Symbiosome Research Articles

Functional Analysis of Nodulin 26, an Aquaporin in Soybean Root Nodule Symbiosomes

Upon infection of soybean roots, nitrogen-fixing bacteria become enclosed in a specific organelle known as the symbiosome. The symbiosome membrane (SM) is a selectively permeable barrier that controls metabolite flux between the plant cytosol and the symbiotic bacterium inside. Nodulin 26 (NOD 26), a member of the aquaporin (AQP) water channel family, is a major protein component of the SM. Expression of NOD 26 in Xenopus oocytes gave a mercury-sensitive increase in osmotic water permeability (Pf). To define the biophysical properties of NOD 26 water channels in their native membranes, symbiosomes were isolated from soybean root nodules and the SM separated as vesicles from the bacteria. Permeabilities were measured using stopped-flow fluorimetry in SM vesicles with entrapped carboxyfluorescein. Osmotic water permeability (Pf) of SM was high, with a value of 0.05 +/- 0.003 cm/s observed at 20 degrees C (mean +/- S.E.; n = 15). Water flow exhibited a low activation energy, was inhibited by HgCl2 (0.1 mM), and exhibited a unit conductance of 3.2 +/- 1.3 x 10(-15) cm3/s, a value 30-fold lower than that of AQP 1, the red blood cell water channel. Diffusive water permeability (Pd) was 0.0024 +/- 0.0002 cm/s, and the resulting Pf to Pd ratio was 18.3, indicating that water crosses the SM in single file fashion via the NOD 26 water channel. In addition to high water permeability, SM vesicles also show high mercury-sensitive permeability to glycerol and formamide, but not urea, suggesting that NOD 26 also fluxes these solutes. Overall, we conclude that NOD 26 acts as a water channel with a single channel conductance that is 30-fold lower than AQP 1. Because the solutes that permeate NOD 26 are far larger than water, and water appears to cross the channel via a single file pathway, solute flux across NOD 26 appears to occur by a pathway that is distinct from that for water.

Comparative study of two cryotechniques to elucidate real functional aspects of legume nodules development

Legume nodules from Lupinus albus L. cv. Multolupa and Phaseolus vulgaris L. cv. Tormesina plants were subjected to two different cryotechniques (High Pressure Freezing, HPF, and Fast Freeze Fixation, FFF) in order to compare the structure obtained with respect to previous features and to deepen the relationships between the microsymbiont and the host cell in the native state. Comparing both cryotechniques, HPF yielded better general nodule ultrastructure than FFF. HPF allowed the observation of the native nodule structure since it permitted an excellent preservation. The result obtained showed that the peribacteroidal membrane was in close contact with the bacteroids, which is in contrast with the observations after chemical fixation. The narrow peribacteroid space observed between bacteroid and peribacteroid membranes appears as a dense matrix which is in agreement with the former knowledge on the existence of some enzymes and proteins in this space. O 2 -released by leghemoglobin will occur in close proximity to the bacteroid membranes. The spatial relationship between Golgi apparatus and ER with the peribacteroidal membrane observed along the development of the nodule indicates that these structures are very much related with the formation of the symbiosome membrane. HPF could be the right technique to continue studying real functional aspects in developing nodules.

Characterization of theGeosiphon pyriforme symbiosome by affinity techniques: confocal laser scanning microscopy (CLSM) and electron microscopy

