Host-plant invasion by Rhizobium: the role of cell-surface components.

Rhizobia are soil bacteria that can become endosymbionts, reducing atmospheric nitrogen within nodules formed on the roots of legume plants. During tissue and cell invasion, bacterial cell-surface components adapt the bacterium to survive as an endophyte without eliciting host-defence responses. The structures of many of these components have been established recently, allowing their possible roles in invasion to be defined more clearly.

Cloning of conserved genes from Zymomonas mobilis and Bradyrhizobium japonicum that function in the biosynthesis of hopanoid lipids.

The squalene-hopene cyclase (SHC) is the only enzyme involved in the biosynthesis of hopanoid lipids that has been characterized on the genetic level. To investigate if additional genes involved in hopanoid biosynthesis are clustered with the shc gene, we cloned and analyzed the nucleotide sequences located immediately upstream of the shc genes from Zymomonas mobilis and Bradyrhizobium japonicum. In Z. mobilis, five open reading frames (ORFs, designated as hpnA-E) were detected in a close arrangement with the shc gene. In B. japonicum, three similarly arranged ORFs (corresponding to hpnC-E from Z. mobilis) were found. The deduced amino acid sequences of hpnC-E showed significant similarity (58-62%) in both bacteria. Similarities to enzymes of other terpenoid biosynthesis pathways (carotenoid and steroid biosynthesis) suggest that these ORFs encode proteins involved in the biosynthesis of hopanoids and their intermediates. Expression of hpnC to hpnE from Z. mobilis as well as expression of hpnC from B. japonicum in Escherichia coli led to the formation of the hopanoid precursor squalene. This indicates that hpnC encodes a squalene synthase. The two additional ORFs (hpnA and hpnB) in Z. mobilis showed similarities to enzymes involved in the transfer and modification of sugars, indicating that they may code for enzymes involved in the biosynthesis of the complex, sugar-containing side chains of hopanoids.

Bacterial and plant glycoconjugates at the Rhizobium-legume interface.

Many classes of bacterial and plant glycoconjugate have been shown to be involved in establishing the Rhizobium root nodule symbiosis with peas (Pisum sativum). It was demonstrated, using techniques of molecular genetics, that a group of Rhizobium nodulation genes (nod genes) co-operate to synthesize a lipo-oligosaccharide signal molecule that specifically initiates nodule development on legume hosts. An additional gene function, encoded by nodX, has been found to extend the host range of Rhizobium leguminosarum bv. viciae to include nodulation of a pea mutant, cultivar Afghanistan; the nodX gene product specifies the addition of an acetyl group to the terminal N-acetylglucosamine residue at the reducing end of the pentasaccharide core of this signal molecule. Several other classes of bacterial glycoconjugate have also been shown by genetic analysis to be essential for normal nodule development and function: these include a capsular extracellular polysaccharide; lipopolysaccharide in the outer membrane; and cyclic glucans present in the periplasmic space. Potential functions for these glycoconjugates are discussed in the context of tissue and cell invasion by Rhizobium. Some plant components involved in symbiotic interactions have been identified by the analysis of nodule-specific gene expression (early nodulins). Several of the cDNA clones encoding these early nodulins specify proline-rich proteins that presumably correspond to cell wall glycoproteins or membrane arabinogalactan proteins. Other plant glycoconjugates have been identified using monoclonal antibodies as probes. A plant glycoprotein present in intercellular spaces has been identified as a component of the luminal matrix of infection threads. Because it attaches to the surface of bacteria and is itself susceptible to oxidative cross-linking, this glycoprotein may be involved in limiting the progress of microbial infections. Endocytosis of bacteria into the plant cytoplasm is apparently driven by direct interactions between the bacterial surface and the plasma membrane that is exposed within an unwalled infection droplet; glycoprotein and glycolipid components of the plant membrane glycocalyx have been defined using monoclonal antibodies. Differentiation of endosymbiotic bacteroids is preceded by differentiation of the plant-derived peribacteroid membrane which encloses the symbiosome compartment. Using a monoclonal antibody that identifies a group of plant membrane-associated, inositol-containing glycolipids, we have identified a very early marker for the differentiation of peribacteroid membrane from plasma membrane.

Expression of a cell surface antigen from Rhizobium leguminosarum 3841 is regulated by oxygen and pH.

