The soybean NRAMP homologue,GmDMT1, is a symbiotic divalent metal transporter capable of ferrous iron transport

Iron is an important nutrient in N2-fixing legume root nodules. Iron supplied to the nodule is used by the plant for the synthesis of leghemoglobin, while in the bacteroid fraction, it is used as an essential cofactor for the bacterial N2-fixing enzyme, nitrogenase, and iron-containing proteins of the electron transport chain. The supply of iron to the bacteroids requires initial transport across the plant-derived peribacteroid membrane, which physically separates bacteroids from the infected plant cell cytosol. In this study, we have identified Glycine max divalent metal transporter 1 (GmDmt1), a soybean homologue of the NRAMP/Dmt1 family of divalent metal ion transporters. GmDmt1 shows enhanced expression in soybean root nodules and is most highly expressed at the onset of nitrogen fixation in developing nodules. Antibodies raised against a partial fragment of GmDmt1 confirmed its presence on the peribacteroid membrane (PBM) of soybean root nodules. GmDmt1 was able to both rescue growth and enhance 55Fe(II) uptake in the ferrous iron transport deficient yeast strain (fet3fet4). The results indicate that GmDmt1 is a nodule-enhanced transporter capable of ferrous iron transport across the PBM of soybean root nodules. Its role in nodule iron homeostasis to support bacterial nitrogen fixation is discussed.     

Characterisation by proteomics of peribacteroid space and peribacteroid membrane preparations from pea (Pisum sativum) symbiosomes

The legume Rhizobium symbiosis leads to the formation of a new compartment in the plant cell, the symbiosome. This compartment harbours the bacteroids surrounded by a peribacteroid membrane (PBM) originating from the plant plasma membrane. The PBM and the space between the PBM and the bacteroid membrane, called peribacteroid space (PS), mediate the exchange of metabolites between the symbionts. Proteome analysis was used as an approach to characterise the proteins in the PBM and the PS. A standard differential centrifugation procedure including a Percoll gradient was used for symbiosome isolation from pea root nodules. Proteins in the PBM and PS fractions obtained from the symbiosomes were separated by two-dimensional gel electrophoresis, and 89 spots were analysed by tandem mass spectrometry. The proteins of 46 spots could be identified by database search. The results showed that PS and even PBM preparations from pea symbiosomes always contain abundant amounts of bacteroid proteins as a contaminate. Interestingly, in addition to a few PS/PBM proteins a number of endomembrane proteins (less likely representing a contaminate), including V-ATPase, BIP, and an integral membrane protein known from COPI-coated vesicles, were found in the PBM fraction, supporting the role of the endomembrane system in PBM biogenesis.     

ATPase activity and anion transport across the peribacteroid membrane of isolated soybean symbiosomes

Addition of ATP to intact symbiosomes isolated from soybean nodules, resulted in generation of a membrane potential (positive inside) across the peribacteroid membrane (PBM). This energisation was monitored as oxonol fluorescence quenching. The rate of fluorescence quenching was inhibited by the inclusion of permeant anions in the reaction medium. Using this inhibition as a measure of anion uptake across the PBM, the presence of a phthalonate-sensitive dicarboxylate carrier on the PBM was confirmed. Following dissipation of the membrane potential by a permeant anion, a pH gradient, measured using [¹⁴C]methylamine uptake, was slowly established across the PBM. This pH was abolished by addition of an uncoupler but was insensitive to inhibitors of bacteroid respiration. The difference in pH between the external medium and the symbiosome interior was estimated to be in the range of 1–1.6 pH units. The magnitude in planta will depend on the concentrations of ATP and permeant anions in the cytosol of the host cell.Abbreviations PBM peribacteroid membrane - electrical membrane potential - MA methylamine The term symbiosome refers to the peribacteroid unit consisting of bacteroids enclosed in the host-derived peribacteroid membrane     

