Structural similarity of the membrane envelopes of rhizobial bacteroids and the host plasma membrane as revealed by freeze-fracturing.

The freeze-fracture technique was used to study the host plasma membrane and the membrane envelope of bacteroids in rhizobial root nodules of three host-rhizobium combinations. In all three combinations studied, the membrane envelopes of bacteroids are structurally similar to their host plasma membrane. However, the membrane appears to be reversed, because the number and arrangement of particles in the outer fractured face (face A, concave) and in the inner fractured face (face B, convex) of the host plasma membrane are seen, respectively, in the inner fractured face (face B, convex) and in the outer fractured face (face A, concave) of the membrane envelope of the bacteroids at an early stage. This reversion of the membrane surface is consistent with the hypothesis that the membrane envelopes of bacteroids are derived from the host plasma membrane during endocytotic engulfment.     

Isolation and characterization of the membrane envelope enclosing the bacteroids in soybean root nodules

The membrane envelope enclosing the bacteroids in soybean root nodules is shown by ultrastructural and biochemical studies to be derived from, and to retain the characteristics of, the host cell plasma membrane. During the early stages of the infection process, which occurs through an invagination, Rhizobium becomes surrounded by the host cell wall and plasma membrane, forming the infection thread. The cell wall of the infection thread is degraded by cellulolytic enzyme(s), leaving behind the enclosed plasma membrane, the membrane envelope. Cellulase activity in young nodules increases two- to threefold as compared to uninfected roots, and this activity is localized in the cell wall matrix of the infection threads. Membrane envelopes were isolated by first preparing bacteroids enclosed in the envelopes on a discontinuous sucrose gradient followed by passage through a hypodermic needle, which released the bacteroids from the membranes. This membrane then sedimented at the interface of 34--45% sucrose (mean density of 1.14 g/cm3). Membranes were characterized by phosphotungstic acid (PTA)-chromic acid staining. ATPase activity, and localization, sensitivity to nonionic detergent Nonidet P-40 (NP-40) and sodium dodecyl sulfate (SDS) gel electrophoresis. These analyses revealed a close similarity between plasma membrane and the membrane envelope. Incorporation of radioactive amino acids into the membrane envelope proteins was sensitive to cycloheximide, suggesting that the biosynthesis of these proteins is primarily under host-cell control. No immunoreactive material to leghemoglobin antibodies was found inside or associated with the isolated bacteroids enclosed in the membrane envelope, and its location is confined to the host cell cytoplasmic matrix.     

超结瘤大豆根瘤的亚显微结构

电镜观察表明,超结瘤大豆未受侵染的宿主细胞中有一明显增大的细胞核。幼年美菌体为椭圆形,里面有个拟核区,正常类菌体有完整的周膜和PHB颗粒。受侵染的寄主细胞中出现类似无效根瘤的异常现象：少数类菌体退化或溶解,还有空周膜及裸露的类菌体,这可能是超结瘤大豆固氮活性较低的原因。     

灰金合欢根瘤类菌体繁殖及其宿主细胞的超微结构

根瘤细胞早期发育阶段,以宿主细胞器和根瘤菌转化类菌体的数量增多为特征。随后类菌体增殖到填满宿主细胞内的大部分区域。各个类菌体周膜内含有1至几个类菌体。晚期共生发育阶段,类菌体细胞结构和宿主细胞器数量发生了变化。文中还讨论了根瘤的共生固氮作用。     

侵染细胞质膜附近小泡的来源及其作用

箭舌豌豆根瘤幼龄侵染细胞的壁和质膜比较光滑,成熟侵染细胞与此不同,不仅细胞壁厚薄均,有较多的胞间连丝,而且质膜常常内陷形成各种突起,然后离质膜形成小泡。这些位于质膜附近的小泡体积较小,多呈圆形,既可单独存在,也可多个聚在一起。在向细胞中央移动中,有的小泡靠近细胞质膜,甚至与细菌周期融合,有的小泡不民附近的小液泡融合变为较大液泡,并常用降解程度不同的细菌位于其中,在衰老侵染细胞中,细胞壁附近有小泡,     

