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 the Rhizobium leguminosarum biovar viciae Ca2+-dependent adhesin, which mediates the first step in attachment of cells of the family Rhizobiaceae to plant root hair tips.

The Ca2+-dependent adhesin which mediates the first step in attachment of bacteria of the family Rhizobiaceae to plant root hair tips was isolated from the surface of Rhizobium leguminosarum biovar viciae cells; its ability to inhibit attachment of R. leguminosarum to pea root hair tips was used as a bioassay. Isolated adhesin was found to be able to inhibit attachment of both carbon-limited and manganese-limited R. leguminosarum cells. A multicolumn purification procedure was developed which resulted in pure adhesin, as judged from silver staining of isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gel electropherograms. The crucial step in purification was the elution of rhizobial proteins by a CaCl2 gradient from a hydroxyapatite matrix. The specific activity increased 1,250 times during purification. The isoelectric point of the adhesin was determined to be 5.1, and the molecular mass was 14 kilodaltons (kDa), as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. By using gel filtration in the presence and absence of Ca2+, the molecular mass of the adhesin was determined to be 15 and 6 kDa, respectively. The adhesin appeared to be a calcium-binding protein. The purified adhesin inhibited attachment of various other rhizobia to pea root hair tips. Also, cell surface preparations of several other rhizobial strains, including Agrobacterium, Bradyrhizobium, and Phyllobacterium spp., showed adhesin activity, suggesting that a common plant receptor is used for attachment of Rhizobiaceae cells and that the adhesin is common among Rhizobiaceae. No attachment-inhibiting activity was detected in cell surface preparations from various other bacterial strains tested. Cell surface preparations from Sym or Ti plasmid-cured Rhizobium and Agrobacterium strains, respectively, also showed adhesin activity, indicating that Sym or Ti plasmid-borne genes are not required for the synthesis and biogenesis of the adhesin. The adhesin was also found to be involved in the attachment of rhizobia to the root hairs of various other legumes and nonlegume plants, including monocotyledonous ones. Since the adhesin appears to be specific for Rhizobiaceae and is Ca2+ dependent, we propose to designate it rhicadhesin. A more detailed model for rhizobial attachment to plant root hairs is discussed.     

Correlation between extracellular fibrils and attachment of Rhizobium leguminosarum to pea root hair tips.

As part of a project meant to characterize molecules involved in nodulation, a semiquantitative microscopic assay was developed for measuring attachment of Rhizobium leguminosarum cells to pea root hair tips, i.e., the site at which R. leguminosarum initiates nodulation. This form of attachment, designated as cap formation, was dependent on the incubation pH and growth phase, with optimal attachment at pH 7.5 and with bacteria in the early stationary phase of growth. Addition of glucose to the growth medium delayed the initiation of the stationary phase and cap formation, suggesting a correlation between cap formation and carbon limitation. Attachment of R. leguminosarum was not inhibited by pea lectin haptens which makes it unlikely that lectins are involved under the tested conditions. Moreover, heterologous fast-growing rhizobia adhered equally well to pea root hair tips. Since the attachment characteristics of a Sym plasmid-cured derivative were indistinguishable from those of the wild-type strain, the Sym plasmidborne nodulation genes are not necessary for attachment. Sodium chloride and various other salts abolished attachment when present during the attachment assay in final concentrations of 100 mM. R. leguminosarum produced extracellular fibrils. A positive correlation between the percentage of fibrillated cells and the ability of the bacteria to form caps and to adhere to glass and erythrocytes was observed under various conditions, suggesting that these fibrils play a role in attachment of the bacteria to pea root hair tips, to glass, and to erythrocytes.     

Role of Ca2+ in the activity of rhicadhesin from Rhizobium leguminosarum biovar viciae,which mediates the first step in attachment of Rhizobiaceae cells to plant root hair tips

The first step in attachment of Rhizobiaceae cells to plant root hair tips is mediated by a Ca²⁺-dependent, Ca²⁺-binding protein, rhicadhesin. The possible role of Ca²⁺ in synthesis, anchoring and activity of rhicadhesin was investigated. Growth of Rhizobium leguminosarum biovar viciae cells under Ca²⁺-limitation was found to result in loss of attachment ability. Under these conditions, rhicadhesin could not be usolated from the bacterial cell surface, but was found to be excreted in the growth medium. Divalent ions appeared to be essential for the ability of purified rhicadhesin to inhibit attachment of R. leguminosarum biovar viciae cells to pea root hair tips. Calcium ions were found not to be involved in binding of rhicadhesin to the plant surface, but appeared to be involved in anchoring of the adhesin to the bacterial cell surface. A model for the role of Ca²⁺ in activity of rhicadhesin is presented.     

Lectin-enhanced accumulation of manganese-limited Rhizobium leguminosarum cells on pea root hair tips.

