35S-labeled Sulfate Research Articles

Metabolic Labeling of Proteoglycans and Analysis of Their Synthesis and Sorting in Filter-Grown and Polarized Epithelial Cells.

Studies of synthesis, turnover, and secretion of macromolecules in cell culture are carried out to address mechanisms of cellular and physiological importance. Culture systems have been developed to mimic the in vivo situation as much as possible. In line with this aim, epithelial and endothelial cells have been grown on filters for more than three decades. Growing such cells on permeable support allows for nutrient uptake via the basolateral membrane of tight epithelial monolayers, from a medium reservoir underneath the filter. While this basolateral medium reservoir resembles the blood supply, the apical medium reservoir resembles the organ lumen. Growing the cells in a polarized manner allows for studies of differential transport and localization of apical and basolateral proteins and of endocytic and secretory transport at both sides of the epithelium. Here we describe how metabolic labeling of proteoglycans (PGs) with 35S-labeled sulfate enables analysis of synthesis of different types of PGs, with respect to size, glycosaminoglycan (GAG) chain length, and charge. We also describe protocols for studies of intracellular PG sorting, in the apical and basolateral direction in polarized epithelial cells, in the absence and presence of inhibitors of synthesis and transport.

Diversity and Activity of Sulfate-Reducing Prokaryotes in Kamchatka Hot Springs.

Microbial communities of the Kamchatka Peninsula terrestrial hot springs were studied using radioisotopic and cultural approaches, as well as by the amplification and sequencing of dsrB and 16S rRNA genes fragments. Radioisotopic experiments with 35S-labeled sulfate showed that microbial communities of the Kamchatka hot springs are actively reducing sulfate. Both the cultivation experiments and the results of dsrB and 16S rRNA genes fragments analyses indicated the presence of microorganisms participating in the reductive part of the sulfur cycle. It was found that sulfate-reducing prokaryotes (SRP) belonging to Desulfobacterota, Nitrospirota and Firmicutes phyla inhabited neutral and slightly acidic hot springs, while bacteria of phylum Thermodesulofobiota preferred moderately acidic hot springs. In high-temperature acidic springs sulfate reduction was mediated by archaea of the phylum Crenarchaeota, chemoorganoheterotrophic representatives of genus Vulcanisaeta being the most probable candidates. The 16S rRNA taxonomic profiling showed that in most of the studied communities SRP was present only as a minor component. Only in one microbial community, the representatives of genus Vulcanisaeta comprised a significant group. Thus, in spite of comparatively low sulfate concentrations in terrestrial hot springs of the Kamchatka, phylogenetically and metabolically diverse groups of sulfate-reducing prokaryotes are operating there coupling carbon and sulfur cycles in these habitats.

Microbial Sulfate Reduction Potential in Coal-Bearing Sediments Down to ~2.5 km below the Seafloor off Shimokita Peninsula, Japan.

Sulfate reduction is the predominant anaerobic microbial process of organic matter mineralization in marine sediments, with recent studies revealing that sulfate reduction not only occurs in sulfate-rich sediments, but even extends to deeper, methanogenic sediments at very low background concentrations of sulfate. Using samples retrieved off the Shimokita Peninsula, Japan, during the Integrated Ocean Drilling Program (IODP) Expedition 337, we measured potential sulfate reduction rates by slurry incubations with 35S-labeled sulfate in deep methanogenic sediments between 1276.75 and 2456.75 meters below the seafloor. Potential sulfate reduction rates were generally extremely low (mostly below 0.1 pmol cm−3 d−1) but showed elevated values (up to 1.8 pmol cm−3 d−1) in a coal-bearing interval (Unit III). A measured increase in hydrogenase activity in the coal-bearing horizons coincided with this local increase in potential sulfate reduction rates. This paired enzymatic response suggests that hydrogen is a potentially important electron donor for sulfate reduction in the deep coalbed biosphere. By contrast, no stimulation of sulfate reduction rates was observed in treatments where methane was added as an electron donor. In the deep coalbeds, small amounts of sulfate might be provided by a cryptic sulfur cycle. The isotopically very heavy pyrites (δ34S = +43‰) found in this horizon is consistent with its formation via microbial sulfate reduction that has been continuously utilizing a small, increasingly 34S-enriched sulfate reservoir over geologic time scales. Although our results do not represent in-situ activity, and the sulfate reducers might only have persisted in a dormant, spore-like state, our findings show that organisms capable of sulfate reduction have survived in deep methanogenic sediments over more than 20 Ma. This highlights the ability of sulfate-reducers to persist over geological timespans even in sulfate-depleted environments. Our study moreover represents the deepest evidence of a potential for sulfate reduction in marine sediments to date.