Geosiphon pyriforme represents a photoautotrophic endosymbiosis of aGlomus-like fungus with the cyanobacteriumNostoc punctiforme. The fungus forms unicellular bladders of up to 2 mm in length and 0.5 mm in diameter growing on the soil surface and harboring the endosymbioticNostoc filaments. The cyanobacteria are located in a compartment (the symbiosome) bordered by a host membrane. The space between this symbiosome membrane (SM) and theNostoc cell wall is filled with an about 30–40 nm thick layer of amorphous material, which is present also in the regions of the symbiosome where noNostoc filaments are located. At these sites the amorphous material consists of a 20–30 nm thick layer separating the SM. The region between the SM and the cyanobacterium is defined as symbiosome space (SS). Fungal bladders, hyphae and free livingNostoc were analyzed by affinity techniques as well as the material occurring in the SS. FITC-coupled lectins with sugar specificity to α-D-mannosyl/α-D-glucosyl (Con A), N-acetyl-β-D-glucosamine oligomers (WGA), α-L-fucosyl (UEA-I), β-D-galactosyl (RCA-120), α-D-galactosyl (BS-I-B4), N-acetyl-α-D-galactosamine (HPA), and sialic acid (EBL) residues were tested. WGA binding and calcofluor white staining demonstrated that the bladder wall as well as the SS contain fibrillar chitin. Of the other lectins only Con A clearly labeled the symbiosome. On the contrary, the lectin binding properties of the slime produced by free livingNostoc-colonies indicate the presence of mannose, fucose, GalNAc, sialic acid, and galactose, while chitin or GlucNAc-oligomers could not be detected. The symbiosome was also investigated electron microscopically. WGA-gold binding confirmed the presence of chitin, while a slight PATAg reaction indicated some polysaccharidic molecules within the SS. Our results show that the amorphous material within the SS contains molecules typical of the fungal cell wall and suggest that the SM is related to the fungal plasma membrane. The applied lectins all bind to the hyphal surface, indicating a high molecular complexity. Mannosyl, β-galactosyl, and sialic acid residues are strongly exposed at the outer cell wall layer, whereas GlucNAc, GalNAc, and α-galactosyl residues seem to be present in smaller amounts. The symbiotic interface established between the fungus andNostoc inGeosiphon shows many similarities to that occurring between fungi and root cells in arbuscular mycorrhizas.

Effect of proline on nitrogenase activity in symbiosomes from root nodules of soybean (Glycine maxL.) subjected to drought stress

Experiments were carried out to investigate if drought stress affects the ability of bacteroids from soybean (Glycine max L.) root nodules to utilize proline and malate to support nitrogenase activity. The bacteroids were isolated in sub-ambient oxygen and nitrogenase activity was measured by acetylene reduction. Nitrogenase activity supported by proline was 8-fold higher in bacteroids from drought-stressed nodules than in bacteroids from control nodules. In contrast to the results with proline there was no significant response to drought stress in the rate of bacteroid nitrogenase activity supported by malate. The effect of drought stress on transport of proline and malate across the symbiosome membrane was investigated by incubation of symbiosomes isolated in sub-ambient oxygen with radioactive tracers. Drought stress tended to increase the rate of proline uptake relative to a minor decrease in malate uptake into symbiosomes in response to drought. There was no indication of a saturable carrier in the symbiosome membrane for either substrate at concentrations in the range 0.1-2 mM. The rate of malate uptake into symbiosomes was twice as high as the rate of proline uptake at all substrate levels tested. The protein composition of the symbiosome membrane was altered in response to drought stress and these changes may relate to the permeability of the symbiosome membrane.

The s29x gene of symbiotic bacteria in Amoeba proteus with a novel promoter

Gram − symbiotic bacteria (called X-bacteria), present in the xD strain of Amoeba proteus as required cell components, synthesize and export a large amount of a 29-kDa protein, S29x. S29x is exported into the host's cytoplasm across the bacterial membranes and the symbiosome membrane. The complete nucleotide (nt) sequence of the s29x gene of X-bacteria has been determined, and the promoter sequence and tsp have also been identified. The gene has a nonconventional promoter with putative nt sequences different from the known consensus sequences. When Escherichia coli cells are transformed with s29x, the gene is expressed and the product is secreted into the culture medium. Functions of S29x are not fully known, but it is suspected that S29x plays an important role in the symbiotic relationship between amoebae and X-bacteria.

Phosphorylation of Nodulin 26 on Serine 262 Affects Its Voltage-sensitive Channel Activity in Planar Lipid Bilayers

