Structural requirements of Rhizobium chitolipooligosaccharides for uptake and bioactivity in legume roots as revealed by synthetic analogs and fluorescent probes.

Rhizobium chitolipooligosaccharides (CLOSs) are heterogeneous fatty acylated N-acetyl glucosamine oligomers with variations in both the polar (hydrophilic) oligosaccharide head group and the non-polar (hydrophobic) fatty acyl chain. They trigger root hair deformation and cortical cell divisions in legume roots during development of the nitrogen-fixing root-nodule symbiosis. It has been proposed that only certain unique molecular species of CLOSs made by a particular rhizobia can elicit these responses on the corresponding legume host, suggesting that receptor-mediated perception of CLOSs serves as a basis of symbiotic specificity. We evaluated the relative symbiotic importance of the hydrophilic and hydrophobic structural domains of CLOSs by comparing the biological activities of CLOSs from wild type R. leguminosarum bv. trifolii ANU843 with that of various synthetic analogs. These tests were performed in axenic bioassays on the compatible symbiotic host, white clover (Trifolium repens) and the incompatible non-host legume, alfalfa (Medicago sativa). Fluorochrome-tagged derivatives of the native CLOSs and the analogs were also prepared in order to evaluate the uptake and localization patterns of these molecules within host root cells. The results indicate a direct link between uptake and biological activities of Rhizobium CLOSs on legume roots. The smallest CLOS analog taken up and biologically active on white clover and alfalfa was a N-fatty acylglucosamine, without an essential requirement of oligomerization, fatty N-acyl unsaturation, or acetate/sulfate functionalization. This suggests that N-fattyacylglucosamine is the common minimum structure required and sufficient for uptake and biological activity of CLOS glycolipids in these legumes, and that the various specific modifications of its polar head group and hydrophobic tail modulate its inherent ability to further express these activities, thus influencing which legumes are capable of responding to CLOSs rather than dictating their biological activities per se.

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 trifolii exoB 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.

Modulation of development, growth dynamics, wall crystallinity, and infection sites in white clover root hairs by membrane chitolipooligosaccharides from Rhizobium leguminosarum biovar trifolii.

We used bright-field, time-lapse video, cross-polarized, phase-contrast, and fluorescence microscopies to examine the influence of isolated chitolipooligosaccharides (CLOSs) from wild-type Rhizobium leguminosarum bv. trifolii on development of white clover root hairs, and the role of these bioactive glycolipids in primary host infection. CLOS action caused a threefold increase in the differentiation of root epidermal cells into root hairs. At maturity, root hairs were significantly longer because of an extended period of active elongation without a change in the elongation rate itself. Time-series image analysis showed that the morphological basis of CLOS-induced root hair deformation is a redirection of tip growth displaced from the medial axis as previously predicted. Further studies showed several newly described infection-related root hair responses to CLOSs, including the localized disruption of the normal crystallinity in cell wall architecture and the induction of new infection sites. The application of CLOS also enabled a NodC- mutant of R. leguminosarum bv. trifolii to progress further in the infection process by inducing bright refractile spot modifications of the deformed root hair walls. However, CLOSs did not rescue the ability of the NodC- mutant to induce marked curlings or infection threads within root hairs. These results indicate that CLOS Nod factors elicit several host responses that modulate the growth dynamics and symbiont infectibility of white clover root hairs but that CLOSs alone are not sufficient to permit successful entry of the bacteria into root hairs during primary host infection in the Rhizobium-clover symbiosis.

Mutation or increased copy number of nodE has no effect on the spectrum of chitolipooligosaccharide nod factors made by Rhizobium leguminosarum bv. trifolii.