Proteins Specified by Herpes Simplex Virus IX. Contiguity of Host and Viral Proteins in the Plasma Membrane of Infected Cells

Artificial mixtures of plasma membrane vesicles produced by microcavitation from infected and uninfected cells band at the same density on isopycnic centrifugation in sucrose density gradient. However, after reaction with antiviral antibody, the density of the infected cell plasma membrane vesicles increases, and the infected and uninfected cell membranes are quantitatively separable on isopycnic centrifugation. Plasma membrane vesicles prepared from cells doubly labeled before and after infection with radioactive amino acids and reacted with antibody banded at a high density. Polyacrylamide gel electropherograms show that the vesicles reacted with antibody consist of both host- and virus-specific membrane proteins. Microcavitation does not disrupt viral envelopes since infectivity is not affected by this procedure. We conclude that viral and cellular proteins in the plasma membrane preparations are contiguous.     

Proteins Specified by Herpes Simplex Virus VI. Viral Proteins in the Plasma Membrane

Previous papers in the series have shown that the surface membranes of herpesvirus-infected cells acquire new immunological specificities and that purified infected cell membrane preparations, characterized by their physical properties rather than topology in the cell, contain new glycoproteins genetically determined by the virus. In this study, we prepared purified plasma membrane identified by its 5' nucleotidase, fucose, and reduced nicotinamide adenine dinucleotide-diaphorase content. Analysis of the membrane proteins and glycoproteins by electrophoresis in acrylamide gels indicated the following. (i) Purified plasma membranes from infected cells contained two sets of proteins, i.e., host proteins were present both before and after infection and viral proteins were present only after infection. (ii) After infection, no appreciable selective or nonselective loss of host proteins from membranes was demonstrable. However, no new host proteins were made. (iii) Electropherograms of plasma membrane proteins from infected cells indicated the presence of at least 12 virus-specific proteins ranging in molecular weight from 25 x 10(3) to 126 x 10(3) daltons. Of these, at least nine were glycosylated. Proteins and glycoproteins with similar electrophoretic mobilities but in somewhat different ratios were also present in preparations of highly purified virions.     

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.     

箭舌豌豆根瘤液泡中细菌周膜来源的研究

电镜观察结果表明,幼龄箭舌豌豆根瘤侵染细胞的细胞质较少,中央是一些体积较大的液泡。细胞质中侵入线经常可见,由侵入线释放出来的细菌均有细菌周膜。这些细菌只位于细胞质中,不出现在液泡里面。成熟根瘤中的侵染细胞与此不同,它们中有大量的成熟侵染细胞,细胞质丰富,里面充满大量细菌,中央常有一个大液泡。当中央液泡发育到一定程度时,位于其附近的细菌可通过液泡膜内吞、液泡膜与细菌周膜融合及液泡膜破裂3种途径进入液泡,后一种途径常伴有寄主细胞质。液泡中的细菌绝大部分裸露在外,只有个别细菌具有细菌周膜且多位于液泡膜的破损处附近,因此细菌周膜可能是原来就有的。     

Lysis of bacterioids in the vicinity of the host cell nucleus in an ineffective (fix-) root nodule of soybean (Glycine max)

In nodules of Glycine max cv. Mandarin infected with a nod ⁺fix^- mutant of Rhizobium japonicum (RH 31-Marburg), lysis of bacteroids was observed 20 d after infection, but occurred in the region around the host cell nucleus, where lytic compartments were formed. Bacteroids, and peribacteroid membranes in other parts of the host cell remained stable until senescence (40d after infection). With two other nod⁺ fix^- mutants of R. japonicum either stable bacteroids and peribacteroid membranes were observed throughout the cell (strain 61-A-165) or a rapid degeneration of bacteroids without an apparent lysis (strain USDA 24) occurred. The size distribution of RH 31-Marburg-infected nodules exhibited only two maxima compared with four in wild-type nodules and nodule leghaemoglobin content was found to be reduced to about one half that of the wild type. The RH 31-Marburg-nodule type is discussed in relation to the stability of the bacteroids and the peribacteroid membrane system in soybean.     