Sulfate Separation by Selective Crystallization with a Bis-iminoguanidinium Ligand

A simple and effective method for selective sulfate separation from aqueous solutions by crystallization with a bis-guanidinium ligand, 1,4-benzene-bis(iminoguanidinium) (BBIG), is demonstrated. The ligand is synthesized as the chloride salt (BBIG-Cl) by in situ imine condensation of terephthalaldehyde with aminoguanidinium chloride in water, followed by crystallization as the sulfate salt (BBIG-SO4). Alternatively, BBIG-Cl is synthesized ex situ in larger scale from ethanol. The sulfate separation ability of the BBIG ligand is demonstrated by selective and quantitative crystallization of sulfate from seawater. The ligand can be recycled by neutralization of BBIG-SO4 with aqueous NaOH and crystallization of the neutral bis-iminoguanidine, which can be converted back into BBIG-Cl with aqueous HCl and reused in another separation cycle. Finally, 35S-labeled sulfate and β liquid scintillation counting are employed for monitoring the sulfate concentration in solution. Overall, this protocol will instruct the user in the necessary skills to synthesize a ligand, employ it in the selective crystallization of sulfate from aqueous solutions, and quantify the separation efficiency.

Sulfur utilization of corals is enhanced by endosymbiotic algae.

ABSTRACTSulfur-containing compounds are important components of all organisms, but few studies have explored sulfate utilization in corals. Our previous study found that the expression of a sulfur transporter (SLC26A11) was upregulated in the presence of Symbiodinium cells in juveniles of the reef-building coral Acropora tenuis. In this study, we performed autoradiography using 35S-labeled sulfate ions (35SO4 2−) to examine the localization and amount of incorporated radioactive sulfate in the coral tissues and symbiotic algae. Incorporated 35SO4 2− was detected in symbiotic algal cells, nematocysts, ectodermal cells and calicoblast cells. The combined results of 35S autoradiography and Alcian Blue staining showed that incorporated 35S accumulated as sulfated glycosaminoglycans (GAGs) in the ectodermal cell layer. We also compared the relative incorporation of 35SO4 2− into coral tissues and endosymbiotic algae, and their chemical fractions in dark versus light (photosynthetic) conditions. The amount of sulfur compounds, such as GAGs and lipids, generated from 35SO4 2− was higher under photosynthetic conditions. Together with the upregulation of sulfate transporters by symbiosis, our results suggest that photosynthesis of algal endosymbionts contributes to the synthesis and utilization of sulfur compounds in corals.

Sulfate reduction and inorganic carbon assimilation in acidic thermal springs of the Kamchatka peninsula

Thermoacidophilic sulfate reduction, which remains a poorly studied process, was investigated in the present work. Radioisotope analysis with 35S-labeled sulfate was used to determine the rates of dissimilatory sulfate reduction in acidic thermal springs of Kamchatka, Russia. Sulfate reduction rates were found to vary from 0.054 to 12.9 nmol SO4/(cm3 day). The Oil Site spring (Uzon caldera, 60°C, pH 4.2) and Oreshek spring (Mutnovskii volcano, 91°C, pH 3.5) exhibited the highest activity of sulfate-reducing prokaryotes. Stable enrichment cultures reducing sulfate at pH and temperature values close to the environmental ones were obtained from these springs. Analysis of the 16S rRNA gene sequences revealed that a chemolithoautotrophic bacterium Thermodesulfobium sp. 3127-1 was responsible for sulfate reduction in the enrichment from the Oil Site spring. A chemoorganoheterotrophic archaeon Vulcanisaeta sp. 3102-1 (phylum Crenarchaeota) was identified in the enrichment from Oreshek spring. Thus, dissimilatory sulfate reduction under thermoacidophilic conditions was demonstrated and the agents responsible for this process were revealed.