Ferrous iron is transported across the peribacteroid membrane of soybean nodules

Symbiosomes and bacteroids isolated from soybean (Glycine max Merr.) nodules are able to take up ferrous iron. This uptake activity was completely abolished in the presence of ferrous-iron chelators. The kinetics of uptake were characterized by initially high rates of iron internalization, but no saturation was observed with increasing iron concentration. This process does not appear to involve the ferric reductase of the peribacteroid membrane. The transport of ferrous iron was inhibited by other transition metals, particularly copper. Ferrous iron was taken up by symbiosomes more efficiently than the ferric form. This indicates that the iron transport from the plant host cell to the microsymbiont in vivo may occur mainly as the ferrous form. Received: 11 February 1998 / Accepted: 29 May 1998     

Localization of H+-ATPases in soybean root nodules

The localization of H⁺-ATPases in soybean (Glycine max L. cv. Stevens) nodules was investigated using antibodies against both P-type and V-type enzymes. Immunoblots of peribacteroid membrane (PBM) proteins using antibodies against tobacco and Arabidopsis H⁺-ATPases detected a single immunoreactive band at approximately 100 kDa. These antibodies recognized a protein of similar relative molecular mass in the crude microsomal fraction from soybean nodules and uninoculated roots. The amount of this protein was greater in PBM from mature nodules than in younger nodules. Immunolocalization of P-type ATPases using silver enhancement of colloidal-gold labelling at the light-microscopy level showed signal distributed around the periphery of non-infected cells in both the nodule cortex and nodule parenchyma. In the central nitrogen-fixing zone of the nodule, staining was present in both the infected and uninfected cells. Examination of nodule sections using confocal microscopy and fluorescence staining showed an immunofluorescent signal clearly visible around the periphery of individual symbiosomes which appeared as vesicles distributed throughout the infected cells of the central zone. Electron-microscopic examination of immunogold-labelled sections shows that P-type ATPase antigens were present on the PBM of both newly formed, single-bacteroid symbiosomes just released from infection threads, and on the PBM of mature symbiosomes containing two to four bacteroids. Immunogold labelling using antibody against the B-subunit of V-type ATPase from oat failed to detect this protein on symbiosome membranes. Only a very faint signal with this antibody was detected on Western blots of purified PBM. During nodule development, fusion of small symbiosomes to form larger ones containing multiple bacteroids was observed. Fusion was preceded by the formation of cone-like extensions of the PBM, allowing the membrane to make contact with the adjoining membrane of another symbiosome. We conclude that the major H⁺-ATPase on the PBM of soybean is a P-type enzyme with homology to other such enzymes in plants. In vivo, this enzyme is likely to play a critical role in the regulation of nutrient exchange between legume and bacteroids. Received: 25 November 1998 / Accepted: 6 January 1999     

Calcium-Based Interactions of Symbiotic Partners in Legumes: Role of Peribacteroid Membrane

Based on experimental evidence, a concept is formulated that mutualistic relationships between pro- and eukaryotic cells during nitrogen-fixing legume–rhizobia symbiosis rely both on selective transfer of metabolites and ion transport, Ca²⁺ in particular, across the peribacteroid membrane (PBM). PBM in the nitrogen-fixing cells of yellow lupine (Lupinus luteus L.) and broad bean (Vicia faba L.) is endowed with a calcium-translocating ATPase that pumps Ca²⁺ into the symbiosome. This pumping ensures, on the one hand, calcium homeostasis in the cytosol of infected plant cells and, on the other hand, it optimizes Ca²⁺ level in symbiosomes, first of all in the bacteroids, because Ca²⁺ is one of the main factors controlling their nitrogenase activity. The balance between the symbiotic partners and the maintenance of optimal Ca²⁺ level in the bacteroids also depends on passive Ca²⁺ efflux from symbiosomes to the plant cell cytosol via calcium channels. The Ca²⁺-transporting mechanisms residing at PBM are characterized.     

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.     

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.     

