Molecular mechanisms of attachment of Rhizobium bacteria to plant roots

Attachment of bacteria to plant cells is one of the earliest steps in many plant-bacterium interactions. This review covers the current knowledge on one of the best-studied examples of bacterium-plant attachment, namely the molecular mechanism by which Rhizobium bacteria adhere to plant roots. Despite differences in several studies with regard to growth conditions of bacteria and plants and to methods used for measuring attachment, an overall consensus can be drawn from the available data. Rhizobial attachment to plant root hairs appears to be a two-step process. A bacterial Ca(2+)-binding protein, designated as rhicadhesin, is involved in direct attachment of bacteria to the surface of the root hair cell. Besides this step, there is another step which results mainly in accumulation and anchoring of the bacteria to the surface of the root hair. This leads to so-called firm attachment. Depending on the growth conditions of the bacteria, the latter step is mediated by plant lectins and/or by bacterial appendages such as cellulose fibrils and fimbriae. The possible role of these adhesions in root nodule formation is discussed.     

The effect of the Agrobacterium tumefaciens attR mutation on attachment and root colonization differs between legumes and other dicots

Infections of wound sites on dicot plants by Agrobacterium tumefaciens result in the formation of crown gall tumors. An early step in tumor formation is bacterial attachment to the plant cells. AttR mutants failed to attach to wound sites of both legumes and nonlegumes and were avirulent on both groups of plants. AttR mutants also failed to attach to the root epidermis and root hairs of nonlegumes and had a markedly reduced ability to colonize the roots of these plants. However, AttR mutants were able to attach to the root epidermis and root hairs of alfalfa, garden bean, and pea. The mutant showed little reduction in its ability to colonize these roots. Thus, A. tumefaciens appears to possess two systems for binding to plant cells. One system is AttR dependent and is required for virulence on all of the plants tested and for colonization of the roots of all of the plants tested except legumes. Attachment to root hairs through this system can be blocked by the acetylated capsular polysaccharide. The second system is AttR independent, is not inhibited by the acetylated capsular polysaccharide, and allows the bacteria to bind to the roots of legumes.     

The Effect of the Agrobacterium tumefaciens attR Mutation on Attachment and Root Colonization Differs between Legumes and Other Dicots

Infections of wound sites on dicot plants by Agrobacterium tumefaciens result in the formation of crown gall tumors. An early step in tumor formation is bacterial attachment to the plant cells. AttR mutants failed to attach to wound sites of both legumes and nonlegumes and were avirulent on both groups of plants. AttR mutants also failed to attach to the root epidermis and root hairs of nonlegumes and had a markedly reduced ability to colonize the roots of these plants. However, AttR mutants were able to attach to the root epidermis and root hairs of alfalfa, garden bean, and pea. The mutant showed little reduction in its ability to colonize these roots. Thus, A. tumefaciens appears to possess two systems for binding to plant cells. One system is AttR dependent and is required for virulence on all of the plants tested and for colonization of the roots of all of the plants tested except legumes. Attachment to root hairs through this system can be blocked by the acetylated capsular polysaccharide. The second system is AttR independent, is not inhibited by the acetylated capsular polysaccharide, and allows the bacteria to bind to the roots of legumes.     

Elaboration of cellulose fibrils by Agrobacterium tumefaciens during attachment to carrot cells.

The attachment of virulent strains of Agrobacterium tumefaciens to plant cells is the first step in the bacterial induction of tumors. Binding of A. tumefaciens to carrot tissue culture cells occurred as a two-step process. The initial step was the attachment of the bacteria to the plant cell wall. Living plant cells were not required. Bacterial attachment to heat-killed or glutaraldehyde-fixed carrot cells proceeded with only slightly altered kinetics and unaltered bacterial strain specificity. After the bacteria bound to the carrot cell surface, scanning electron microscopy showed that fibrils developed, surrounded the bacteria, and anchored them to the plant cell surface. These fibrils were synthesized by the bacteria and not by the plant cell since they were also made after the attachment of A. tumefaciens to dead carrot cells and since under some conditions the bacteria synthesized fibrils in the absence of plant cells. Calcofluor staining, acid hydrolysis, enzymatic digestion studies, and infrared spectroscopy showed that the fibrils were composed of cellulose. The formation of these cellulose fibrils occurred during the attachment of virulent strains of A. tumefaciens to plant cells in vitro. The fibrils anchored the bacteria to the plant cell surface and entrapped additional bacteria. The multiplication of entrapped and attached bacteria resulted in the formation of large clusters of bacteria held close to the plant cell wall and plasma membrane by cellulose fibrils. This high concentration of bacteria may facilitate transfer of Ti plasmid deoxyribonucleic acid to the plant cell resulting in the formation of tumors.     