The bacterial gene nodE is the key determinant of host specificity in the Rhizobium leguminosarum-legume symbiosis and has been proposed to determined unique polyunsaturated fatty acyl moieties in chitolipooligosaccharides (CLOS) made by the bacterial symbiont. We evaluated nodE function by examining CLOS structures made by wild-type R. leguminosarum bv. trifolii ANU843, an isogenic nodE::Tn5 mutant, and a recombinant strain containing multiple copies of the pSym nod region of ANU843. 1H-NMR, electrospray ionization mass spectrometry, fast atom bombardment mass spectrometry, flame ionization detection-gas chromatography, gas chromatography/mass spectrometry, and high performance liquid chromatography/UV photodiode array analyses revealed that these bacterial strains made the same spectrum of CLOS species. We also found that ions in the mass spectra which were originally assigned to nodE-dependent CLOS species containing unique polyunsaturated fatty acids (Spaink, H. P., Bloemberg, G. V., van Brussel, A. A. N., Lugtenberg, B. J. J., van der Drift, K. M. G. M., Haverkamp, J., and Thomas-Oates, J. E. (1995) Mol. Plant-Microbe Interact. 8, 155-164) were actually due to sodium adducts of the major nodE-independent CLOS species. No evidence for nodE-dependent CLOSs was found for these strains. These results indicate a need to revise the current model to explain how nodE determines host range in the R. leguminosarum-legume symbiosis.

Flavone-enhanced accumulation and symbiosis-related biological activity of a diglycosyl diacylglycerol membrane glycolipid from Rhizobium leguminosarum biovar trifolii.

Rhizobium leguminosarum bv. trifolii is the bacterial symbiont which induces nitrogen-fixing root nodules on the leguminous host, white clover (Trifolium repens L.). In this plant-microbe interaction, the host plant excretes a flavone, 4',7-dihydroxyflavone (DHF), which activates expression of modulation genes, enabling the bacterial symbiont to elicit various symbiosis-related morphological changes in its roots. We have investigated the accumulation of a diglycosyl diacylglycerol (BF-7) in wild-type R. leguminosarum bv. trifolii ANU843 when grown with DHF and the biological activities of this glycolipid bacterial factor on host and nonhost legumes. In vivo labeling studies indicated that wild-type ANU843 cells accumulate BF-7 in response to DHF, and this flavone-enhanced alteration in membrane glycolipid composition was suppressed in isogenic nodA::Tn5 and nodD::Tn5 mutant derivatives. Seedling bioassays performed under microbiologically controlled conditions indicated that subnanomolar concentrations of purified BF-7 elicit various symbiosis-related morphological responses on white clover roots, including thick short roots, root hair deformation, and foci of cortical cell divisions. Roots of the nonhost legumes alfalfa and vetch were much less responsive to BF-7 at these low concentrations. A structurally distinct diglycosyl diacylglycerol did not induce these responses on white clover, indicating structural constraints in the biological activity of BF-7 on this legume host. In bioassays using aminoethoxyvinylglycine to suppress plant production of ethylene, BF-7 elicited a meristematic rather than collaroid type of mitogenic response in the root cortex of white clover. These results indicate an involvement of flavone-activated nod expression in membrane accumulation of BF-7 and a potent ability of this diglycosyl diacylglycerol glycolipid to perform as a bacterial factor enabling R. leguminosarum bv. trifolii to activate segments of its host's symbiotic program during early development of the root nodule symbiosis.

Evaluation of acidic heteropolysaccharide structures in Rhizobium leguminosarum biovars altered in nodulation genes and host range.

1H-NMR spectroscopy showed that the extracellular heterpolysaccharides (EPS) from derivatives of Rhizobium leguminosarum bv. trifolii ANU843 altered in pSym nod composition or function (transposon insertions, deletion of pSym, induction by flavone, and introduction of cloned pSym nod regions from ANU843 and R. l. bv. viciae 248 on recombinant plasmids into the pSym-cured background of ANU843) differed only in 3-hydroxybutyrate stoichiometry per octaglycosyl unit. This change in EPS was likely to be an indirect effect of altered growth during expression of pSym nod genes in the presence of the flavone. No modifications were found in EPS made by R. l. bv. phaseoli 8002 when its resident pSym was deleted or replaced with pSym from R. l. bv. viciae 248, or with a derivative of this pSym lacking the host-specific nodulation genes nodFELMNTO. Thus, although certain O-acyl noncarbohydrate substitutions in EPS are affected by pSym nod genes (including the ones that determine host range) in certain backgrounds of R. leguminosarum, this change does not occur universally among all strains of R. leguminosarum. We conclude that the structure of the acidic EPS does not control host-specific nodulation of white clover, hairy vetch, and beans for the strains of R. leguminosarum tested here.