Nodulin 26 is an symbiosome membrane protein of soybean nodules that shows ion channel activity in planar lipid bilayers. Serine 262 of nodulin 26 is phosphorylated by calmodulin-like domain protein kinase. To study the effects of phosphorylation, nodulin 26 with Ser, Ala, or Asp at position 262 were expressed in Escherichia coli. The expressed protein possessed a histidine-rich leader sequence for purification by Ni2+ chelate fast protein liquid chromatography. Upon reconstitution into planar lipid bilayers, the recombinant proteins showed a large single channel conductance (3.1 nanosiemens (nS) in cis0.2M/trans1.0 M KCl and 1.6 nS in cis 0.2M/trans0.2 M KCl) and weak anion selectivity, similar to native soybean nodulin 26. Nodulin 26 with Ser- or Ala-262 occupied the maximal open conductance state greater than 97% of the time (3.1 nS in cis0.2M/trans1.0 M KCl) regardless of applied voltage. However, nodulin 26 with Asp-262 showed increased gating and preferential occupancy of lower subconductance states (1.8 and 0.6 nS in cis0.2M/trans1.0 M KCl) at high applied voltages (e.g. 70 mV). In situ phosphorylation of Ser-262 of nodulin 26 by calmodulin-like domain protein kinase also resulted in increased voltage-dependent gating and preferential occupancy of lower subconductance states. These results suggest that phosphorylation of serine 262 of nodulin 26 modulates channel activity by conferring voltage sensitivity.

Expression of soybean nodulin 26 in transgenic tobacco. Targeting to the vacuolar membrane and effects on floral and seed development.

Nodulin 26 is an integral membrane protein of the symbiosome membrane of nitrogen-fixing soybean nodules. We expressed a nodulin 26 cDNA in transgenic tobacco (TN26 tobacco) under the control of the cauliflower mosaic virus 35S promoter to study subcellular targeting and the physiological effect(s) of its expression. Based on Northern and Western blots, the expression of nodulin 26 mRNA and protein in transgenic plants is high in apical shoot sections, flowers, and stems, low in mature leaves, and absent in roots. Western blot analysis revealed high levels of transgenic nodulin 26 protein in tonoplast membranes. In contrast, nodulin 26 protein was not found in isolated plasma membranes, the soluble fraction, nor in chloroplast and mitochondria-enriched membrane fractions. About 50-60% of the flowers and pods from TN26 tobacco plants abscised prematurely. Seed capsule size and seed fill per capsule from the remainder of surviving flowers were about 50% of that of control plants. Pollen viability was found to be normal, but flowers from TN26 tobacco plants showed shorter anther filaments compared with control plants. Normal seed production and capsule size was restored by manually crossing the stigmas from TN26 plants with isolated pollen from either transgenic or control plants. Thus, the aberrant filament growth could have resulted in the reproductive defects associated with the plants.

Artificially Induced Senescence of Soybean Root Nodules Affects Different Polypeptides and Nodulins in the Symbiosome Membrane Compared to Physiological Ageing

Summary The central unit of all nitrogen-fixing nodules is the symbiosome, which can be considered as a temporary but independent nitrogen-fixing endosymbiotic organelle consisting of the bacteroid, the peribacteroid space and the surrounding symbiosome membrane. Ageing and induced senescence of this membrane were investigated with respect to its protein components. No qualitative changes in the protein and nodulin composition could be detected in mature and ageing root nodules. When root nodule senescence was induced by plant decapitation, the symbiosome membrane persisted as a structure over the examined period. However, in the symbiosome membrane some nodule-specific proteins (nodulins) were extensively degraded, suggesting the presence of selective proteolytic activities against the symbiosome membrane nodulins. Enhanced proteolytic activities were observed during induced nodule senescence but not during physiological ageing. During induced senescence, activities and protein levels of glutamine synthetase (GS) and nitrogenase (N 2 ase) were significantly reduced. Glutamine synthetase activity decreased to 50% of the initial activity, while the immunodetectable amount of the respective polypeptide disappeared completely.

Nodulin 26, a nodule-specific symbiosome membrane protein from soybean, is an ion channel.

Nodulin 26 is an integral symbiosome membrane protein of nitrogen-fixing soybean nodules. Nodulin 26 is a member of a family of structurally homologous membrane proteins with diverse transport functions. Thus, it has been proposed to be involved in symbiosome membrane transport. Despite this claim, there has not been any evidence that nodulin 26 has a transport activity. In this study, nodulin 26 was purified from soybean nodules by a non-denaturing protocol and was reconstituted into liposomes for channel studies in planar lipid bilayers. Nodulin 26 readily incorporated into bilayers, forming single channels with a maximum unitary conductance of 3.1 nanosiemens (nS) in a recording buffer of 20 mM 3-(N-morpholino)propanesulfonate-NaOH, pH 7.4, 1 M KCl. Nodulin 26 also exhibited multiple, discreet lower conductance states ranging from 0.5 to 2.5 nS. Nodulin 26 channels were voltage-sensitive. The maximal 3.1-nS state was preferentially occupied at lower applied voltages, whereas the lower conductance states were more frequently occupied at higher voltage potentials. Nodulin 26 channels transported both cations and anions, but showed a weak selectivity for anions. These results represent the first purification and functional characterization of the nodulin 26 channel and support a role for this protein in symbiosome membrane transport.