The effect of cellulose overproduction on binding and biofilm formation on roots by Agrobacterium tumefaciens

Agrobacterium tumefaciens growing in liquid attaches to the surface of tomato and Arabidopsis thaliana roots, forming a biofilm. The bacteria also colonize roots grown in sterile quartz sand. Attachment, root colonization, and biofilm formation all were markedly reduced in celA and chvB mutants, deficient in production of cellulose and cyclic beta-(1,2)-D-glucans, respectively. We have identified two genes (celG and cell) in which mutations result in the overproduction of cellulose as judged by chemical fractionation and methylation analysis. Wild-type and chvB mutant strains carrying a cDNA clone of a cellulose synthase gene from the marine urochordate Ciona savignyi also overproduced cellulose. The overproduction in a wild-type strain resulted in increased biofilm formation on roots, as evaluated by light microscopy, and levels of root colonization intermediate between those of cellulose-minus mutants and the wild type. Overproduction of cellulose by a nonattaching chvB mutant restored biofilm formation and bacterial attachment in microscopic and viable cell count assays and partially restored root colonization. Although attachment to plant surfaces was restored, overproduction of cellulose did not restore virulence in the chvB mutant strain, suggesting that simple bacterial binding to plant surfaces is not sufficient for pathogenesis.     

Role of bacterial cellulose fibrils in Agrobacterium tumefaciens infection.

During the attachment of Agrobacterium tumefaciens to carrot tissue culture cells, the bacteria synthesize cellulose fibrils. We examined the role of these cellulose fibrils in the attachment process by determining the properties of bacterial mutants unable to synthesize cellulose. Such cellulose-minus bacteria attached to the carrot cell surface, but, in contrast to the parent strain, with which larger clusters of bacteria were seen on the plant cell, cellulose-minus mutant bacteria were attached individually to the plant cell surface. The wild-type bacteria became surrounded by fibrils within 2 h after attachment. No fibrils were seen with the cellulose-minus mutants. Prolonged incubation of wild-type A. tumefaciens with carrot cells resulted in the formation of large aggregates of bacteria, bacterial fibrils, and carrot cells. No such aggregates were formed after the incubation of carrot cells with cellulose-minus A. tumefaciens. The absence of cellulose fibrils also caused an alteration in the kinetics of bacterial attachment to carrot cells. Cellulose synthesis was not required for bacterial virulence; the cellulose-minus mutants were all virulent. However, the ability of the parent bacterial strain to produce tumors was unaffected by washing the inoculation site with water, whereas the ability of the cellulose-minus mutants to form tumors was much reduced by washing the inoculation site with water. Thus, a major role of the cellulose fibrils synthesized by A. tumefaciens appears to be anchoring the bacteria to the host cells, thereby aiding the production of tumors.     

Attachment of Streptomyces coelicolor is mediated by amyloidal fimbriae that are anchored to the cell surface via cellulose

The chaplin proteins ChpA-H enable the filamentous bacterium Streptomyces coelicolor to form reproductive aerial structures by assembling into surface-active amyloid-like fibrils. We here demonstrate that chaplins also mediate attachment of S. coelicolor to surfaces. Attachment coincides with the formation of fimbriae, which are connected to the cell surface via spike-shaped protrusions. Mass spectrometry, electron microscopy and Congo red treatment showed that these fimbriae are composed of bundled amyloid fibrils of chaplins. Attachment and fimbriae formation were abolished in a strain in which the chaplin genes chpA–H were inactivated. Instead, very thin fibrils emerged from the spike-shaped protrusions in this mutant. These fibrils were susceptible to cellulase treatment. This enzymatic treatment also released wild-type fimbriae from the cell surface, thereby abolishing attachment. The reduced attachment of a strain in which the gene of a predicted cellulose synthase was inactivated also indicates a role of cellulose in surface attachment. We propose that the mechanism of attachment via cellulose-anchored amyloidal fimbriae is widespread in bacteria and may function in initiation of infection and in formation of biofilms.     