Development of a 14C detectable real-time radioisotope imaging system for plants under intermittent light environment

A new real-time radioisotope imaging system (RRIS) to study the kinetics of nutrient uptake and transfer of photosynthetic products in a living plant was developed and evaluated through a test run. 14C is a common radioisotope of carbon and useful to trace the photosynthetic products as well as a low energy beta emitter. The rationale of this study was to develop a RRIS that has the ability to detect low energy beta emitters, such as 14C, 35S, and 45Ca. To achieve compatibility between the detection of low energy beta emitters and irradiation of the test plant, an intermittent lighting system was added to the RRIS. Furthermore, a commercially available digital camera was added to the RRIS for acquisition of photographic images of the test plants. The capabilities of the new RRIS were evaluated through a test run by using seedlings of rice plants and 35S-labeled sulfate. It was shown that the new RRIS was able to detect 35S absorbed by rice plant seedlings, and it was able to acquire photon-counting images and photographic images of the test plants simultaneously. Despite some limitations, the new RRIS provides a means to study the kinetics of elements in plants by utilizing low energy beta emitters.

Heparan Sulfate Chain Valency Controls Syndecan-4 Function in Cell Adhesion

Fibroblasts null for the transmembrane proteoglycan, syndecan-4, have an altered actin cytoskeleton, compared with matching wild-type cells. They do not organize α-smooth muscle actin into bundles, but will do so when full-length syndecan-4 is re-expressed. This requires the central V region of the core protein cytoplasmic domain, though not interactions with PDZ proteins. A second key requirement is multiple heparan sulfate chains. Mutant syndecan-4 with no chains, or only one chain, failed to restore the wild-type phenotype, whereas those expressing two or three were competent. However, clustering of one-chain syndecan-4 forms with antibodies overcame the block, indicating that valency of interactions with ligands is a key component of syndecan-4 function. Measurements of focal contact/adhesion size and focal adhesion kinase phosphorylation correlated with syndecan-4 status and α-smooth muscle actin organization, being reduced where syndecan-4 function was compromised by a lack of multiple heparan sulfate chains.

Production of methanethiol and volatile sulfur compounds by the archaeon “Ferroplasma acidarmanus”

Acidophiles are typically isolated from sulfate-rich ecological niches yet the role of sulfur metabolism in their growth and survival is poorly defined. Studies of heterotrophically grown "Ferroplasma acidarmanus" showed that its growth requires a minimum of 100 mM of a sulfate-containing salt. Headspace gas analyses by GC/MS determined that the volatile sulfur compound emitted by active "F. acidarmanus" cultures is methanethiol. In "F. acidarmanus" cultures grown either heterotrophically or chemolithotrophically, methanethiol was produced constitutively. Radiotracer studies with (35)S-labeled methionine, cysteine, and sulfate showed that all three were used in methanethiol production. Additionally, (3)H-labeled methionine was incorporated into methanethiol and was probably used as a methyl-group donor. Methanethiol production in whole cell lysates supplied with SO (3) (2-) indicated that NADPH-dependant sulfite reductase and methyltransferase activities were present. Cell lysates also contained enzymatic activity for methionine-gamma-lyase that cleaved the side chain of either methionine to form methanethiol or cysteine to produce H(2)S. Since methanethiol was detected from the degradation of cysteine, it is likely that sulfide was methylated by a thiol methyltransferase. Collectively, these data demonstrate that "F. acidarmanus" produces methanethiol through the metabolism of methionine, cysteine, or sulfate. This is the first report of a methanethiol-producing acidophile, thus identifying a new contributor to the global sulfur cycle.

Substrate Specificity and Domain Functions of Extracellular Heparan Sulfate 6-O-Endosulfatases, QSulf1 and QSulf2

The extracellular sulfatases (Sulfs) are an evolutionally conserved family of heparan sulfate (HS)-specific 6-O-endosulfatases. These enzymes remodel the 6-O-sulfation of cell surface HS chains to promote Wnt signaling and inhibit growth factor signaling for embryonic tissue patterning and control of tumor growth. In this study we demonstrate that the avian HS endosulfatases, QSulf1 and QSulf2, exhibit the same substrate specificity toward a subset of trisulfated disaccharides internal to HS chains. Further, we show that both QSulfs associate exclusively with cell membrane and are enzymatically active on the cell surface to desulfate both cell surface and cell matrix HS. Mutagenesis studies reveal that conserved amino acid regions in the hydrophilic domains of QSulf1 and QSulf2 have multiple functions, to anchor Sulf to the cell surface, bind to HS substrates, and to mediate HS 6-O-endosulfatase enzymatic activity. Results of our current studies establish the hydrophilic domain (HD) of Sulf enzymes as an essential multifunctional domain for their unique endosulfatase activities and also demonstrate the extracellular activity of Sulfs for desulfation of cell surface and cell matrix HS in the control of extracellular signaling for embryonic development and tumor progression.

