology with translation initiation and elongation factors. The consensus sequence involved in the GTP-binding domain is conserved.     

Nitrate reductase activities of rhizobia and the correlation between nitrate reduction and nitrogen fixation.

All species of Rhizobium except R. lupini had nitrate reductase activity. Only R. lupini was incapable of growth with nitrate as the sole source of nitrogen. However, the conditions necessary for the induction of nitrate reductase varied among species of Rhizobium. Rhizobium japonicum and some Rhizobium species of the cowpea strains expressed nitrate reductase activities both in the root nodules of appropriate leguminous hosts and when grown in the presence of nitrate. Rhizobium trifolii, R. phaseoli, and R. leguminosarum did not express nitrate reductase activities in the root nodules, but they did express them when grown in the presence of nitrate. In bacteroids of R. japonicum and some strains of cowpea Rhizobium, high N2 fixation activities were accompanied by high nitrate reductase activities. In bacteroids of R. trifolii, R. leguminosarum, and R. phaseoli, high N2 fixation activities were not accompanied by high nitrate reductase activities.     

Root colonization of different plants by plant-growth-promoting Rhizobium leguminosarum bv. trifolii R39 studied with monospecific polyclonal antisera.

Monospecific polyclonal antisera raised against Rhizobium leguminosarum bv. trifolii R39, a bacterium which was isolated originally from red clover nodules, were used to study the colonization of roots of leguminous and nonleguminous plants (Pisum sativum, Lupinus albus, Triticúm aestivum, and Zea mays) after inoculation. Eight weeks after inoculation of soil-grown plants, between 0.1 and 1% of the total bacterial population in the rhizospheres of all inoculated plants were identified as R. leguminosarum bv. trifolii R39. To characterize the associative colonization of the nonleguminous plants by R.leguminosarum bv. trifolii R39 in more detail, a time course study was performed with inoculated roots of Z. mays. R. leguminosarum bv. trifolii R39 was found almost exclusively in the rhizosphere soil and on the rhizoplane 4 weeks after inoculation. Colonization of inner root tissues was detected only occasionally at this time. During the process of attachment of R. leguminosarum bv. trifolii R39 to the rhizoplane, bacterial lipopolysaccharides were overexpressed, and this may be important for plant-microbe interaction. Fourteen weeks after inoculation, microcolonies of R. leguminosarum bv. trifolii R39 were detected in lysed cells of the root cortex as well as in intracellular space of central root cylinder cells. At the beginning of flowering (18 weeks after inoculation), the number of R. leguminosarum bv. trifolii R39 organisms decreased in the rhizosphere soil, rhizoplane, and inner root tissue.     

Bacteriophage-induced acidic heteropolysaccharide lyases that convert the acidic heteropolysaccharides of Rhizobium trifolii into oligosaccharide units

Acidic heteropolysaccharide lyases from lysates of phages 4S and BY15 grown on Rhizobium trifolii 4S and R. trifolii 0403, respectively, were used to analyze the capsular and excreted extracellular acidic polysaccharides of R. trifolii 0403. The activities of the enzymes as measured by viscometry were enhanced by the addition of calcium. The oligosaccharide products obtained by depolymerase digestion of the polysaccharides isolated from cells grown on agar plates for 5 days were isolated by gel filtration and had a glycosyl composition of glucose, galactose, glucuronic acid, and alpha-linked 4-deoxy-L-threo-hex-4-enopyranosyluronic acid in an approximate molar ratio of 5:1:1:1. This latter component was identified by 1H-nuclear magnetic resonance spectroscopy and confirmed by UV spectroscopy, ozonolysis, and its reactivity with thiobarbituric acid. The oligosaccharide had glucose as the reducing terminus, 4-deoxy-L-threo-hex-4-enopyranosyluronic acid as the enzymatically generated nonreducing terminus, and galactose as the terminus of the branched chain. The noncarbohydrate components of the oligosaccharides were acetate, ketal-linked pyruvate, and ether-linked 3-hydroxybutyrate. The mode of action of the enzymes was by beta-elimination from a uronic acid residue with concomitant loss of the glycosyl component substituted at C-4. The 235-nm absorbing properties of the resulting terminal unsaturated sugar were used to study the kinetics of depolymerization of the capsular and excreted extracellular acidic polysaccharides, using the enzyme from phage BY15. The two substrates exhibited different kinetics of depolymerization, and the oligosaccharide products differed in the amount of noncarbohydrate substituents, indicating that the acidic capsular and excreted extracellular polysaccharides from 5-day-old cultures of R. trifolii 0403 were different.     

A cultivar-specific interaction between Rhizobium leguminosarum bv. trifolii and subterranean clover is controlled by nodM, other bacterial cultivar specificity genes, and a single recessive host gene.

Insertion mutagenesis identified two negatively acting gene loci which restrict the ability of Rhizobium leguminosarum bv. trifolii TA1 to infect the homologous host Trifolium subterraneum cv. Woogenellup. One locus was confirmed by DNA sequence analysis as the nodM gene, while the other locus, designated csn-1 (cultivar-specific nodulation), is not located on the symbiosis plasmid. The presence of these cultivar specificity loci could be suppressed by the introduction of the nodT gene from ANU843, a related R. leguminosarum bv. trifolii strain. Other nod genes, present in R. leguminosarum bv. viciae (including nodX) and R. meliloti, were capable of complementing R. leguminosarum bv. trifolii TA1 for nodulation on cultivar Woogenellup. Nodulation studies conducted with F2 seedlings from a cross between cultivar Geraldton and cultivar Woogenellup indicated that a single recessive gene, designated rwt1, is responsible for the Nod- association between strain TA1 and cultivar Woogenellup. Parallels can be drawn between this association and gene-for-gene systems common in interactions between plants and biotrophic pathogens.     

Structure and role in symbiosis of the exoB gene of Rhizobium leguminosarum bv trifolii

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 trifoliiexoB disruption mutant differed 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. Received: 31 May 1996 / Accepted: 18 December 1996