银合欢根瘤细胞的光学显微镜和电子显微镜研究

银合欢接种根瘤菌形成根瘤后,应用光镜和电镜技术观察。银合欢根瘤由分生组织细胞、皮层组织细胞、维管束系统和侵染细胞区域四个不同部分组成。根瘤菌借助于侵染线侵染细胞,释放进入宿主细胞质中,转变成固氮类菌体。最初每个包被膜内只含单独的类菌体,随后较老的侵染细胞中,每个包被膜内含有一个以上的类菌体。因此,成熟根瘤的侵染细胞可见有2~5个类菌体群集包被膜里,并且明显地累积PHB物质,显示电子染色透明颗粒。本文还讨论了上述变化的意义与银合欢根瘤细胞结构和功能的关系。     

Cell wall (outer membrane) of bacteroids in nitrogen-fixing peanut nodules

Rhizobia in peanut nodules are transformed from rod-shaped cells to extremely large spherical bacteroids. The invading rhizobia shed their outer membranes soon after their release into the host cells. The outer membranes are seen to be peeled off and replaced by new outer membranes before rhizobia are differentiated into nitrogen-fixing bacteroids. Evidence is presented that peanut bacteroids, in spite of their spheroplast-or protoplast-like appearance, do possess both the inner and the outer membrane.     

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.     

Properties of the Peribacteroid Membrane ATPase of Pea Root Nodules and Its Effect on the Nitrogenase Activity

Peribacteroid membrane vesicles from pea (Pisum sativum) root nodules were isolated from membrane-enclosed bacteroids by an osmotic shock. The ATPase activity associated with this membrane preparation was characterized, and its electrogenic properties were determined. The pH gradient was measured as a change of the fluorescence intensity of 9-amino-6-chloro-2-methoxyacridine and the membrane potential as a shift of absorbance of bis-(3-propyl-5-oxoisoxazol-4-yl)pentamethine oxonol. It was demonstrated that the ATPase generates a pH gradient as well as a membrane potential across the peribacteroid membrane. The reversibility of the ATPase was demonstrated by a light-dependent ATP synthesis by peribacteroid membrane vesicles fused with bacteriorhodopsin-phospholipid vesicles. The light-driven ATP synthesis by the peribacteroid membrane ATPase was completely inhibited by a proton-conducting ionophore. The proton-pumping activity of the peribacteroid membrane ATPase could also be demonstrated with peribacteroid membrane-enclosed bacteroids, and effects on nitrogenase activity were established. At pH values below 7.5, an active peribacteroid membrane ATPase inhibited the nitrogenase activity of peribacteroid membrane-enclosed bacteroids. At pH values above 8, at which whole cell nitrogenase activity was inhibited, the protonpumping activity of the peribacteroid membrane ATPase could partially reverse the pH inhibition. Vanadate, an inhibitor of plasma membrane and peribacteroid membrane ATPases, stimulated nodular nitrogenase activity. It will be proposed that the proton-pumping activity of the peribacteroid membrane ATPase in situ is a possible regulator of nodular nitrogenase activity.     

Membrane production and yolk degradation in the early fly embryo (Calliphora erythrocephala meig.): An ultrastructural analysis