Bacterial endosymbiont-derived lipopolysaccharides and a protein on symbiosome membranes in newly infected amoebae and their roles in lysosome-symbiosome fusion.

Experimental results are presented to support the view that symbiont-derived lipopolysaccharides are involved in the prevention of lysosome-symbiosome fusion in xD amoebae harboring bacterial endosymbionts. Monoclonal antibodies against lipopolysaccharides and a 96-kDa protein present on symbiosome membranes of amoebae were used to monitor the appearance of the membrane-specific components in newly infected amoebae with endosymbionts from xD amoebae. The lipopolysaccharides and protein appeared on the newly forming symbiosome membranes within 3 to 7 days, as detected by indirect immunofluorescence staining with monoclonal antibodies. The lysosome-symbiosome fusion was followed by double staining of two antigens with different monoclonal antibodies applied to the same amoeba. Antilipopolysaccharide monoclonal antibodies were detected by staining with a fluorescein isothiocyanate-conjugated secondary antibody, and a biotinylated anti-lysosomal protein monoclonal antibody was detected by staining with Texas Red-conjugated streptavidin. In xD amoebae injected with an antilipopolysaccharide antibody, lysosomes fused with some of the symbiosomes that did not fuse with lysosomes in noninjected cells.

Bacterial endosymbiont-derived lipopolysaccharide and a protein on symbiosome membranes in newly infected amoeba and their roles in lysosome-symbiosome fusion

Bacterial endosymbiont-derived lipopolysaccharide and a protein on symbiosome membranes in newly infected amoeba and their roles in lysosome-symbiosome fusion

The symbiotic interface in an alga—invertebrate symbiosis

Both the cell membranes of the symbiotic alga Symbiodinium and the surrounding membranes of the sea anemone host Anemonia viridis have ATPase activity, as determined by enzyme cytochemistry. The interface between Symbiodinium and Anemonia also bears phosphatase activity, with substrate specificity for B glycerophosphate and a-naphthylphosphate. An immunocytochemical analysis of the distribution of the weak base -N-(3-[2,4-dinitrophenyl-amino]propyl)-N-(3-aminopropyl)methylamine dihydrochloride (DAMP) suggests that the membrane-bound compartment surrounding Symbiodinium cells is not markedly acidic. These data suggest that the symbiosome membrane of A. viridis may control the availability of phosphorous compounds and other nutrients to Symbiodinium . They are not readily compatible with the diffusion-depletion hypothesis, by which symbiont demand is hypothesized to be the primary determinant of the nutrient supply to the symbiont cells.

An Auxin Binding Protein is Localized in the Symbiosome Membrane of Soybean Nodules

Binding of tritiated indole-3-acetic acid ([3H]IAA) to symbiosome membranes of soybean nodules occurred in a protein-dependent manner and was competitively inhibited by unlabeled indole-3-acetic acid (IAA), 1-naphthaleneacetic acid (1-NAA) and dithiothreitol (DTT), but not by tryptophan and benzoic acid. The symbiosome membranes bound IAA with a KD of 1 × 10-6 m. Photoaffinity labeling identified an auxin-binding protein (ABP) in the symbiosome membrane with an apparent molecular mass of 23 kDa. This 23 kDa protein was labeled either with 5-azido-[7-3H]indole-3-acetic acid ([3H]N3IAA) or with 5′-azido-[3,6-3H2]-1-naphthylphthalamic acid ([3H2]N3NPA). Labeling of the 23 kDa protein with [3H]N3IAA was competitively inhibited by unlabeled IAA and 1-NAA. NPA and quercetin, inhibitors of polar auxin transport, as well as rutin, a glycosylated derivative of quercetin, competed with IAA for binding. Conversely, [3H2]N3NPA labeling was inhibited by unlabeled IAA and NPA. The 23 kDa symbiosome membrane protein was partially solubilized with Triton X-100 and nearly completely using Triton X-114. The observation that auxin transport inhibitors compete with IAA for binding suggests that the symbiosome membrane ABP could be part of an auxin efflux carrier system required to control the auxin concentration in infected soybean nodule cells.