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.

N-Acetylglutamic acid: an extracellular nod signal of Rhizobium trifolii ANU843 that induces root hair branching and nodule-like primordia in white clover roots.

An extracellular metabolite purified from Rhizobium trifolii ANU843 was established as N-acetylglutamic acid (GluNAc) by 1H NMR and Fourier transform IR spectroscopy, gas chromatography/mass spectrometry of its methylated product, and organic synthesis. TLC analyses indicated that extracellular accumulation of GluNAc by R. trifolii ANU843 grown in defined BIII culture medium was dependent on induction of its bacterial nodulation (nod) genes and the positive regulatory gene nodD on its symbiotic plasmid. 1H NMR analyses showed less GluNAc in fractionated culture supernatants of nodL and nodM mutant derivatives of R. trifolii ANU843. GluNAc induced three morphological responses on axenic roots of white clover seedlings: (i) root hair branching; (ii) tip swelling followed by resumed elongation of root hairs; and (iii) a slight increase in foci of cortical cell divisions, which developed into nodule-like primordia. These biological activities of extracellular GluNAc from R. trifolii ANU843 were confirmed with authentic standards of GluNAc. These results indicate that extracellular accumulation of N-acetylglutamic acid is linked to flavone-dependent metabolism involving nodD, nodL, and nodM in R. trifolii ANU843. This constitutes the first report on the structure of a nod-dependent extracellular signal from R. trifolii that can affect root hair and nodule development in white clover and whose biological activity on this host has been confirmed with authentic standards.

The effect of interspecies transfer of Rhizobium host-specific nodulation genes on acidic polysaccharide structure and in situ binding by host lectin.

Host specificity in the Rhizobium-legume symbiosis is controlled in the bacterium by host specific nodulation (hsn) genes residing on its symbiotic plasmid. We have examined the structure of the major acidic heteropolysaccharide produced by recombinant hybrid strains of Rhizobium leguminosarum carrying cloned R. trifolii hsn genes with those produced by the parent donor and recipient strains. Alteration of the nod gene composition of R. leguminosarum strain 300 by introduction of an 8-kilobase set of hsn genes (nodFERL and nodMN) from R. trifolii strain ANU843, resulted in a hybrid strain which conferred efficient white clover infection and nodulation, production of the R. trifolii-type acidic polysaccharide, and an increased proportion of bacterial cells which bound to the white clover lectin, trifoliin A, in the external root environment. 1H NMR studies indicated that the structure of the polysaccharide from the hybrid recombinant differed from that of the R. leguminosarum strain 300 recipient in site and stoichiometry of acetate and stoichiometry of 3-hydroxybutyrate substituents. In contrast, the polysaccharide from a different hybrid recombinant strain containing only R. trifolii nodFERL genes had the acetylation pattern of the R. leguminosarum recipient but was substituted with 3-hydroxybutyrate at a level between that made by R. trifolii and R. leguminosarum. This latter recombinant strain displays sparse infection and nodulation of white clover roots. Immunofluorescence studies indicated that the R. leguminosarum recombinant strain containing the full complement of R. trifolii hsn genes (nodFERL and nodMN) gained the ability to interact with the excreted lectin, trifoliin A, in the white clover root environment, whereas the recombinant strain containing R. trifolii nodFERL only, was significantly attenuated in this cell-lectin interaction. These results indicate that the acylation pattern of the acidic polysaccharide synthesized by these hybrid recombinants of R. leguminosarum is influenced by the introduced hsn genes of R. trifolii and suggest that the acidic polysaccharide of R. trifolii and the interaction of these bacteria with the host lectin may contribute to host specificity in the white clover-R. trifolii symbiosis.

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)