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.     

Common components of the infection thread matrix and the intercellular space identified by immunocytochemical analysis of pea nodules and uninfected roots

Three rat hybridoma cell lines have been isolated which produce monoclonal antibodies identifying a noduleenhanced, soluble component of Pisum sativum root nodules. These antibodies each recognized a protease-sensitive band (M_r 95K) on SDS-polyacrylamide gels. The 95K antigen was resolved by isoelectric focusing into acidic and neutral components which were separately detected by AFRC MAC 236 and MAC 265 respectively. The third antibody (MAC 204) reacted with both acidic and neutral components through an epitope that was sensitive to periodate oxidation. These monoclonal antibodies were used for immunogold localizations at light and electron microscopic levels. In each case, the antigen was shown to be present in the matrix that surrounds the invading rhizobia in infection threads and infection droplets, as well as in the intercellular spaces between plant cell walls of nodules and also of uninfected roots. By contrast, a fourth monoclonal antibody, AFRC JIM 5, labelled a pectic component in the walls of infection threads, and JIM 5 was also found to label the middle lamella of plant cell walls, especially at three-way junctions between cells. The composition and structure of the infection thread lumen is thus comparable to that of an intercellular space.     

Immunocytological evidence for abnormal symbiosome development in nodules of the pea mutant line Sprint2Fix− (sym31)

Summary Using a series of antibody probes as markers of symbiosome development, we have investigated the impaired development of symbiosomes in nodules formed by the plant mutant line Sprint2Fix⁻ (sym31). In wild-type pea (Pisum sativum L.) nodules, bacteria differentiate into large pleiomorphic, nitrogen-fixing bacteroids and are singly enclosed within a peribacteroid membrane. In thesym31 mutant, several small undifferentiated bacteroids were often enclosed within one peribacteroid membrane, or were found within a vacuole-like compartment. In wild-type nodules, the monoclonal antibody JIM18, which recognizes a plasmalemma glycolipid antigen, bound to the juvenile peribacteroid membrane, and did not recognize the mature peribacteroid membrane. However, in the mutant, the antibody bound to all peribacteroid membranes within the nodule, suggesting that differentiation of the peribacteroid membrane was arrested. Another antibody, MAC266, recognized plant glycoproteins which normally accumulate in symbiosomes at a late stage of nodule development. Binding of this antibody was much reduced within mutant nodules, labelling only a few mature cells. Similarly, MAC301, which normally recognizes a lipopolysaccharide epitope expressed on differentiated bacteroids prior to the induction of nitrogenase, failed to react with rhizobial cell extracts isolated from nodules of thesym31 mutant. On the basis of these developmental markers, the symbiosomes ofsym31 nodules appeared to be blocked at an early stage of development. The distribution of infection structures was also found to be abnormal in the mutant nodules. Models of symbiosome development are presented and discussed in relation to the morphological and developmental lesions observed in thesym31 mutant.     

Chemical characterization of pH-dependent structural epitopes of lipopolysaccharides from Rhizobium leguminosarum biovar phaseoli.

Lipopolysaccharide (LPS) was isolated from free-living Rhizobium leguminosarum bv. phaseoli CE3 cells grown at pH 4.8 (antigenically similar to bacteroid LPS) and compared with that from cells grown at pH 7.2 (free-living bacteria). Composition analysis revealed that pH 7.2 LPS differs from pH 4.8 LPS in that 2,3,4-tri-O-methylfucose is replaced by 2,3-di-O-methylfucose. The amount of 2-O-methylrhamnose is greater in the pH 4.8 LPS than in the pH 7.2 LPS. Analysis of the structural components of LPS (O-chain polysaccharide, core oligosaccharides, and the lipid A) revealed that all the composition differences in the various LPSs occur in the O-chain polysaccharide. These structural variations between pH 4.8 and pH 7.2 LPSs provide a chemical basis for the observed lack of cross-reactivity with pH 4.8 LPS of two monoclonal antibodies, JIM28 and JIM29, raised against free-living bacteria grown at pH 7.2. An LPS preparation isolated from bacteroids contained both 2,3,4-tri-O- and 2,3-di-O-methylfucose residues. This result is consistent with the finding that the two monoclonal antibodies react weakly with bacteroid LPS. It is concluded that methylation changes occur on the LPS O-chain of R. leguminosarum bv. phaseoli when the bacteria are grown at low pH and during nodule development.     