Mass spectrometric identification of ethyl sulfate as an ethanol metabolite in humans.

After alcohol ingestion, the bulk of ethanol ingested (≥95%) is rapidly eliminated in the liver in a two-stage oxidation process: first to acetaldehyde by alcohol dehydrogenase and then to acetic acid by aldehyde dehydrogenase. The remainder is excreted mainly unchanged in urine and expired air. However, another small fraction of the ingested ethanol dose (<0.1%) (1) undergoes a phase II conjugation reaction catalyzed by UDP-glucuronosyltransferase (UGT) to produce ethyl glucuronide (EtG), which is eventually excreted in the urine (2)(3)(4). Because EtG has a longer period of elimination than the parent compound, the interest in EtG has largely focused on its use as a sensitive and specific biomarker of recent alcohol intake with clinical and forensic applications (5)(6). Animal studies have indicated that ethanol may also undergo sulfate conjugation through the action of sulfotransferase to produce ethyl sulfate (EtS) (7)(8)(9). After an oral dose of ethanol and injection of 35S-labeled sulfate in rats, EtS was apparently excreted in urine mainly during the first 24 h (10). However, a general limitation in these studies was the lack of reliable methods for unequivocal identification of EtS and precise quantification. In the present study on humans, we used a sensitive and specific liquid chromatographic–mass spectrometric (LC-MS) method to determine whether EtS is formed after …

Phloem-localizing sulfate transporter, Sultr1;3, mediates re-distribution of sulfur from source to sink organs in Arabidopsis.

For the effective recycling of nutrients, vascular plants transport pooled inorganic ions and metabolites through the sieve tube. A novel sulfate transporter gene, Sultr1;3, was identified as an essential member contributing to this process for redistribution of sulfur source in Arabidopsis. Sultr1;3 belonged to the family of high-affinity sulfate transporters, and was able to complement the yeast sulfate transporter mutant. The fusion protein of Sultr1;3 and green fluorescent protein was expressed by the Sultr1;3 promoter in transgenic plants, which revealed phloem-specific expression of Sultr1;3 in Arabidopsis. Sultr1;3-green fluorescent protein was found in the sieve element-companion cell complexes of the phloem in cotyledons and roots. Limitation of external sulfate caused accumulation of Sultr1;3 mRNA both in leaves and roots. Movement of (35)S-labeled sulfate from cotyledons to the sink organs was restricted in the T-DNA insertion mutant of Sultr1;3. These results provide evidence that Sultr1;3 transporter plays an important role in loading of sulfate to the sieve tube, initiating the source-to-sink translocation of sulfur nutrient in Arabidopsis.

Specific Adsorption of Simple Organic Acids on Metal(hydr)oxides: A Radiotracer Approach

The pH dependence of adsorption of 14C-labeled benzoic and oxalic acids on γ-Al 2O 3 and hematite was studied in acid medium in the presence of 0.5 mol dm −3 NaClO 4 supporting electrolyte. It was found that the adsorption of the organic species starts at pH values where the protonation of the oxide surface takes place. In the case of benzoic acid the extent of adsorption with decreasing pH goes through a sharp maximum at a pH value not far from the pK (4.2) of the acid, while in the case of oxalic acid only a small decrease can be observed at very low pH values (pH<1). In indirect radiotracer studies using 35S-labeled sulfate ions it was shown that the competitive adsorption of formic, malonic, maleic, and oxalic acids with sulfate ions depends on pH and the effect of the organic acid on the anion adsorption becomes pronounced at pH values about and above the pK of the acid. On the basis of these observations and considerations concerning the dissociation of the organic acids studied it is assumed that the specific adsorption of the anionic form of the acids takes place. It is, however, emphasized that the negative charge of the anions, consequently the electrostatic forces, do not play significant role in the adsorption.