Calcium Status of Yellow Lupin Symbiosomes as a Potential Regulator of Their Nitrogenase Activity: The Role of the Peribacteroid Membrane

The capacity of symbiosomes from yellow lupin root nodules for active Ca²⁺uptake and the sensitivity of their nitrogenase activity to a disturbance of the symbiotic Ca partition were investigated. The experiments carried out on the isolated symbiosomes and the peribacteroid membrane (PBM) vesicles, using Ca²⁺indicators arsenazo III and chlorotetracycline, and the cytochemical Ca visualization with potassium pyroantimonate (PA) provided evidence that an Mg-ATP-energized pump, most likely Mg²⁺-dependent Ca²⁺-ATPase catalyzing the active transport of Ca²⁺from the cytosol of the plant cell into the symbiosomes across the PBM, functions on this membrane. Depleting the symbiosomes of Ca both in vivoandin vitroby treating the intact nodules of yellow lupin root or the purified symbiosomes isolated from the latter with EGTA and Ca²⁺-ionophore A23187 substantially decreased their nitrogenase activity. The inhibitory effect of calcium deficit in the symbiosomes was not reversed by the addition of calcium to the incubation medium containing the plant tissues under study and was even enhanced under these conditions. The nitrogenase activity of the isolated symbiosomes not experiencing calcium deficit was also inhibited by the addition of relatively high concentrations of exogenous calcium to the incubation medium. These results seem to give evidence that the calcium status of nodule symbiosomes from yellow lupin roots controls their nitrogenase activity. The data obtained suggest that both Ca²⁺transport on PBM and the low passive permeability of this membrane for the given cation play the key role in such a control.     

The galactolipid digalactosyldiacylglycerol accumulates in the peribacteroid membrane of nitrogen-fixing nodules of soybean and Lotus

The peribacteroid membrane (PBM) surrounding nitrogen fixing rhizobia in the nodules of legumes is crucial for the exchange of ammonium and nutrients between the bacteria and the host cell. Digalactosyldiacylglycerol (DGDG), a galactolipid abundant in chloroplasts, was detected in the PBM of soybean (Glycine max) and Lotus japonicus. Analyses of membrane marker proteins and of fatty acid composition confirmed that DGDG represents an authentic PBM lipid of plant origin and is not derived from the bacteria or from plastid contamination. In Arabidopsis, DGDG is known to accumulate in extraplastidic membranes during phosphate deprivation. However, the presence of DGDG in soybean PBM was not restricted to phosphate limiting conditions. Complementary DNA sequences corresponding to the two DGDG synthases, DGD1 and DGD2 from Arabidopsis, were isolated from soybean and Lotus. The two genes were expressed during later stages of nodule development in infected cells and in cortical tissue. Because nodule development depends on the presence of high amounts of phosphate in the growth medium, the accumulation of the non-phosphorus galactolipid DGDG in the PBM might be important to save phosphate for other essential processes, i.e. nucleic acid synthesis in bacteroids and host cells.     

Presence of Host-Plasma Membrane Type H-ATPase in the Membrane Envelope Enclosing the Bacteroids in Soybean Root Nodules

An improved method is described for the isolation of membrane envelope enclosing the bacteroids (peribacteroid membrane) from soybean (Glycine max L.) root nodules. The ATPase activity of the peribacteroid membrane from infected roots is compared with that of the plasma membrane from uninfected roots. The two ATPases are similar in terms of their vanadate sensitivities, pH optima, and mineral cation requirements, and show antigenic cross-reactivity. However, the ATPase of peribacteroid membrane is more sensitive to stimulation by NH₄⁺. ATP-dependent proton translocation across the peribacteroid membrane was demonstrated in broken protoplasts of infected cells, by the use of fluorescence microscopy with acridine orange. It is suggested that acidification of the peribacteroid space by the peribacteroid membrane ATPase results in the conversion of NH₃ to NH₄⁺ in this space and thereby facilitates the removal of fixed-nitrogen from the bacteroid.     