The ability of Rhizobium leguminosarum 248 to attach to developing Pisum sativum root hairs was investigated during various phases of bacterial growth in yeast extract-mannitol medium. Direct cell counting revealed that growth of the rhizobia transiently stopped three successive times during batch culture in yeast extract-mannitol medium. These interruptions of growth, as well as the simultaneous autoagglutination of the bacteria, appeared to be caused by manganese limitation. Rhizobia harvested during the transient phases of growth inhibition appeared to have a better attachment ability than did exponentially growing rhizobia. The attachment characteristics of these manganese-limited rhizobia were compared with those of carbon-limited rhizobia (G. Smit, J. W. Kijne, and B. J. J. Lugtenberg, J. Bacteriol. 168:821-827, 1986, and J. Bacteriol. 169:4294-4301, 1987). In contrast to the attachment of carbon-limited cells, accumulation of manganese-limited rhizobia (cap formation) was already in full progress after 10 min of incubation; significantly delayed by 3-O-methyl-D-glucose, a pea lectin haptenic monosaccharide; partially resistant to sodium chloride; and partially resistant to pretreatment of the bacteria with cellulase. Binding of single bacteria to the root hair tips was not inhibited by 3-O-methyl-D-glucose. Whereas attachment of single R. leguminosarum cells to the surface of pea root hair tips seemed to be similar for both carbon- and manganese-limited cells, the subsequent accumulation of manganese-limited rhizobia at the root hair tips is apparently accelerated by pea lectin molecules. Moreover, spot inoculation tests with rhizobia grown under various culture conditions indicated that differences in attachment between manganese- and carbon-limited R. leguminosarum cells are correlated with a significant difference in infectivity in that manganese-limited rhizobia, in contrast to carbon-limited rhizobia, are infective. This growth-medium-dependent behavior offers and explanation for the seemingly conflicting data on the involvement of host plant lectins in attachment of rhizobia to root hairs of leguminous plants. Sym plasmid-borne genes do not play a role in manganese-limitation-induced attachment of R. leguminosarum.     

Identification of a NodD repressible gene adjacent to nodM in Rhizobium leguminosarum biovar viciae

The nodFEL and nodMNT operons in Rhizobium leguminosarum biovar viciae are transcribed in the same orientation and induced by NodD in response to flavonoids secreted by legumes. In the narrow intergenic region between nodFEL and nodMNT, we identified a small gene divergently transcribed from nodM to the 3' end of nodL. Unlike the promoters upstream of nodF and nodM, the promoter of this gene is constitutively expressed. It appeared that its promoter might partially overlap with that of nodM and its expression was repressed by nodD. A deletion mutation was made and proteins produced by the mutant were compared with those by wild-type using 2D gel electrophoresis. Several protein differences were identified suggesting that this small gene influences the expression or stability of these proteins. However, the mutant nodulated its host plant (pea) normally.     

Plant phenology and genetic variability in root and nodule development strongly influence genetic structuring of Rhizobium leguminosarum biovar viciae populations nodulating pea

The symbiotic relationships between legumes and their nitrogen (N(2))-fixing bacterial partners (rhizobia) vary in effectiveness to promote plant growth according to both bacterial and legume genotype. To assess the selective effect of host plant on its microsymbionts, the influence of the pea (Pisum sativum) genotype on the relative nodulation success of Rhizobium leguminosarum biovar viciae (Rlv) genotypes from the soil populations during plant development has been investigated. Five pea lines were chosen for their genetic variability in root and nodule development. Genetic structure and diversity of Rlv populations sampled from nodules were estimated by molecular typing with a marker of the genomic background (rDNA intergenic spacer) and a nodulation gene marker (nodD region). Differences were found among Rlv populations related to pea genetic background but also to modification of plant development caused by single gene mutation. The growth stage of the host plant also influenced structuring of populations. A particular nodulation genotype formed the majority of nodules during the reproductive stage. Overall, modification in root and nodule development appears to strongly influence the capacity of particular rhizobial genotypes to form nodules.     

Glucomannan-mediated attachment of Rhizobium leguminosarum to pea root hairs is required for competitive nodule infection

The Rhizobium leguminosarum biovar viciae genome contains several genes predicted to determine surface polysaccharides. Mutants predicted to affect the initial steps of polysaccharide synthesis were identified and characterized. In addition to the known cellulose (cel) and acidic exopolysaccharide (EPS) (pss) genes, we mutated three other loci; one of these loci (gmsA) determines glucomannan synthesis and one (gelA) determines a gel-forming polysaccharide, but the role of the other locus (an exoY-like gene) was not identified. Mutants were tested for attachment and biofilm formation in vitro and on root hairs; the mutant lacking the EPS was defective for both of these characteristics, but mutation of gelA or the exoY-like gene had no effect on either type of attachment. The cellulose (celA) mutant attached and formed normal biofilms in vitro, but it did not form a biofilm on root hairs, although attachment did occur. The cellulose-dependent biofilm on root hairs appears not to be critical for nodulation, because the celA mutant competed with the wild-type for nodule infection. The glucomannan (gmsA) mutant attached and formed normal biofilms in vitro, but it was defective for attachment and biofilm formation on root hairs. Although this mutant formed nodules on peas, it was very strongly outcompeted by the wild type in mixed inoculations, showing that glucomannan is critical for competitive nodulation. The polysaccharide synthesis genes around gmsA are highly conserved among other rhizobia and agrobacteria but are absent from closely related bacteria (such as Brucella spp.) that are not normally plant associated, suggesting that these genes may play a wide role in bacterium-plant interactions.     