Rhizobium leguminosarum bv. viciae 3841 was grown in liquid suspension culture to investigate how culture conditions could affect the expression of a developmentally regulated cell surface antigen associated with lipopolysaccharide. The antigen, which is recognized by monoclonal antibody AFRC MAC 203, was expressed when cultures were grown at neutral pH under low-oxygen conditions (less than 7.5% [vol/vol] O2 in the gas phase). Antigen was also expressed in aerobically grown cultures at pH values below 5.3. The nature of the nitrogen and the carbon sources had no effect on antigen expression except by indirect changes on the pH of the culture medium; similarly, growth in 0.3 M NaCl did not result in antigen expression. The induction of MAC 203 antigen by low-oxygen or low-pH culture conditions is discussed in the context of tissue-specific expression within the legume root nodule.

Lipid A and O-chain modifications cause Rhizobium lipopolysaccharides to become hydrophobic during bacteroid development.

Modifications to the lipopolysaccharide (LPS) structure caused by three different growth conditions were investigated in the pea-nodulating strain Rhizobium leguminosarum 3841. The LPSs extracted by hot phenol-water from cultured cells fractionated into hydrophilic water and/or hydrophobic phenol phases. Most of the LPSs from cells grown under standard conditions extracted into the water phase, but a greater proportion of LPSs were extracted into the phenol phase from cells grown under acidic or reduced-oxygen conditions, or when isolated from root nodules as bacteroids. Compared with the water-extracted LPSs, the phenol-extracted LPSs contained greater degrees of glycosyl methylation and O-acetylation, increased levels of xylose, glucose and mannose and increased amounts of long-chain fatty acids attached to the lipid A moiety. The water- and phenol-phase LPSs also differed in their reactivity with monoclonal antibodies and in their polyacrylamide gel electrophoretic banding patterns. Phenol-extracted LPSs from rhizobia grown under reduced-oxygen conditions closely resembled the bulk of LPSs isolated from pea nodule bacteria (i.e. mainly bacteroids) in their chemical properties, reactivities with monoclonal antibodies and extraction behaviour. This finding suggests that, during symbiotic bacteroid development, reduced oxygen tension induces structural modifications in LPSs that cause a switch from predominantly hydrophilic to predominantly hydrophobic molecular forms. Increased hydrophobicity of LPSs was also positively correlated with an increase in the surface hydrophobicity of whole cells, as shown by the high degree of adhesion to hydrocarbons of bacterial cells isolated from nodules or from cultures grown under low-oxygen conditions. The implications of these LPS modifications are discussed for rhizobial survival and function in different soil and in planta habitats.

Developmental regulation of a Rhizobium cell surface antigen during growth of pea root nodules.

A monoclonal antibody, AFRC MAC 203, was used to examine the expression of a nodule-induced cell surface antigen associated with lipopolysaccharide in Rhizobium leguminosarum bv. viciae 3841. Silver-enhanced immunogold-labeled tissue sections revealed that, in very young tissues of pea root nodules, the nodule-induced form of lipopolysaccharide antigen was not expressed either by rhizobia in the infection thread or by bacteria recently released into the plant cell cytoplasm. In the more mature regions of the nodule, the antigen was expressed by membrane-enclosed bacteroids, including immature forms that had not yet expressed the enzyme nitrogenase and were not yet Y shaped. Immunogold labeling of thin sections revealed that the MAC 203 antigen, but not the nitrogenase, was also expressed by bacteria in infection threads situated in and between bacteroid-containing plant cells in mature nodule tissue.

Immunochemical analysis of lipopolysaccharides from free-living and endosymbiotic forms of Rhizobium leguminosarum.