Role of cellulose fibrils and exopolysaccharides of Rhizobium leguminosarum in attachment to and infection of Vicia sativa root hairs

Infection and subsequent nodulation of legume host plants by the root nodule symbiote Rhizobium leguminosarum usually require attachment of the bacteria to root-hair tips. Bacterial cellulose fibrils have been shown to be involved in this attachment process but appeared not to be essential for successful nodulation. Detailed analysis of Vicia sativa root-hair infection by wild-type Rhizobium leguminosarum RBL5523 and its cellulose fibril-deficient celE mutant showed that wild-type bacteria infected elongated growing root hairs, whereas cellulose-deficient bacteria infected young emerging root hairs. Exopolysaccharide-deficient strains that retained the ability to produce cellulose fibrils could also infect elongated root hairs but infection thread colonization was defective. Cellulose-mediated agglutination of these bacteria in the root-hair curl appeared to prevent entry into the induced infection thread. Infection experiments with V sativa roots and an extracellular polysaccharide (EPS)- and cellulose-deficient double mutant showed that cellulose-mediated agglutination of the EPS-deficient bacteria in the infection thread was now abolished and that infection thread colonization was partially restored. Interestingly, in this case, infection threads were initiated in root hairs that originated from the cortical cell layers of the root and not in epidermal root hairs. Apparently, surface polysaccharides of R. leguminosarum, such as cellulose fibrils, are determining factors for infection of different developmental stages of root hairs.     

A novel polar surface polysaccharide from Rhizobium leguminosarum binds host plant lectin

Rhizobium bacteria produce different surface polysaccharides which are either secreted in the growth medium or contribute to a capsule surrounding the cell. Here, we describe isolation and partial characterization of a novel high molecular weight surface polysaccharide from a strain of Rhizobium leguminosarum that nodulates Pisum sativum (pea) and Vicia sativa (vetch) roots. Carbohydrate analysis showed that the polysaccharide consists for 95% of mannose and glucose, with minor amounts of galactose and rhamnose. Lectin precipitation analysis revealed high binding affinity of pea and vetch lectin for this polysaccharide, in contrast to the other known capsular and extracellular polysaccharides of this strain. Expression of the polysaccharide was independent of the presence of a Sym plasmid or the nod gene inducer naringenin. Incubation of R. leguminosarum with labelled pea lectin showed that this polysaccharide is exclusively localized on one of the poles of the bacterial cell. Vetch roots incubated with rhizobia and labelled pea lectin revealed that this bacterial pole is involved in attachment to the root surface. A mutant strain deficient in the production of this polysaccharide was impaired in attachment and root hair infection under slightly acidic conditions, in contrast to the situation at slightly alkaline conditions. Our data are consistent with the hypothesis that rhizobia can use (at least) two mechanisms for docking at the root surface, with use of a lectin-glycan mechanism under slightly acidic conditions.     

Surface properties and motility of rhizobium and azospirillum in relation to plant root attachment

Plant growth promotion by rhizobacteria is a widely spread phenomenon. However only a few rhizobacteria have been studied thoroughly. Rhizobium is the best-studied rhizobacterium. It forms a symbiosis with a restricted host range. Azospirillum is another plant-growth-promoting rhizobacterium which forms rhizocoenoses with a wide range of plants. In both bacteria, the interaction with the plant involves the attraction toward the host plant and the attachment to the surface of the root. Both bacteria are attracted to plant roots, but differ in specificity. Attachment to plant roots occurs in two steps for both bacteria: a quick, reversible adsorption, and a slow, irreversible anchoring to the plant root surface. However, for the two systems under study, the bacterial surface molecules involved in plant root attachment are not necessarily the same. Correspondence to: J. Vanderleyden.     

The role of polysaccharide-containing components of the Azospirillum brasilense capsule in adsorbing bacteria on wheat seedling roots

Azospirillum brasilense cells deprived of capsular exopolysaccharides completely lost their ability to bind wheat germ agglutinin (WGA) and much of their ability to attach to wheat seedling roots. The decapsulation of bacterial cells by washing them with a NaCl solution led to an increase in the relative hydrophobicity of the cell surface. The pretreatment of wheat seedling roots with N-acetyl-D-glucosamine (GlcNAc) or the GlcNAc-containing polysaccharide complexes stripped from Azospirillum cells reduced their attachment to the roots. Under the experimental conditions, 3-h incubation of wheat seedling roots with exponential-phase azospirilla, bacterial adsorption is mainly driven by the attachment of the cells to the roots, whose operation is due to the capsular polysaccharide components and the WGA present on the wheat seedling roots.     

Role of the Polysaccharide Components of Azospirillum brasilense Capsules in Bacterial Adsorption on Wheat Seedling Roots

Azospirillum brasilense cells deprived of capsular exopolysaccharides completely lost their ability to bind wheat germ agglutinin (WGA) and much of their ability to attach to wheat seedling roots. The decapsulation of bacterial cells by washing them with a NaCl solution led to an increase in the relative hydrophobicity of the cell surface. The pretreatment of wheat seedling roots with N-acetyl-D-glucosamine (GlcNAc) or the GlcNAc-containing polysaccharide complexes stripped from Azospirillum cells reduced their attachment to the roots. Under the experimental conditions used (3-h incubation of wheat seedling roots with exponential-phase azospirilla), bacterial adsorption is mainly driven by the specific mechanisms attachment of the cells to the roots, whose operation is due to the capsular polysaccharide components and the WGA present on the wheat seedling roots.     