Engineering the Rhizobium leguminosarum bv. viciae hydrogenase system for expression in free-living microaerobic cells and increased symbiotic hydrogenase activity

Rhizobium leguminosarum bv. viciae UPM791 induces hydrogenase activity in pea (Pisum sativum L.) bacteroids but not in free-living cells. The symbiotic induction of hydrogenase structural genes (hupSL) is mediated by NifA, the general regulator of the nitrogen fixation process. So far, no culture conditions have been found to induce NifA-dependent promoters in vegetative cells of this bacterium. This hampers the study of the R. leguminosarum hydrogenase system. We have replaced the native NifA-dependent hupSL promoter with the FnrN-dependent fixN promoter, generating strain SPF25, which expresses the hup system in microaerobic free-living cells. SPF25 reaches levels of hydrogenase activity in microaerobiosis similar to those induced in UPM791 bacteroids. A sixfold increase in hydrogenase activity was detected in merodiploid strain SPF25(pALPF1). A time course induction of hydrogenase activity in microaerobic free-living cells of SPF25(pALPF1) shows that hydrogenase activity is detected after 3 h of microaerobic incubation. Maximal hydrogen uptake activity was observed after 10 h of microaerobiosis. Immunoblot analysis of microaerobically induced SPF25(pALPF1) cell fractions indicated that the HupL active form is located in the membrane, whereas the unprocessed protein remains in the soluble fraction. Symbiotic hydrogenase activity of strain SPF25 was not impaired by the promoter replacement. Moreover, bacteroids from pea plants grown in low-nickel concentrations induced higher levels of hydrogenase activity than the wild-type strain and were able to recycle all hydrogen evolved by nodules. This constitutes a new strategy to improve hydrogenase activity in symbiosis.     

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.     

A comparison of DNA from free living and endosymbiotic Rhizobium leguminosarum (strain PRE).

1. Bacteroids of Rhizobium leguminosarum (strain PRE) purified from root nodules of Pisum sativum (var. 'Rondo') by the standard procedure of differential centrifugation contained considerable contamination of mitochondrial material. This could be removed by incubation of the bacteroid preparation with 1 M KCl/1% deoxycholate. 2. The DNA content of bacteroid cells of R. leguminosarum was found to have increased about three fold in comparison with the DNA content of free living R. leguminosarum bacteria. 3. No significant difference in DNA composition of free living R. leguminosarum bacteria and bacteroids could be detected by CsCl equilibrium centrifugation, RNA - DNA hybridization and DNA - DNA reassociation studies.     

DNA content of free living rhizobia and bacteroids of various Rhizobium-legume associations

The DNA content of bacteroids from 22 different Rhizobium-legume associations was compared to that of the corresponding free living Rhizobium species using laser flow microfluorometry. In all 18 effective associations, the bacteroids had either similar or higher DNA content than the free living rhizobia. Bacteroid populations isolated from effective clover (Trifolium repens) and alfalfa (Medicago sativa) nodules had an average DNA content of >1.5-fold higher than free living R. trifolii and R. meliloti. These populations also contained a significant number of bacteroids with more than 3-fold the DNA content of the free living rhizobia. Populations isolated from effective nodules of winged beans (Psophocarpus tetragonolobus), peas (Pisum sativum), and mung beans (Phaseolus aureus) had an average DNA content of 1.1- to 1.5-fold higher than free living R. “cowpeas” and R. leguminosarum. Bacteroids from nodules of lupins (Lupinus angustifolius and L. minaretta), kidney beans (Phaseolus vulgaris), and soybeans (Glycine max), however, had similar DNA content to the free living forms. Two of the four associations which formed ineffective nodules contained bacteroids with lower DNA content than the free living rhizobia. The other two associations contained bacteroids with slightly higher or similar DNA content to the free living rhizobia. Nodules of the ineffective associations also did not contain leghemoglobin.     

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.     

Immunological characterization of Rhizobium leguminosarum outer membrane antigens by use of polyclonal and monoclonal antibodies.