Rhizobium leguminosarum B556 and 8002 differ only with respect to carrying symbiotic plasmids with specificity for Pisum or Phaseolus hosts, respectively. Protease-treated samples derived from free-living cultures of both strains revealed a ladder of lipopolysaccharide (LPS-1) bands after periodate-silver staining of sodium dodecyl sulfate-polyacrylamide gels. These bands were arranged as doublets. After Western (immuno-) blotting, all LPS-1 bands reacted with monoclonal antibody JIM 21, whereas monoclonal antibody MAC 57 reacted only with the upper (slower-migrating) band and monoclonal antibody MAC 114 reacted only with the lower band of each doublet pair. Preparations obtained from bacteroids of Pisum or Phaseolus nodules showed significant differences in the size distribution and antigenicity of LPS. In bacteroids from Phaseolus sp., JIM 21 and MAC 57 each stained a ladder of LPS-1 bands on sodium dodecyl sulfate-polyacrylamide gels which corresponded in mobility to the upper band of each doublet pair seen in free-living cultures. MAC 114 did not react with the LPS from Phaseolus sp.-derived bacteroids. In bacteroids from Pisum sp., only fast-migrating (lower-molecular-weight) forms of LPS-1 could be visualized on gels, but both upper and lower bands of each doublet were still present and could be stained by the appropriate monoclonal antibody, MAC 57 or MAC 114, respectively. Similarly, bacteroids from R. leguminosarum 3841, which nodulates Pisum species, differed with respect to the structure and antigenicity of their LPS-1 from bacteroids of a related strain, B625, which nodulates Phaseolus species. Physiological factors were investigated that could account for these differences between the structures of LPS-1 from free-living cultures of B556 and 8002 and that from bacteroids. The following modifications in growth conditions each tended to reduce the expression of MAC 114 antigen and enhance the expression of MAC 57 antigen: succinate rather than glucose as the carbon source; microaerobic (2.5%, vol/vol) oxygen concentrations; and acidic (pH 5 to 6) culture medium. When all three of these conditions were combined, the LPS-1 that resulted was very similar to that in bacteroids from Pisum nodules. However, it was not possible to reproduce the LPS-1 pattern observed for bacteroids from Phaseolus nodules, which maintained a ladder of LPS bands reacting with MAC 57 antibody.

Molecular dissection of structure and function in the lipopolysaccharide of Rhizobium leguminosarum strain 3841 using monoclonal antibodies and genetic analysis.

Following treatment with nitrosoguanidine, mutant derivatives of Rhizobium leguminosarum strain 3841 were isolated which failed to react with AFRC MAC 203. This monoclonal antibody normally recognizes a strain-specific lipopolysaccharide epitope which is developmentally regulated during legume nodule differentiation. Structural modification of lipopolysaccharide (LPS) was analysed by examining reactivity with a range of monoclonal antibodies with different epitope specificities, and also by analysis of LPS mobility changes after electrophoresis on polyacrylamide gels. One class of these LPS-defective mutants induced normal nitrogen-fixing (Fix+) nodules on peas (Pisum sativum), while another two classes of Fix- mutants were also identified, suggesting that a component of the LPS antigen that is part of the MAC 203 epitope is essential for normal nodule development leading to symbiotic nitrogen fixation. When grown under low-oxygen or low-pH culture conditions, one class of Fix- mutants completely lacked LPS-1 (the species that carries O antigen) and a second class showed a modified and truncated form of LPS-1. Mutants with defective LPS structure were also obtained after Tn5 mutagenesis of R. leguminosarum 3841 and all nine Fix- mutants were also found to lack the MAC 203 epitope. Three of these transposon-induced mutants synthesized a truncated form of LPS-1 that was structurally similar to that of the class of the NTG-induced mutants described above. These transposon-induced mutations, and the nitrosoguanidine-induced Fix- mutations, were closely linked and could be suppressed by the same cloned fragment of chromosomal DNA. The data presented here suggest that a precondition for normal nodule development of R. leguminosarum 3841 within pea nodules is the ability to synthesize relatively long-chain LPS-1 macromolecules under the physiological conditions encountered within the nodule. All mutants that lacked the ability to elongate LPS-1 macromolecules also failed to express the MAC 203 epitope.

Lipopolysaccharide epitope expression of Rhizobium bacteroids as revealed by in situ immunolabelling of pea root nodule sections.

To investigate the in situ expression of lipopolysaccharide (LPS) epitopes on nodule bacteria of Rhizobium leguminosarum, monoclonal antibodies recognizing LPS macromolecules were used for immunocytochemical staining of pea nodule tissue. Many LPS epitopes were constitutively expressed, and the corresponding antibodies reacted in nodule sections with bacteria at all stages of tissue infection and cell invasion. Some antibodies, however, recognized epitopes that were only expressed in particular regions of the nodule. Two general patterns of regulated LPS epitope expression could be distinguished on longitudinal sections of nodules. A radial pattern probably reflected the local physiological conditions experienced by endosymbiotic bacteria as a result of oxygen diffusion into the nodule tissue. The other pattern of expression, which followed a linear axis of symmetry along a longitudinal section of the pea nodule, was apparently associated with the differentiation of nodule bacteria and the development of the nitrogen-fixing capacity in bacteroids. Basically similar patterns of LPS epitope expression were observed for pea nodules harboring either of two immunologically distinct strains of R. leguminosarum bv. viciae, although these epitopes were recognized by different sets of strain-specific monoclonal antibodies. Furthermore, LPS epitope expression of rhizobia in pea nodules was compared with that of equivalent strains in nodules of French bean (Phaseolus vulgaris). From these observations, it is suggested that structural modifications of Rhizobium LPS may play an important role in the adaptation of endosymbiotic rhizobia to the surrounding microenvironment.