A 96-well dot-blot assay for carbohydrate sulfotransferases

Here we describe an efficient dot-blot assay for high-throughput screening of two enzymes, heparan sulfate N-deacetylase/ N-sulfotransferase (NDST-1) and high-endothelial cell GlcNAc-6-sulfotransferase (HEC-GlcNAc-6-ST). The assay proceeds by transfer of 35S-labeled sulfate from [ 35S]-3 ′-phosphoadenosine-5 ′-phosphosulfate (PAPS) to the free amino groups of de- N-sulfated heparin (NDST-1), or the 6-hydroxyl groups of N-acetylglucosamine residues linked to a polyacrylamide scaffold (HEC-GlcNAc-6-ST). The 35S-labeled products are then captured on an appropriate membrane, taking advantage of their polymeric architecture. In one step, 35S-labeled by-products are then eluted from the membrane, leaving spatially separated 35S-labeled product “dots” for subsequent quantification. This assay allows for direct product detection on the membrane, obviating excessive washing and elution steps endemic to other assays. The assay was validated by measuring K M values for PAPS and K I values for PAP, the product of sulfuryl transfer. The assay method should be useful for inhibitor screens for both enzymes. In addition, the general assay architecture should be readily applicable to high-throughput screens of other carbohydrate sulfotransferases.

Sulfur Assimilation in Developing Lupin Cotyledons Could Contribute Significantly to the Accumulation of Organic Sulfur Reserves in the Seed

It is currently assumed that the assimilation of sulfur into reduced forms occurs predominantly in the leaves of plants. However, developing seeds have a strong requirement for sulfur amino acids for storage protein synthesis. We have assessed the capacity of developing seeds of narrow-leaf lupin (Lupinus angustifolius) for sulfur assimilation. Cotyledons of developing lupin seeds were able to transfer the sulfur atom from 35S-labeled sulfate into seed proteins in vitro, demonstrating the ability of the developing cotyledons to perform all the steps of sulfur reduction and sulfur amino acid biosynthesis. Oxidized sulfur constituted approximately 30% of the sulfur in mature seeds of lupins grown in the field and almost all of the sulfur detected in phloem exuded from developing pods. The activities of three enzymes of the sulfur amino acid biosynthetic pathway were found in developing cotyledons in quantities theoretically sufficient to account for all of the sulfur amino acids that accumulate in the protein of mature lupin seeds. We conclude that sulfur assimilation by developing cotyledons is likely to be an important source of sulfur amino acids for the synthesis of storage proteins during lupin seed maturation.

The Interaction between Basic Fibroblast Growth Factor and Heparan Sulfate Can Prevent the in Vitro Degradation of the Glycosaminoglycan by Chinese Hamster Ovary Cell Heparanases

Heparan sulfate proteoglycans on Chinese hamster ovary (CHO) cell surfaces can bind and internalize basic fibroblast growth factor (bFGF). We have investigated whether this interaction affects heparan sulfate catabolism in vitro by measuring the ability of partially purified CHO heparanase activities to degrade 35S-labeled heparan sulfate glycosaminoglycans in the absence or presence of bFGF. Our studies show that the presence of the growth factor prevents partially purified heparanases from degrading the nascent 81-kDa chains to short 6-kDa products, whether the glycosaminoglycan is free in solution or covalently bound to core proteins. A 30-60 molar excess of the growth factor is required to inhibit completely chain degradation by heparanases, implying that multiple bFGF molecules must be bound to the glycosaminoglycan to prevent heparanase-catalyzed catabolism. This hypothesis is supported by protection studies indicating that nascent CHO heparan sulfate glycosaminoglycans have at least four to eight bFGF binding sites/chain. It does not appear, however, that the growth factor inhibits heparanase-catalyzed degradation of the glycosaminoglycan by binding to the sequence cleaved by the enzyme. Both the nascent and short chains bind bFGF with similar affinity (Kd values of 27.0 +/- 3.5 and 38.9 +/- 5.1 nM, respectively), indicating that heparanase activities do not destroy the bFGF binding sites. Rather, our results suggest that the growth factor interferes sterically with heparanase action by binding the heparan sulfate chain at a sequence next to the cleavage site or at a secondary site recognized by the enzyme.

