Erosion of root epidermal cell walls by Rhizobium polysaccharide-degrading enzymes as related to primary host infection in the Rhizobium-legume symbiosis.

A central event of the infection process in the Rhizobium-legume symbiosis is the modification of the host cell wall barrier to form a portal of entry large enough for bacterial penetration. Transmission electron microscopy (TEM) indicates that rhizobia enter the legume root hair through a completely eroded hole that is slightly larger than the bacterial cell and is presumably created by localized enzymatic hydrolysis of the host cell wall. In this study, we have used microscopy and enzymology to further clarify how rhizobia modify root epidermal cell walls to shed new light on the mechanism of primary host infection in the Rhizobium-legume symbiosis. Quantitative scanning electron microscopy indicated that the incidence of highly localized, partially eroded pits on legume root epidermal walls that follow the contour of the rhizobial cell was higher in host than in nonhost legume combinations, was inhibited by high nitrate supply, and was not induced by immobilized wild-type chitolipooligosaccharide Nod factors reversibly adsorbed to latex beads. TEM examination of these partially eroded, epidermal pits indicated that the amorphous, noncrystalline portions of the wall were disrupted, whereas the crystalline portions remained ultrastructurally intact. Further studies using phase-contrast and polarized light microscopy indicated that (i) the structural integrity of clover root hair walls is dependent on wall polymers that are valid substrates for cell-bound polysaccharide-degrading enzymes from rhizobia, (ii) the major site where these rhizobial enzymes can completely erode the root hair wall is highly localized at the isotropic, noncrystalline apex of the root hair tip, and (iii) the degradability of clover root hair walls by rhizobial polysaccharide-degrading enzymes is enhanced by modifications induced during growth in the presence of chitolipooligosaccharide Nod factors from wild-type clover rhizobia. The results suggest a complementary role of rhizobial cell-bound glycanases and chitolipooligosaccharides in creating the localized portals of entry for successful primary host infection.

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.

Surface polysaccharide mutants of Rhizobium sp. (Acacia) strain GRH2: major requirement of lipopolysaccharide for successful invasion of Acacia nodules and host range determination.

Two transposon Tn5-induced mutants of wild-type broad-host-range Rhizobium sp. GRH2 were isolated and found to harbour different alterations in surface polysaccharides. These mutants, designated GRH2-14 and GRH2-50, induced a few, empty nodules on Acacia and lost the ability to nodulate most host herbaceous legumes. Whereas mutant GRH2-14 produces an acidic exopolysaccharide (EPS) similar to the wild-type, the acidic EPS of mutant GRH2-50 lacks galactose and the pyruvyl and 3-hydroxybutyryl substituents attached to this sugar moiety. In addition, both mutants GRH2-50 and GRH2-14 were altered in smooth lipopolysaccharides (LPS). DNA sequence analyses of the corresponding Tn5 insertions revealed that strain GRH2-50 was mutated in a DNA locus homologous to galE, and in vitro enzyme assays indicated that the UDPglucose 4-epimerase (GalE) activity was missing in this mutant strain. DNA hybridization studies showed that the GRH2-50 mutant DNA has homologous sequences within the different biovars of Rhizobium leguminosarum. However, no DNA homology to GRH2-14 altered DNA was found in those rhizobial strains, indicating that it represents a new chromosomal lps locus in Rhizobium sp. (Acacia) involved in symbiotic development.

Structurally diverse chitolipooligosaccharide nod factors accumulate primarily in membranes of wild type Rhizobium leguminosarum biovar trifolii.

The general view on Rhizobium chitolipooligosaccharides (CLOS) is that they are made in very low levels as diffusible molecules and are primarily secreted by the bacteria into the extracellular milieu where they interact with the host. However, the structural and predicted physicochemical properties of these amphiphilic molecules led us to postulate that they should normally be targeted to bacterial membranes after synthesis. Thus, we analyzed membrane lipid extracts of Rhizobium leguminosarum bv. trifolii wild-type strain ANU843 cells and the corresponding culture supernatants for CLOS-type glycolipids. As predicted, fractionation of the membrane extracts from pelleted cells led to the isolation of a diverse family of CLOS in high yield (> or = 15 mg/L of culture), whereas all attempts to isolate CLOS from the corresponding culture supernatant failed. Structural analyses reveal that the membrane CLOS of ANU843 consist of a complex mixture of O-acetylated or non-O-acetylated chito- tri-, -tetra-, and pentasaccharides bearing an N-acyl moiety at the nonreducing glucosamine residue. cis-Vaccenic acid was the predominant acyl substituent (> 70%), but several other saturated, unsaturated, and 3-hydroxy fatty acids were found in the CLOS glycolipids. Membrane accumulation of CLOS in ANU843 is promoted by the presence of 4',7-dihydroxyflavone and pSym nod genes. Potential host-selective biological activity host-selective biological activity of the purified membrane CLOS fraction from ANU843 was indicated by its ability to elicit meristems resembling rudimentary nodule primordia in the root cortex of axenic seedlings of the host legume, white clover, but not of the nonhost legumes hairy vetch or alfalfa.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Phospholipid and fatty acid compositions of Rhizobium leguminosarum biovar trifolii ANU843 in relation to flavone-activated pSym nod gene expression.