Involvement of both cellulose fibrils and a Ca2+-dependent adhesin in the attachment of Rhizobium leguminosarum to pea root hair tips.

We have previously described an assay for the attachment of Rhizobium bacteria to pea root hair tips (cap formation) which was used as a model to study the attachment step in the nodulation process. Under all conditions tested, a positive correlation was observed between the percentage of fibrillated cells and the ability of these bacteria to form caps and to adhere to glass, suggesting that fibrils play a role in the attachment of Rhizobium leguminosarum to pea root hair tips and to glass (G. Smit, J. W. Kijne, and B. J. J. Lugtenberg, J. Bacteriol. 168:821-827, 1986). In the present paper the chemical and functional characterization of the fibrils of R. leguminosarum is described. Characterization of purified fibrils by infrared spectroscopy and cellulase treatment followed by thin-layer chromatography showed that the fibrils are composed of cellulose. Purified cellulose fibrils, as well as commercial cellulose, inhibited cap formation when present during the attachment assay. Incubation of the bacteria with purified cellulase just before the attachment assay strongly inhibited cap formation, indicating that the fibrils are directly involved in the attachment process. Tn5-induced fibril-overproducing mutants showed a greatly increased ability to form caps, whereas Tn5-induced fibril-negative mutants lost this ability. None of these Tn5 insertions appeared to be located on the Sym plasmid. Both types of mutants showed normal nodulation properties, indicating that cellulose fibrils are not a prerequisite for successful nodulation under the conditions used. The ability of the fibril-negative mutants to attach to glass was not affected by the mutations, indicating that attachment to pea root hair tips and attachment to glass are (partly) based on different mechanisms. However, growth of the rhizobia under low Ca2+ conditions strongly reduced attachment to glass and also prevented cap formation, although it had no negative effect on fibril synthesis. This phenomenon was found for several Rhizobium spp. It was concluded that both cellulose fibrils and a Ca2+ -dependent adhesin(s) are involved in the attachment of R. leguminosarum to pea root hair tips. A model cap formation as a two-step process is discussed.     

Purification and partial characterization of a glycoprotein from pea (Pisum sativum) with receptor activity for rhicadhesin,an attachment protein of Rhizobiaceae

Attachment of Rhizobium and Agrobacterium bacteria to cells of their host plants is a two-step process. The first step, direct attachment of bacteria to the plant cell wall, is mediated by the bacterial protein rhicadhesin. A putative plant receptor molecule for rhicadhesin was purified from cell walls of pea roots using a bioassay based on suppression of rhicadhesin activity. This molecule appeared to be sensitive to treatments with pronase or glycosidase. Its isoelectric point is 6.4, and its apparent molecular mass was estimated to be 32 kDa before and 29 kDa after glycosidase treatment, as determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and ultrafiltration. The sequence of the first 29 N-terminal amino acids was determined: A-D-A-D-A-L-Q-D-L-C(?)-V-A-D-Y-A-S-V-I-L-V-N-G-F-A-S-K(Q)-(P/Q)-(L)-(I). No homology with known proteins was found. In the course of this research project the extracellular matrix protein vitronectin was reported to inhibit attachment of A. tumefaciens to carrot cells [29]. A variety of adhesive proteins, including vitronectin, contain a common cell attachment determinant with the sequence R-G-D. Since we could not detect other cell wall components able to suppress rhicadhesin activity, and since an R-G-D containing hexapeptide was also active as a receptor, we speculate that the plant receptor for rhicadhesin is a glycoprotein containing an R-G-D attachment site.     

Observation by SEM of the attachment ofAgrobacterium tumefaciens to the surface of vinca,asparagus and rice cells

Transformation of vinca cells was performed by the co-cultivation of cell-wall regenerated vinca protoplasts withAgrobacterium tumefaciens. Using thisin vitro and single cell system, attachment of the bacteria to the surface of vinca cells was observed by scanning electron microscopy (SEM). Figures of the bacteria polarly binding to the plant cell wall were often observed. AsEscherichia coli does not attach to the plant cells at all, the observed attachment ofA. tumefaciens is suggested as a characteristic feature in crown gall induction. Even though no evidence of transformation was obtained by the co-cultivation methods, a similar attachment was observed in the cell-wall regenerated protoplasts of rice. The bacteria also attached to the surface of isolated mesophyll cells of asparagus and root hairs of rice. From these observation, we concluded that the attachment is not the limiting step of crown gall induction byA. tumefaciens in monocotyledonous plants. Extracellular fibrils like pili were observed with a few strains of A.tumefaciens for the first time. These fibrils were observed regardless of their ability of attachment and infectivity.     