Characterization and Hormonal Modulation of Anticoagulant Heparan Sulfate Proteoglycans Synthesized by Rat Ovarian Granulosa Cells

Anticoagulant heparan sulfate proteoglycans endow the vascular endothelium with antithrombotic properties, but their role outside the vascular bed is unknown. Granulosa cells form an avascular compartment in the ovarian follicle, in which a heparin-like activity has been described. At ovulation extravascular coagulation occurs around ovulatory follicles, and after expulsion of the oocyte, a fibrin clot forms in the antral cavity. Granulosa cells synthesize two major heparan sulfate proteoglycans, whose anticoagulant nature has not been investigated. The purpose of this study was to characterize anticoagulant heparan sulfate proteoglycans synthesized by rat ovarian granulosa cells. Affinity purified 35S-labeled anticoagulant heparan sulfate glycosaminoglycans represent 6.5% of the total heparan sulfate synthesized, and they contain 13% 3-O-sulfated disaccharides that are markers of the antithrombin-binding site of heparin. The biological activity of granulosa cell heparan sulfate proteoglycans was demonstrated by their ability to bind antithrombin and to accelerate the formation of thrombin-antithrombin complexes. The impact of hormonal stimulation on granulosa cell anticoagulant heparan sulfate proteoglycans was studied using 125I-antithrombin binding assays. Follicle-stimulating hormone induced a redistribution of anticoagulant heparan sulfate proteoglycans from the granulosa cell layer to the culture medium, indicating that their distribution could be modulated according to the stage of follicular development. These results suggest that anticoagulant heparan sulfate might be critically located in the follicle to maintain fluidity around the oocyte until its expulsion at ovulation.

Characteristics of assimilatory sulfate transport in Rhodobacter sulfidophilus

Sulfate uptake was investigated with four species of phototrophic sulfur bacteria. Rhodobacter sulfidophilus and Chromatium vinosum took up 35S-labeled sulfate added in micromolar concentrations. Sulfate uptake by C. vinosum was expressed only under sulfate starvation. R. sulfidophilus took up 10 μM sulfate almost completely and accumulated it up to 5300-fold, also when grown with excess sulfate. Sulfite (1 mM) as an intermediate of sulfate assimilation inhibited sulfate uptake completely within 1 min. Moderate inhibition was observed with cysteine (1 mM) and none with sulfide (1 mM). Transport was not dependent on the cations K +, Na +, Li + or protons, but was sensitive to uncouplers and to the ATPase inhibitor dicyclohexylcarbodiimide (DCCD). The accumulation of sulfate correlated with the ATP concentration in the cells, indicating an ATP-dependent uptake mechanism.

Characteristics of assimilatory sulfate transport inRhodobacter sulfidophilus

Sulfate uptake was investigated with four species of phototrophic sulfur bacteria. Rhodobacter sulfidophilus and Chromatium vinosum took up 35S-labeled sulfate added in micromolar concentrations. Sulfate uptake by C. vinosum was expressed only under sulfate starvation. R. sulfidophilus took up 10 μM sulfate almost completely and accumulated it up to 5300-fold, also when grown with excess sulfate. Sulfite (1 mM) as an intermediate of sulfate assimilation inhibited sulfate uptake completely within 1 min. Moderate inhibition was observed with cysteine (1 mM) and none with sulfide (1 mM). Transport was not dependent on the cations K+, Na+, Li+ or protons, but was sensitive to uncouplers and to the ATPase inhibitor dicyclohexylcarbodiimide (DCCD). The accumulation of sulfate correlated with the ATP concentration in the cells, indicating an ATP-dependent uptake mechanism.

Occurrence of sulfoquinovosyl diacylglycerol in some members of the family Rhizobiaceae.

A radiolabeled component of a membrane extract of Rhizobium meliloti 2011 cells grown in the presence of 35S-labeled sulfate was isolated by silica flash chromatography and purified by high performance liquid chromatography (HPLC). Based on 1- and 2-dimensional nuclear magnetic resonance (NMR) spectroscopic and mass spectrometric analyses, the structure of the compound was determined to be sulfoquinovosyl diacylglycerol (SQDG). NMR analyses indicated substantial heterogeneity in the fatty acid composition and that an important group was the cyclopropyl fatty acids. This first report of the occurrence of SQDG outside of the plant kingdom, photosynthetic bacteria or diatoms deserves special attention as, in this case, the bacterium is one that can fix nitrogen in symbiosis with plants. The origins of the bacterium's ability to synthesize this class of membrane lipids is an important question. Membrane extracts of other strains of the family Rhizobiaceae were screened for the presence of SQDG. The occurrence of SQDG in the symbiotic organisms was confirmed, while no SQDG was detected in either the Agrobacterium tumefaciens or the Escherichia coli strains tested. The current function of these lipids in symbiosis and the commonality of the ability of bacteria that function as plant symbionts to synthesize such molecules are all germane to studies of the Rhizobium/legume symbiosis.