Surface antigens of Rhizobium leguminosarum biovar viciae strain 248 were characterized by using polyclonal and monoclonal antibodies. With Western immunoblotting as the criterion, an antiserum raised against living whole cells recognized mainly flagellar antigens and the O-antigen-containing part of the lipopolysaccharide (LPS). Immunization of mice with a peptidoglycan-outer membrane complex yielded eight monoclonal antibodies, of which three reacted with LPS and five reacted with various sets of outer membrane protein antigens. The observation that individual monoclonal antibodies react with sets of related proteins is discussed. Studies of the influence of calcium deficiency and LPS alterations on surface antigenicity showed that in normally grown wild-type cells, the O-antigenic side chain of LPS blocks binding of an antibody to a deeper-lying antigen. This antigen is accessible to antibodies in cells grown under calcium limitation as well as in O-antigen-lacking mutant cells. Two of the antigen groups which can be distinguished in cell envelopes of free-living bacteria were depleted in cell envelopes of isolated bacteroids, indicating that the monoclonal antibodies could be useful tools for studying the differentiation process from free-living bacteria to bacteroids.     

Rhizobium etli CE3 bacteroid lipopolysaccharides are structurally similar but not identical to those produced by cultured CE3 bacteria

Rhizobium etli CE3 bacteroids were isolated from Phaseolus vulgaris root nodules. The lipopolysaccharide (LPS) from the bacteroids was purified and compared with the LPS from laboratory-cultured R. etli CE3 and from cultures grown in the presence of anthocyanin. Comparisons were made of the O-chain polysaccharide, the core oligosaccharide, and the lipid A. Although LPS from CE3 bacteria and bacteroids are structurally similar, it was found that bacteroid LPS had specific modifications to both the O-chain polysaccharide and lipid A portions of their LPS. Cultures grown with anthocyanin contained modifications only to the O-chain polysaccharide. The changes to the O-chain polysaccharide consisted of the addition of a single methyl group to the 2-position of a fucosyl residue in one of the five O-chain trisaccharide repeat units. This same change occurred for bacteria grown in the presence of anthocyanin. This methylation change correlated with the inability of bacteroid LPS and LPS from anthocyanin-containing cultures to bind the monoclonal antibody JIM28. The core oligosaccharide region of bacteroid LPS and from anthocyanin-grown cultures was identical to that of LPS from normal laboratory-cultured CE3. The lipid A from bacteroids consisted exclusively of a tetraacylated species compared with the presence of both tetra- and pentaacylated lipid A from laboratory cultures. Growth in the presence of anthocyanin did not affect the lipid A structure. Purified bacteroids that could resume growth were also found to be more sensitive to the cationic peptides, poly-l-lysine, polymyxin-B, and melittin.     

Symbiotic properties of C4-dicarboxylic acid transport mutants of Rhizobium leguminosarum.

The transport of succinate was studied in bacteroids of an effective, streptomycin-resistant strain (GF160) of Rhizobium leguminosarum. High levels of succinate transport occurred, and the kinetics, specificity, and sensitivity to metabolic inhibitors were similar to those previously described for free-living cells. The symbiotic properties of two transposon (Tn5)-mediated C4-dicarboxylate transport mutants (strains GF31 and GF252) were determined. Strain GF31 formed ineffective nodules, and bacteroids from these nodules showed no succinate transport activity. Strain GF252 formed partially effective nodules, and bacteroids from these nodules showed about 50% of the succinate transport activity of the parent bacteroids. Another dicarboxylic acid transport mutant (Dct-), strain GFS5, isolated after N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis, formed ineffective nodules. The ability to form ineffective nodules in strains GF31 and GFS5 was shown to correlate with the Dct- phenotype. The data indicate that the presence of a functional C4-dicarboxylic acid transport system is essential for N2 fixation to occur in pea nodules.     

Isolation of monoclonal antibodies reacting with peribacteriod membranes and other components of pea root nodules containing Rhizobium leguminosarum