Changes in the Light Scattering by Symbiosomes from Vicia faba Root Nodules as Indicator of Ion and Metabolite Transport across the Peribacteroid Membrane

Passive transport of ions and metabolites across the peribacteroid membrane (PBM) was investigated on symbiosome preparations isolated from the broad bean (Vicia faba L.) root nodules and suspended in a potassium-free medium. Optical density of the symbiosome suspension at 546 nm was monitored as an indicator of light-scattering changes. Depolarization of the PBM with tetraphenylphosphonium cation (TPP⁺) caused an increase in light scattering of symbiosome suspension. This effect was enhanced after adding a K⁺ ionophore valinomycin to the incubation medium. A similar effect was observed after supplementing the symbiosome suspension with nigericin, a K⁺/H⁺ antiporter. Similar experiments on bacteroid suspensions prepared from isolated symbiosomes did not reveal any appreciable changes in light scattering in the presence of the same membrane-active substances. The light scattering by symbiosome suspensions decreased after adding malate or succinate, while the subsequent addition of centimolar concentrations of K⁺ substantially accelerated this process. Light scattering by the symbiosome suspension was insensitive to the addition of glutamate, a substance normally impermeant through the PBM of legume root nodules. These results suggest that the changes in light scattering by symbiosomes reflect the osmotically induced changes of symbiosome volume. These volume changes were assigned to alteration of the peribacteroid space (PBS). The incubation of symbiosomes in a potassium-free medium acidified their the PBS; this acidification was accelerated by valinomycin, carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP), and nigericin, and it was abolished in the presence of comparatively high concentrations of K⁺ in the incubation medium. The results indicate a relatively high permeability of the PBM to K⁺ ions.     

Identification with proteomics of novel proteins associated with the peribacteroid membrane of soybean root nodules

Soybean peribacteroid membrane (PBM) proteins were isolated from nitrogen-fixing root nodules and subjected to N-terminal sequencing. Sequence data from 17 putative PBM proteins were obtained. Six of these proteins are homologous to proteins of known function. These include three chaperones (HSP60, BiP [HSP70], and PDI) and two proteases (a serine and a thiol protease), all of which are involved in some aspect of protein processing in plants. The PBM homologs of these proteins may play roles in protein translocation, folding, maturation, or degradation in symbiosomes. Two proteins are homologous to known, nodule-specific proteins from soybean, nodulin 53b and nodulin 26B. Although the function of these nodulins is unknown, nodulin 53b has independently been shown to be associated with the PBM. All of the eight proteins with identifiable homologs are likely to be peripheral rather than integral membrane proteins. Possible reasons for this apparent bias are discussed. The identification of homologs of HSP70 and HSP60 associated with the PBM is the first evidence that the molecular machinery for co- or post-translational import of cytoplasmic proteins is present in symbiosomes. This has important implications for the biogenesis of this unique, nitrogen-fixing organelle.     

The effect of metabolites on the pH gradient and membrane potential of the bean peribacteroid membrane

The effects of malate, succinate, and glutamate on the kinetics of changes in the pH gradient (delta pH) and membrane potential (delta psi) on the peribacteroid membrane (PBM) of the symbiosomes of bean root nodules varying in age were recorded spectrophotometrically. Addition of all the tested metabolites to potassium-free incubation medium stimulated a passive acidification of the peribacteroid space (PBS) and dissipation of delta psi in PBM of young developing nodules in the presence of the K+/H+ antiporter nigericin in the medium. However, in mature nodules with a high nitrogen-fixing activity, only malate and succinate (but not glutamate) increased delta pH during both passive and ATP-dependent PBS acidification. Dicarboxylates also caused dissipation of both delta pH in the presence of nigericin in the medium and delta psi generated on PBM by H+-ATPase. A decrease in the effects of metabolites on delta pH and the absent activity of the PBM H+ pump were observed in the aging nodules. The obtained data on the changes in deltapH and dlta psi caused by the metabolites in question suggest that PBM is permeable for all these metabolites only in young nodules. Only malate and succinate (but not glutamate) are transported through PBM in mature nodules; and the rate of metabolite translocation through PBM in aging nodules is decreased.     