Capsular polysaccharide surrounds smooth and rugose types of Salmonella enterica serovar Typhimurium DT104

The biofilms and rugose colony morphology of Salmonella enterica serovar Typhimurium strains are usually associated with at least two different exopolymeric substances (EPS), curli and cellulose. In this study, another EPS, a capsular polysaccharide (CP) synthesized constitutively in S. enterica serovar Typhimurium strain DT104 at 25 and 37 degrees C, has been recognized as a biofilm matrix component as well. Fluorophore-assisted carbohydrate electrophoresis (FACE) analysis indicated that the CP is comprised principally of glucose and mannose, with galactose as a minor constituent. The composition differs from that of known colanic acid-containing CP that is isolated from cells of Escherichia coli and other enteric bacteria grown at 37 degrees C. The reactivity of carbohydrate-specific lectins conjugated to fluorescein isothiocyanate or gold particles with cellular carbohydrates demonstrated the cell surface localization of CP. Further, lectin binding also correlated with the FACE analysis of CP. Immunoelectron microscopy, using specific antibodies against CP, confirmed that CP surrounds the cells. Confocal microscopy of antibody-labeled cells showed greater biofilm formation at 25 degrees C than at 37 degrees C. Since the CP was shown to be produced at both 37 degrees C and 25 degrees C, it does not appear to be significantly involved in attachment during the early formation of the biofilm matrix. Although the attachment of S. enterica serovar Typhimurium DT104 does not appear to be mediated by its CP, the capsule does contribute to the biofilm matrix and may have a role in other features of this organism, such as virulence, as has been shown previously for the capsules of other gram-negative and gram-positive bacteria.     

细菌荚膜多糖的化学结构

荚膜是一些细菌所具有的表层结构,与多种疾病有着密切联系。细菌荚膜多糖不仅结构复杂,而且在免疫活性方面发挥着重要的作用。同一种细菌根据其荚膜多糖的抗原性不同可分为不同的血清型,不同血清型细菌荚膜多糖的化学结构也存在差异。以荚膜多糖为基础的疫苗正在积极研究开发当中,对不同致病细菌荚膜多糖具体化学结构的掌握是疫苗得到许可的必备条件之一。本文对致病细菌荚膜多糖的化学结构进行了归纳和总结,以期为荚膜多糖的化学结构研究和疫苗开发提供参考。     

细菌荚膜多糖的分离纯化研究进展

荚膜多糖是细菌的保护性抗原和毒力因子,也是细菌疫苗最重要的靶抗原之一,其分离纯化是制作疫苗的首要步骤。本文从去除菌体、收集总糖、去除菌体核酸和蛋白质、去除内毒素等基本工艺步骤,对现有的工艺和目前的工艺进展进行了综述,重点阐述了中空纤维、深层过滤、超滤、酶水解、柱层析等方法在荚膜多糖分离纯化中的应用进展。     

Cellular interactions between arbuscular mycorrhizal fungi and rhizosphere bacteria

Summary We have investigated whether direct physical interactions occur between arbuscular mycorrhizal (AM) fungi and plant growth promoting rhizobacteria (PGPRs), some of which are used as biocontrol agents. Attachment of rhizobia and pseudomonads to the spores and fungal mycelium ofGigaspora margarita has been assessed in vitro and visualized by a combination of electron and confocal microscopy. The results showed that both rhizobia and pseudomonads adhere to spores and hyphae of AM fungi germinated under sterile conditions, although the degree of attachment depended upon the strain.Pseudomonas fluorescens strain WCS 365 andRhizobium leguminosarum strains B556 and 3841 were the most effective colonizers. Extracellular material of bacterial origin containing cellulose produced around the attached bacteria may mediate fungal/bacterial interactions. These results suggest that antagonistic and synergistic interactions between AM fungi and rhizosphere bacteria may be mediated by soluble factors or physical contact. They also support the view that AM fungi are a vehicle for the colonization of plant roots by soil rhizobacteria.Abbreviations AM arbuscular mycorrhiza - PGPR plant growth promoting rhizobacteria - CBH cellobiohydrolase - DAPG 2,4-(diacetyl-phloroglucinol - TY triptone-yeast - LB Lauria-Bertani Dedicated to Prof. Dr. Dr. h.c. Eberhard Schnepf on the occasion of his retirement