Determination of the site of phosphorylation of nodulin 26 by the calcium-dependent protein kinase from soybean nodules.

Nodulin 26 is a nodule-specific protein that is associated with the symbiosome membrane of soybean root nodules. Nodulin 26 is an endogenous substrate for a novel calcium-dependent protein kinase (CDPK) of soybean root nodules. By phosphopeptide mapping of endoproteinase Lys-C-digested nodulin 26 and automated and manual peptide sequence analyses, we have identified the site on nodulin 26 phosphorylated by CDPK. We have also established that the phosphorylation site of nodulin 26 is identical to the phosphorylation site of CK-15, a synthetic peptide with the carboxyl-terminal sequence of nodulin 26. The phosphorylation of nodulin 26 occurs at position Ser262, and the phosphorylation of CK-15 occurs at the analogous position, Ser,6 in vitro. Thus, the CK-15 sequence apparently contains sufficient structural features of the phosphorylation site of nodulin 26 to be recognized by CDPK. On the basis of peptide mapping analysis of nodulin 26 from nodules that are metabolically labeled with [32P]phosphate, it appears that the site of nodulin 26 that is phosphorylated in vitro is also labeled in vivo. The data indicate that the carboxyl terminus of nodulin 26 is phosphorylated by CDPK and provide initial sequence data for the phosphorylation site of an endogenous substrate for a plant CDPK.

Formation of Novel Polysaccharides by Bradyrhizobium japonicum Bacteroids in Soybean Nodules

Certain strains of Bradyrhizobium japonicum form a previously unknown polysaccharide in the root nodules of soybean plants (Glycine max (L.) Merr.). The polysaccharide accumulates inside of the symbiosome membrane-the plant-derived membrane enclosing the bacteroids. In older nodules (60 days after planting), the polysaccharide occupies most of the symbiosome volume and symbiosomes become enlarged so that there is little host cytoplasm in infected cells. The two different groups of B. japonicum which produce different types of polysaccharide in culture produce polysaccharides of similar composition in nodules. Polysaccharide formed by group I strains (e.g., USDA 5 and USDA 123) is composed of rhamnose, galactose, and 2-O-methylglucuronic acid, while polysaccharide formed by group II strains (e.g., USDA 31 and USDA 39) is composed of rhamnose and 4-O-methylglucuronic acid. That the polysaccharide is a bacterial product is indicated by its composition plus the fact that polysaccharide formation is independent of host genotype but is dependent on the bacterial genotype. Polysaccharide formation in nodules is common among strains in serogroups 123, 127, 129, and 31, with 27 of 39 strains (69%) testing positive. Polysaccharide formation in nodules is uncommon among other B. japonicum serogroups, with only 1 strain in 18 (6%) testing positive.

Macromolecules Involved in the Amoeba‐Bacteria Symbiosis1

ABSTRACT. In the Amoeba‐bacteria symbiosis, rod‐shaped Gram‐negative bacterial endosymbionts reside within symbiosomes in the host cytoplasm, and the host and symbionts are mutually dependent for survival. Three proteins and one group of lipopolysaccharides (LPS) synthesized by the bacterial endosymbionts and two proteins derived from the host cells have been found to be involved in the host‐symbiont interactions, although their respective roles are not yet fully known. The symbiont‐derived molecules included proteins with molecular weights of 29 kDa, 67 kDa and 96 kDa and LPS. The 29‐kDa protein was most abundant in the host cytoplasm, while the 96‐kDa protein and LPS were found mostly on the symbiosome membranes. The 67‐kDa protein was a GroEL analog and stayed within the symbionts. The host‐derived 43‐kDa protein, actin, was selectively accumulated by the symbionts, while the 220/225‐kDa protein, spectrin, was attached to the symbiosome membranes. The symbiont genes coding for the 29‐kDa and 67‐kDa proteins were cloned and sequenced. The 29‐kDa protein gene was unique with no relation to any known DNA sequences but has a leucine zipper‐like motif, suggesting a possible DNA‐binding function. The DNA sequence of the 67‐kDa protein gene showed a 70% identity with heat‐shock‐protein genes of Escherichia coli and Coxiella burnetii.