Genetic derepression of a developmentally regulated lipopolysaccharide antigen from Rhizobium leguminosarum 3841.

Monoclonal antibody AFRC MAC 203 recognizes a developmentally regulated lipopolysaccharide antigen in Rhizobium leguminosarum bv. viciae 3841. Transposon-induced mutants that constitutively expressed MAC 203 antigen were isolated. These strains were morphologically normal, showed no gross abnormalities in lipopolysaccharide size distribution on sodium dodecyl sulfate-polyacrylamide gels, and induced normal nitrogen-fixing nodules. However, the mutants lacked lipopolysaccharide epitopes recognized by another rat monoclonal antibody, AFRC MAC 281, suggesting that the corresponding epitopes may be interconverted or share a common precursor. In conjugational crosses, the transposon insertion associated with both the loss of MAC 281 antigen and the constitutive expression of MAC 203 antigen showed linkage to the chromosomal rif allele. A derivative of strain 3841 with a deletion spanning the nod-fix region of the symbiotic plasmid showed no altered expression pattern for MAC 203 antigen, suggesting that the relevant genetic determinants map to genomic sites that are not associated with nifA or any known genes on the symbiotic plasmid.

Isolation of monoclonal antibodies reacting with the core component of lipopolysaccharide from Rhizobium leguminosarum strain 3841 and mutant derivatives.

Monoclonal antibodies reacting with the core oligosaccharide or lipid A component of Rhizobium lipopolysaccharide (LPS) could be useful for the elucidation of the structure and biosynthesis of this group of macromolecules. Mutant derivatives of Rhizobium leguminosarum 3841 with LPS structures lacking the major O-antigen moiety were used as immunogens, and eight antibodies were selected for further study. All the antibodies reacted with the fast-migrating species known as LPS-2 following gel electrophoresis of Rhizobium cell extracts. For four of these antibodies, reactivity with affinity-purified LPS was lost after mild acid hydrolysis, indicating that they probably recognized the core oligosaccharide component. The four other antibodies still reacted with acid-treated LPS and may recognize the lipid A moiety, which is stable to mild acid hydrolysis. The pattern of antibody staining after gel electrophoresis revealed differences in LPS-2 epitope structure between each of the mutants and the wild type. Furthermore, for each of the mutants the antibodies crossreacted with a minor band that migrated more slowly than LPS-2; we have termed this more slowly migrating form LPS-3. The majority of the antibodies also reacted with LPS from strain CE109, a derivative of Rhizobium etli CE3, confirming that the LPS core antigens can be relatively conserved between strains of different Rhizobium species. One of the antibodies isolated in this study (JIM 32) was unusual because it appeared to react with all forms of LPS from strain 3841 (namely, LPS-1, LPS-2, and LPS-3). Furthermore, JIM 32 reacted positively with the LPS from many strains of Rhizobium tested (excluding the Rhizobium meliloti subgroup). JIM 32 did not react with representative strains from Bradyrhizobium, Azorhizobium or other related bacterial species.

Novel methylated triterpenoids of the gammacerane series from the nitrogen-fixing bacterium Bradyrhizobium japonicum USDA 110.

The nitrogen-fixing, symbiotic root-nodule forming bacterium Bradyrhizobium japonicum USDA 110 contained gammacerane derivatives next to triterpenoids of the hopane series. Diploptene, diplopterol, 2 beta-methyldiplopterol, aminobacteriohopanetriol and adenosylhopane were accompanied by tetrahymanol and the corresponding novel methylated homologues 2 beta-methyltetrahymanol, 20 alpha-methyltetrahymanol, and 2 beta,20 alpha-dimethyltetrahymanol. Incorporation of [(2)H(3)]methyl-L-methionine indicated that the additional methyl groups originated from methionine, probably with S-adenosylmethionine acting as methyl donor, with retention of the three deuterium atoms. The simultaneous presence of hopane and gammacerane derivatives seems a characteristic feature of the genus Bradyrhizobium and the phylogenetically closely related Rhodopseudomonas palustris.