Plant and bacterial antigens contributing to nodule development and symbiosis in pea (Pisum sativum L.) roots were identified after isolation of a set of monoclonal antibody (McAb)-producing hybridoma lines. Rats were immunised with the peribacteriod material released by mild osmotic shock treatment from membrane-enclosed bacteroids of Rhizobium leguminosarum bv. viceae. In order to diversify the range of McAb specificities, this material was either used as immunogen directly (method 1), or after immunodepletion of a set of glycoprotein and lipopolysaccharide antigens (method 2), or after deglycosylation (method 3). After fusion and screening of cloned hybridoma lines, these three immunisation methods gave respectively 4, 2 and 1 classes of McAb with unique antigen specificities. Ultrastructural immunogold localisation studies showed four different antigens to be present on peribacteriod and plasma membranes (identified by MAC 64, 202, 206 or 209); in addition, a glycoprotein of plant origin but present in the infection-thread matrix was identified by MAC 204. Although none of the epitopes recognised by these McAb was nodule-specific, several were found to be more abundant in extracts of nodule tissue than in uninfected roots (MAC 64, 202, 204, 206). Two McAb reacted with new bacterial antigens: MAC 203 identified a bacterial antigen expressed upon infection but not in free-living cultures of Rhizobium, and MAC 115 identified a bacterial polypeptide (55 kdaltons) that was present in both free-living and bacteroid forms. There were also some McAb of broader specificity that react with antigens present in both plant and bacterial cytoplasms.Abbreviations ELISA enzyme-linked immunosorbent assay - Ig inmunoglobulin - kDa kilodalton - LPS lipopolysaccharide - McAb monoclonal antibody - PBM peribacteroid membrane - SDS-PAGE sodium dodecyl sulfate-polyacryl-amide gel electrophoresis - TFMS trifluoromethane sulfonic acid     

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.     

Ammonia assimilation by rhizobium cultures and bacteroids.

The enzymes involved in the assimilation of ammonia by free-living cultures of Rhizobium spp. are glutamine synthetase (EC. 6.o.I.2), glutamate synthase (L-glutamine:2-oxoglutarate amino transferase) and glutamate dehydrogenase (ED I.4.I.4). Under conditions of ammonia or nitrate limitation in a chemostat the assimilation of ammonia by cultures of R. leguminosarum, R. trifolii and R. japonicum proceeded via glutamine synthetase and glutamate synthase. Under glucose limitation and with an excess of inorganic nitrogen, ammonia was assimilated via glutamate dehydrogenase, neither glutamine synthetase nor glutamate synthase activities being detected in extracts. The coenzyme specificity of glutamate synthase varied according to species, being linked to NADP for the fast-growing R. leguminosarum, R. melitoti, R. phaseoli and R. trifolii but to NAD for the slow-growing R. japonicum and R. lupini. Glutamine synthetase, glutamate synthase and glutamate dehydrogenase activities were assayed in sonicated bacteroid preparations and in the nodule supernatants of Glycine max, Vicia faba, Pisum sativum, Lupinus luteus, Medicago sativa, Phaseolus coccineus and P. vulgaris nodules. All bacteroid preparations, except those from M. sativa and P. coccineus, contained glutamate synthase but substantial activities were found only in Glycine max and Lupinus luteus. The glutamine synthetase activities of bacteroids were low, although high activities were found in all the nodule supernatants. Glutamate dehydrogenase activity was present in all bacteroid samples examined. There was no evidence for the operation of the glutamine synthetase/glutamate synthase system in ammonia assimilation in root nodules, suggesting that ammonia produced by nitrogen fixation in the bacteroid is assimilated by enzymes of the plant system.     

Isoenzymes of Superoxide Dismutase in Nodules of Phaseolus vulgaris L., Pisum sativum L., and Vigna unguiculata (L.) Walp

The activity and isozymic composition of superoxide dismutase (SOD; EC 1.15.1.1) were determined in nodules of Phaseolus vulgaris L., Pisum sativum L., and Vigna unguiculata (L.) Walp. formed by Rhizobium phaseoll 3622, R. Ieguminosarum 3855, and Bradyrhizobium sp. BR7301, respectively. A Mn-SOD was present in Rhizobium and two in Bradyrhizobium and bacteroids. Nodule mitochondria from all three legume species had a single Mn-SOD with similar relative mobility, whereas the cytosol contained several CuZn-SODs: two in Phaseolus and Pisum, and four in Vigna. In the cytoplasm of V. unguiculata nodules, a Fe-containing SOD was also present, with an electrophoretic mobility between those of CuZn- and Mn-SODs, and an estimated molecular weight of 57,000. Total SOD activity of the soluble fraction of host cells, expressed on a nodule fresh weight basis, exceeded markedly that of bacteroids. Likewise, specific SOD activities of free-living bacteria were superior or equal to those of their symbiotic forms. Soluble extracts of bacteria and bacteroids did not show peroxidase activity (EC 1.11.1.7), but the nodule cell cytoplasm contained diverse peroxidase isozymes which were readily distinguishable from leghemoglobin components by electrophoresis. Data indicated that peroxidases and leghemoglobins did not significantly interfere with SOD localization on gels. Treatment with chloroform-ethanol scarcely affected the isozymic pattern of SODs and peroxidases, and had limited success in the removal of leghemoglobin.     