Plasma membrane ferric reductase activity of iron-limited algal cells is inhibited by ferric chelators

Iron-limited cells of the green alga Chlorella kesslerii use a reductive mechanism to acquire Fe(III) from the extracellular environment, in which a plasma membrane ferric reductase reduces Fe(III)-chelates to Fe(II), which is subsequently taken up by the cell. Previous work has demonstrated that synthetic chelators both support ferric reductase activity (when supplied as Fe(III)-chelates) and inhibit ferric reductase. In the present set of experiments we extend these observations to naturally-occurring chelators and their analogues (desferrioxamine B mesylate, schizokinen, two forms of dihydroxybenzoic acid) and also two formulations of the commonly-used herbicide N-(phoshonomethyl)glycine (glyphosate). The ferric forms of the larger siderophores (desferrioxamine B mesylate, schizokinen) and Fe(III)-N-(phoshonomethyl)glycine (as the isopropylamine salt) all supported rapid rates of ferric reductase activity, while the iron-free forms inhibited reductase activity. The smaller siderophores/siderophore precursors, 2,3- and 3,4-dihydroxybenzoic acids, did not support high rates of reductase in the ferric form but did inhibit reductase activity in the iron-free form. Bioassays indicated that Fe(III)-chelates that supported high rates of ferric reductase activity also supported a large stimulation in the growth of iron-limited cells, and that an excess of iron-free chelator decreased the growth rate. With respect to N-(phosphonomethyl)glycine, there were differences between the pure compound (free acid form) and the most common commercial formulation (which also contains isopropylamine) in terms of supporting and inhibiting ferric reductase activity and growth. Overall, these results suggest that photosynthetic organisms that use a reductive strategy for iron acquisition both require, and are potentially simultaneously inhibited by, ferric chelators. Furthermore, these results also may provide an explanation for the frequently contradictory results of N-(phosphonomethyl)glycine application to crops: we suggest that low concentrations of this molecule likely solubilize Fe(III), making it available for plant growth, but that higher (but sub-lethal) concentrations decrease iron acquisition by inhibiting ferric reductase activity.     

Siderophore-bound iron in the peribacteriod space of soybean root nodules

Water-soluble, non-leghemoglobin iron (125 µmol kg^-1 wet weight nodule) is found in extracts of soybean root nodules. This iron is probably confined to the peribacteroid space of the symbiosome, where its estimated concentration is 0.5 – 2.5 mM. This iron is bound by siderophores (compounds binding ferric iron strongly) which are different for each of the three strains of Bradyrhizobium japonicum with which the plants were inoculated. One of these, that from nodules inoculated with strain CC 705, is tentatively identified as a member of the pseudobactin family of siderophores. Leghemoglobin is present in only very small amounts in the peribacteroid space of symbiosomes isolated from soybean root nodules, and may be absent from the peribacteroid space of the intact nodule.     

Developmentally regulated membrane glycoproteins sharing antigenicity with rhamnogalacturonan II are not detected in nodulated boron deficient Pisum sativum