Bacterial‐Endosymbiote‐Derived Lipopolysaccharides on Amoeba Symbiosome Membranes

ABSTRACT. Monoclonal antibodies were obtained against lipopolysaccharides of Gram‐negative bacterial endosymbiotes in amoebae and were used to determine the origin and distribution of the antigens. The lipopolysaccharides were located on endosymbiote's surfaces as determined by indirect immunofluorescence staining under both light and electron microscopes. In addition, host symbiosome membranes enclosing the endosymbiotes also contained the same lipopolysaccharides on the cytoplasmic side as shown by indirect immunofluorescence staining after intracellular injection of anti‐lipopolysaccharide antibodies. This is the first report for the presence of symbiote‐derived lipopolysaccharides on the host symbiosome membranes, and their possible roles are discussed in relation to the prevention of lysosomal fusion with symbiosomes.

Are symbiotic algae nutrient deficient?

The evidence for the hypothesis that symbionts in alga−invertebrate associations are nutrient deficient is critically evaluated. It is suggested that the evidence is not consistent with algal symbionts being nutrient deficient, but is consistent with the host regulating its population of symbionts by restricting their supply of nutrients. This distinction is due to the substantial release of photosynthate by symbiotic algae and suggests that there are (at least) two distinct regulatory components involved in the characteristics of nutrient-deficient algal cells.

Calcium-Dependent Phosphorylation of Symbiosome Membrane Proteins from Nitrogen-Fixing Soybean Nodules

By using a peptide (CK-15) based on the COOH-terminal sequence of nodulin-26, we have demonstrated the presence of a Ca(2+)-dependent protein kinase in soluble as well as particulate fractions of nitrogen-fixing soybean (Glycine max) root nodules. Substantial enzyme activity was found in symbiosome membranes. The soluble enzyme was purified 1570-fold. The enzyme was fractionated from endogenous calmodulin and yet was fully activated by Ca(2+) (K(0.5) = 0.4 micromolar) in the absence of exogenous calmodulin, phosphatidylserine and 1,2-dioleylglycerol, oleic acid, and platelet activating factor. CK-15 was used to generate a site-specific antibody to nodulin-26. The antibody reacted with a protein in the symbiosome membrane with an apparent molecular mass of 27,000 daltons, consistent with the molecular mass predicted for nodulin-26 from the deduced amino acid sequence. A symbiosome membrane protein with an identical electrophoretic mobility was phosphorylated in vitro in a Ca(2+)-dependent manner. Additionally, this symbiosome membrane protein was phosphorylated when nodules were incubated with (32)P-phosphate. Overall, the results show the existence of a Ca(2+)-dependent and calmodulin/lipid-independent enzyme in nitrogen-fixing soybean root nodules and suggest that nodulin-26 is a substrate for Ca(2+)-dependent phosphorylation.

A spectrin-like protein present on membranes of Amoeba proteus as studied with monoclonal antibodies

Monoclonal antibodies against a spectrin-like membrane-associated protein of xD amoebae ( Amoeba proteus) were used to determine the distribution of the protein and some of its characteristics. A total of 34 monoclonal antibodies recognizing different epitopes of the protein were obtained, of which seven stained cell membranes by indirect immunofluorescence. The spectrin-like protein had two subtypes of 225 and 220 kDa and several monoclonal antibodies cross-reacted with human erythrocyte spectrin when checked by indirect immunofluorescence staining and immunoblotting. Some of the antibodies also cross-reacted with antigens in HeLa cells and chick embryo fibroblasts. Polyclonal and monoclonal antibodies against Drosophila and human erythrocyte spectrins cross-reacted with the spectrin-like protein from amoebae. On the basis of these results, it was concluded that the protein is a spectrin. The protein was found on most cellular membranes of amoebae, including the plasma, nuclear, and phagosomal membranes, as well as symbiosome membranes.