The phospholipid and associated fatty acid compositions of the bacterial symbiont of clover, Rhizobium leguminosarum biovar trifolii wild-type ANU843, was analyzed by two-dimensional silica thin-layer chromatography, fast atom bombardment-mass spectrometry, flame-ionization detection gas-liquid chromatography and combined gas-liquid chromatography/mass spectrometry. The phospholipid composition included phosphatidylethanolamine (15%), N-methylphosphatidylethanolamine (47%), N,N-dimethylphosphatidylethanolamine (9%), phosphatidylglycerol (19%), cardiolipin (5%) and phosphatidylcholine (2%). Fatty acid composition included predominantly cis-11-octadecenoic acid, lower levels of cis-9-hexadecenoic acid, hexadecanoic acid, 11-methyl-11-octadecenoic acid, octadecanoic acid, 11,12-methyleneoctadecanoic acid, eicosanoic acid and traces of branched, and di- and triunsaturated fatty acids. The influence of expression of the "nodulation" genes encoding symbiotic functions on the composition of these membrane lipids was examined in wild-type cells grown with or without the flavone inducer, 4',7-dihydroxyflavone and in mutated cells lacking the entire symbiotic plasmid where these genes reside, or containing single transposon insertions in selected nodulation genes. No significant changes in phospholipid or associated fatty acid compositions were detected by the above methods of analysis.

Methoxylated fatty acids reported in Rhizobium isolates arise from chemical alterations of common fatty acids upon acid-catalyzed transesterification procedures.

We obtained from a phospholipid extract of wild-type Rhizobium leguminosarum bv. trifolii ANU843 methoxylated fatty acids that had been previously reported as constitutive unusual Rhizobium fatty acids. The use of deuterated reagents and subsequent gas-liquid chromatography-mass spectrometry analyses showed that these methoxylated fatty acid derivatives are the products of chemical alterations of common cyclopropane-containing and unsaturated fatty acids occurring during various acid-catalyzed transesterification treatments aimed at producing the methyl ester derivatives. Similar results were obtained from a phospholipid extract of Escherichia coli K-12. In contrast, these chemical alterations were not induced by an alkaline methanolysis method of transesterification. If an acidic treatment is needed to release the fatty acids from the source molecule, the finding of unusual methoxylated fatty acids should be carefully confirmed with deuterated reagents.

Characterization and symbiotic importance of acidic extracellular polysaccharides of Rhizobium sp. strain GRH2 isolated from acacia nodules.

Rhizobium sp. wild-type strain GRH2 was originally isolated from root nodules of the leguminous tree Acacia cyanophylla and has a broad host range which includes herbaceous legumes, e.g., Trifolium spp. We examined the extracellular exopolysaccharides (EPSs) produced by strain GRH2 and found three independent glycosidic structures: a high-molecular-weight acidic heteropolysaccharide which is very similar to the acidic EPS produced by Rhizobium leguminosarum biovar trifolii ANU843, a low-molecular-weight native heterooligosaccharide resembling a dimer of the repeat unit of the high-molecular-weight EPS, and low-molecular-weight neutral beta (1,2)-glucans. A Tn5 insertion mutant derivative of GRH2 (exo-57) that fails to form acidic heteropolysaccharides was obtained. This Exo- mutant formed nitrogen-fixing nodules on Acacia plants but infected a smaller proportion of cells in the central zone of the nodules than did wild-type GRH2. In addition, the exo-57 mutant failed to nodulate several herbaceous legume hosts that are nodulated by wild-type strain GRH2.

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.

Structural characterization of a novel diglycosyl diacylglyceride glycolipid from Rhizobium trifolii ANU843.

A novel glycolipid was isolated by chloroform-methanol extraction of Rhizobium trifolii ANU843 cells. Compositional analysis, methylation studies, 1H NMR and spectroscopies led to the identification of a diglycosyl diacylglyceride: 1,2-di-O-acyl-3-O-[alpha-D-glucopyranosyl-(1----3)-O-alpha-D- mannopyranosyl]glycerol. Iso-hexadecanoic and anteiso-heptadecanoic acids were the predominant fatty acids esterifying the glyceryl moiety, but a microheterogeneity in fatty acid composition was found, resulting in at least five distinct molecular species of the glycolipid. Although widespread in plants, animals and Gram-positive bacteria, glycosyl glycerides have been seldom reported in Gram-negative bacteria and this work is the first evidence of their occurrence in the bacterial family Rhizobiaceae.

High heterogeneity of the exopolysaccharides of Pseudomonas solanacearum strain GMI 1000 and the complete structure of the major polysaccharide.

The exopolysaccharide of Pseudomonas solanacearum, which is believed to play an important role in bacterial virulence, was considered by most authors as a homogeneous entity essentially composed of N-acetylgalactosamine. The present work demonstrates the high degree of heterogeneity of this exopolysaccharidic material, which consists of a high molecular weight acidic polysaccharide and a mainly noncarbohydrate structure as major subfractions. Rhamnose-rich polyoside and glucan fractions are also present as minor components. We report the complete structure of the acidic heteropolymer involving, in addition to N-acetylgalactosamine, equimolar ratios of two rare amino sugars, 2-N-acetyl-2-deoxy-L-galacturonic acid and 2-N-acetyl-4-N-(3-hydroxybutanoyl)-2,4,6-trideoxy-D-glucose. The structure of this acidic exopolysaccharide provides the first precise basis for the analysis of the correlation exopolysaccharide structure with pathogenicity in P. solanacearum.

Electrofusion of Chinese hamster ovary cells after ethanol incubation.

Electrofusion of Chinese hamster ovary cells is obtained with cells growing in monolayers on a culture dish. The cells were incubated with increasing amounts of ethanol before the field pulsation. The electroperforation was apparently not affected by the incubation but the threshold field intensity required to induce the fusion was shifted to higher values. This effect is tentatively explained by a decrease in size of the field-induced pores as a consequence of an increase in the lipid matrix fluidity.