The peribacteroid membrane (PBM) of symbiosomes from pea root nodules developed in the presence of boron (+B) was labelled by anti-rhamnogalacturonan II (RGII) (anti-rhamnogalacturonan II pectin polysaccharide) antiserum. However, in nodules from plants grown at low boron (-B), anti-RGII pectin polysaccharide did not stain PBMs. Given that RGII pectin binds to borate, and that symbiosomes differentiate aberrantly in -B nodules because of abnormal vesicle traffic, anti-RGII pectin polysaccharide antigens were further analysed. Following electrophoresis and electroblotting, anti-RGII pectin polysaccharide immunostained three bands in +B but not in -B nodule-derived PBMs. A similar banding pattern was observed after the immunostaining of membrane fractions from uninfected roots, indicating that anti-RGII pectin polysaccharide antigens are common to both peribacteroid and plasma membranes. Protease treatment of samples led to disappearance of anti-RGII pectin polysaccharide labelling, indicating that the three immunostained bands correspond to proteins or glycoproteins. The immunochemical study of RGII antigen distribution during nodule development showed that it is strongly present on the PBM of dividing (undifferentiated) symbiosomes but progressively disappeared during symbiosome maturation. In B-deficient nodules, PBMs were never decorated with RGII antigens, and there was an abnormal targeting of vesicles containing pectic polysaccharide (homogalacturanan) to cell membranes. Overall, these results indicate that RGII, boron and certain membrane (glyco)-proteins may interact closely and function cooperatively in membrane processes associated with symbiosome division and general cell growth.     

Rhizobium colonization induced changes in membrane-bound and soluble hydroxyproline-rich glycoprotein composition in pea

Abundance and distribution of plant cell surface proteins of the hydroxyproline-rich glycoprotein (HRGP) class were studied in the pea- Rhizobium symbiosis using immunoblot analysis. The MAC 265-epitope was especially abundant in pea root nodules containing nitrogen-fixing Rhizobium bacteria. A 180-kDa MAC 265-HRGP dominated in pea shoot plasma membranes, while almost no MAC 265-HRGP was detected in root plasma membranes. We show here that a major difference between the plant-derived peribacteroid membrane of the symbiosomes and the root plasma membrane was the presence of a 100-kDa MAC 265-HRGP in the former. Arabinogalactan proteins (AGPs), as recognized by the monoclonal antibodies MAC 207 and JIM 8, were not detected in the peribacteroid membrane, while two isoforms (100 and 220 kDa) were detected in shoot and root plasma membranes. Specific MAC 265-HRGP isoforms were found in the peribacteroid space fraction of the symbiosomes and thus as soluble proteins in the interface between the symbionts. The abundance of the MAC 265-epitope was much reduced in non-nitrogen-fixing nodules when this phenotype resulted from a dicarboxylate transport mutation in Rhizobium . There was no reduction in the abundance of the MAC 265-epitope in non-fixing phenotypes resulting from a mutation in the plant. The results suggest that bacterial signals related to the bacterial ability to fix nitrogen, might be responsible for the regulation of HRGP expression in root nodules.     

Ammonia (C-Methylamine) Transport across the Bacteroid and Peribacteroid Membranes of Soybean Root Nodules

[¹⁴C]Methylamine (MA; an analog of ammonia) was used to investigate ammonia transport across the bacteroid and peribacteroid membranes (PBM) from soybean (Glycine max) root nodules. Free-living Bradyrhizobium japonicum USDA110 grown under nitrogen-limited conditions showed rapid MA uptake with saturation kinetics at neutral pH, indicative of a carrier. Exchange of accumulated MA for added ammonia occurred, showing that the carrier recognized both NH₄⁺ and CH₃NH₃⁺. MA uptake by isolated bacteroids, on the other hand, was very slow at low concentrations of MA and increased linearly with increasing MA concentration up to 1 millimolar. Ammonia did not inhibit MA by isolated bacteroids and did not cause efflux of accumulated MA. PBM-enclosed bacteroids (peribacteroid units [PBUs]) were qualitatively similar to free bacteroids with respect to MA transport. The rates of uptake and efflux of MA by PBUs were linearly dependent on the imposed concentration gradient and unaffected by NH₄Cl. MA uptake by PBUs increased exponentially with increasing pH, confirming that the rate increased linearly with increasing CH₃NH₂ concentration. The results are consistent with other evidence that transfer of ammonia from the nitrogen-fixing bacteroid to the host cytosol in soybean root nodules occurs solely by simple diffusion of NH₃ across both the bacteroid and peribacteroid membranes.