Molecular analysis of a microaerobically induced operon required for hydrogenase synthesis in Rhizobium leguminosarum biovar viciae

The nucleotide sequence (6138 bp) of a microaerobically inducible region (hupV/VI) from the Rhizobium leguminosarum bv. viciae hydrogenase gene cluster has been determined. Six genes, arranged as a single operon, were identified, and designated hypA, B, F, C, D and E based on the sequence similarities of all of them, except hypF, to genes from the hydrogenase pleiotropic operon (hyp) from Escherichia coli. The gene products from hypBFCDE were identified by in vivo expression analysis in E. coli, and their molecular sizes were consistent with those predicted from the nucleotide sequence. Transposon Tn5 insertions into hypB, hypF, hypD and hypE resulted in R. leguminosarum mutants that lacked any hydrogenase activity in symbiosis with peas, but still were able to synthesize the polypeptide for the hydrogenase large subunit. The gene products HypA, HypB, HypF and HypD contained CX₂C motifs characteristic of metal-binding proteins. In addition, HypB bore a long histidine-rich stretch of amino acids near the N-terminus, suggesting a possible role in nickel binding for this protein. The gene product HypF, which was translationally coupled to HypB, presented two cysteine motifs (CX₂CX₈₁CX₂C) with a capacity to form zinc finger-like structures in the N-terminal third of the protein. A role in nickel metabolism in relation to hydrogenase synthesis is postulated for proteins HypB and HypF.     

Construction and characterization of a Rhizobium leguminosarum biovar viciae strain designed to assess horizontal gene transfer in the environment

Abstract An integration vector was developed which inserts cloned DNA in a non-essential site of the Rhizobium leguminosarum biovar viciae chromosome. The expression of integrated genes is under the control of the constitutive neomycin phosphotransferase II ( npt II) promotor of transposon Tn5. The design of the vector ensures that loss of vector sequences can be detected, enabling selection of progeny containing only the requisite DNA. The newly constructed vector was employed to insert the Escherichia coli gusA gene conferring GUS activity into R. leguminosarum bv. viciae strain LRS39401 which is cured of its symbiotic plasmid (pSym). One GUS-positive transconjugant, strain CT0370, was shown to have lost all vector sequences. Conjugal transfer of pSym2004 (a Tn5-tagged derivative of symbiotic plasmid pRL1JI, which specifies pea nodulation and symbiotic nitrogen fixation) to CT0370, restored the GUS-positive strain's symbiotic proficiency. Strain CT0370 is presently being used in a field release experiment in order to assess the extent of pSym transfer in a natural R. leguminosarum bv. viciae population under environmental conditions.     

Protein composition of the peribacteriod space in root nodules of Pisum sativum and Vicia faba induced by Rhizobium leguminosarum biovar viciae

Proteins in the peribacteroid space (PBS) between the bacteroid outer membrane and the peribacteroid membrane in root nodules of Pisum sativum and Vicia faba induced by Rhizobium leguminosarum PRE were analysed by two-dimensional (2-D) gel electrophoresis. Most of the detectable proteins were found to migrate to identical positions; however the level of accumulation of some of these appear to be determined by the host plant. When a different R. leguminosarum strain (RB1) was used to inoculate P. sativum , the majority of the isolated PBS proteins were found to migrate in the 2-D gel to identical positions as those of the other two combinations ( R. leguminosarum PRE x P. sativum and R. leguminosarum PRE x V. faba ).     

Rhizobium leguminosarum bv. viciae genotypes interact with pea plants in developmental responses of nodules, roots and shoots

The variability of the developmental responses of two contrasting cultivars of pea (Pisum sativum) was studied in relation to the genetic diversity of their nitrogen-fixing symbiont Rhizobium leguminosarum bv. viciae. A sample of 42 strains of pea rhizobia was chosen to represent 17 genotypes predominating in indigenous rhizobial populations, the genotypes being defined by the combination of haplotypes characterized with rDNA intergenic spacer and nodD gene regions as markers. We found contrasting effects of the bacterial genotype, especially the nod gene type, on the development of nodules, roots and shoots. A bacterial nod gene type was identified that induced very large, branched nodules, smaller nodule numbers, high nodule biomass, but reduced root and aerial part development. The plants associated with this genotype accumulated less N in shoots, but N concentration in leaves was not affected. The results suggest that the plant could not control nodule development sustaining the energy demand for nodule functioning and its optimal growth. The molecular and physiological mechanisms that may be involved are discussed.