Cloning and Mutagenesis of a Cytochrome P-450 Locus from Bradyrhizobium japonicum That Is Expressed Anaerobically and Symbiotically

Cytochromes P-450, which in many organisms participate in the metabolism of a variety of endobiotic and xenobiotic substances, are synthesized by symbiotic bacteroids of Bradyrhizobium japonicum. Polyclonal antibodies were raised against two cytochromes P-450 (CYP112 and CYP114) purified from bacteroids. A lambda gt11 expression clone of B. japonicum USDA 110 DNA that reacted with the anti-CYP112 antibody was obtained and was used to screen a library of USDA 110 genomic DNA in pLAFR1 for a clone of the P-450 locus. Forced expression of subclones of the P-450 locus in Escherichia coli produced polypeptides that reacted with either the anti-CYP112 antibody or the anti-CYP114 antibody; no cross-reactivity was evident. A Western blot (immunoblot) analysis showed that neither protein was present in free-living aerobically grown B. japonicum cells, but that both proteins were present in cells grown anaerobically, as well as in bacteroids. A mutant strain disrupted in the CYP112 locus produced neither CYP112 nor CYP114, indicating that the mutation was polar for CYP114. The mutant produced effective nodules on soybeans, even though the bacteroids contained no detectable P-450. This suggests that the cytochromes P-450 which we examined are not involved in an essential symbiotic function.     

Cell surface interactions of Rhizobium bacteroids and other bacterial strains with symbiosomal and peribacteroid membrane components from pea nodules

Samples of Rhizobium bacteroids isolated from pea nodule symbiosomes reacted positively with a monoclonal antibody recognizing N-linked glycan epitopes on plant glycoproteins associated with the peribacteroid membrane and peribacteroid fluid. An antiserum recognizing the symbiosomal lectin-like glycoprotein PsNLEC-1 also reacted positively. Samples of isolated bacteroids also reacted with an antibody recognizing a glycolipid component of the peribacteroid membrane and plasma membrane. Bacterial cells derived from free-living cultures then were immobilized on nitrocellulose sheets and tested for their ability to associate with components of plant extracts derived from nodule fractionation. A positive antibody-staining reaction indicated that both PsNLEC-1 and membrane glycolipid had become associated with the bacterial surface. A range of rhizobial strains with mutants affecting cell surface polysaccharides all showed similar interactions with PsNLEC-1 and associated plant membranes, with the exception of strain B659 (a deep-rough lipopolysaccharide mutant of Rhizobium leguminosarum). However, the presence of a capsule of extracellular polysaccharide apparently prevented interactions between rhizobial cells and these plant components. The importance of a close association between peribacteroid membranes, PsNLEC-1, and the bacterial surface is discussed in the context of symbiosome development.     

The effect of ammonium nitrate on the synthesis of nitrogenase and the concentration of leghemoglobin in pea root nodules induced by Rhizobium leguminosarum

The effects of NH4NO3 on the development of root nodules of Pisum sativum after infection with Rhizobium leguminosarum (strain PRE) and on the nitrogenase activity of the bacteroids in the nodule tissue were studied. The addition of NH4NO3 decreased the nitrogenase activity measured on intact nodules. This reduction of nitrogen fixation did not result from a reduced number of bacteroids or a decreased amount of bacteroid proteins per gram of nodule. The synthesis of nitrogenase, measured as the relative amount of incorporation of [35S]sulfate into the components I and II of nitrogenase was similarly not affected. The addition of NH4NO3 decreased the amount of leghemoglobin in the nodules and there was a quantitative correlation between the leghemoglobin content and the nitrogen-fixing capacity of the nodules. The conclusion is that the decrease of nitrogen-fixing capacity is caused by a decrease of the leghemoglobin content of the root nodules and not by repression of the nitrogenase synthesis.