Formation of nuclear envelopes during the last cleavage mitosis and the formation of the cell membranes during the cellularization of the blastoderm have been studied ultrastructurally in the blowfly egg. Dense bodies arising from yolk granules by budding could contain membrane material destined to be incorporated into the new membranes of the blastoderm. The presence of transitional structures indicates that these bodies can be converted into dark multivesicular bodies. Large amounts of endoplasmic reticulum are found around the mitotic nuclei. Clusters or branched chains of vesicles associated with this are interpreted as evidence for the formation of endoplasmic reticulum by the breakdown of dark multivesicular bodies. Nuclear envelopes of mitotic daughter nuclei probably originate from endoplasmic reticulum. The egg contains both intranuclear and extranuclear annulate lamellae. The main events of cytokinesis are furrow initiation and cell membrane growth during the slow first phase, but probably only cytokinetic movement during the rapid second phase. On the assumption that cell membrane growth occurs by incorporation of complete membrane pieces, the addition of coated vesicles and/or light multivesicular bodies is definitely most probable. Some intermediate profiles indicate that light and dark multivesicular bodies are related. The membrane needed for second phase cytokinesis could well be provided by the unfolding of surface microvilli and protuberances of the furrow canal.     

Heterogeneous Distribution of Membrane Cholesterol at the Attachment Site of Cryptosporidium muris to Host Cells

ABSTRACT. Distribution of membrane cholesterol at the attachment site of Cryptosporidium muris was investigated by freeze-fracture cytochemistry using a polyene antibiotic filipin. Since the host plasma membrane enveloped C. muris , the inner and outer membranes were continuous with the parasite plasma membrane at the annular ring and with host membrane at the dense band, respectively. Although many filipin-cholesterol complexes were observed on the plasma membrane of host cells and parasites, a line showing no complexes was evident at the above two membrane junctures. These observations indicate that parasitic infection of C. muris altered the organization of membrane cholesterol.     

Ultrastructural characteristics of the host-symbiont interface in nitrogen-fixing peanut nodules

Summary Nitrogen-fixing peanut root nodules are characterized by their unique structural organization, distinct from other legume nodules. The focus of this study has been in and around the hostsymbiont interface, where the bacterioid and the host cell surface (peribacteroid membrane envelope) interact during symbiosis. The infected nodule cells have revealed the presence of lipid bodies (oleosomes) in intimate association with the peribacteroid membrane, which encloses the large spherical bacteroids with a relatively narrow peribacteroid space. Electron dense structures, referred to as dense bodies have been found attached to the bacteroid outer membranes at the host-symbiont interface. The dense bodies are osmiophilic, amorphous and 3,3-diaminobenzidine positive. The isolated intact bacteroids with dense bodies attached to their cell wall showed significant catalase activity. Many microbodies showing DAB-positive reaction have been found in the host cytoplasm, associated closely with the peribacteroid membrane. These ultrastructural and cytochemical characteristics of peanut root nodules suggest that lipids are utilized during symbiosis and the dense bodies and microbodies may be involved in the catabolic process.Abbreviation DAB 3,3-diaminobenzidine     

Membrane interaction of Pasteurella multocida toxin involves sphingomyelin

Pasteurella multocida toxin (PMT) is an AB toxin that causes pleiotropic effects in targeted host cells. The N-terminus of PMT (PMT-N) is considered to harbor the membrane receptor binding and translocation domains responsible for mediating cellular entry and delivery of the C-terminal catalytic domain into the host cytosol. Previous studies have implicated gangliosides as the host receptors for PMT binding. To gain further insight into the binding interactions involved in PMT binding to cell membranes, we explored the role of various membrane components in PMT binding, utilizing four different approaches: (a) TLC-overlay binding experiments with (125) I-labeled PMT, PMT-N or the C-terminus of PMT; (b) pull-down experiments using reconstituted membrane liposomes with full-length PMT; (c) surface plasmon resonance analysis of PMT-N binding to reconstituted membrane liposomes; (d) and surface plasmon resonance analysis of PMT-N binding to HEK-293T cell membranes without or with sphingomyelinase, phospholipase D or trypsin treatment. The results obtained revealed that, in our experimental system, full-length PMT and PMT-N did not bind to gangliosides, including monoasialogangliosides GM(1) , GM(2) or GM(3) , but instead bound to membrane phospholipids, primarily the abundant sphingophospholipid sphingomyelin or phosphatidylcholine with other lipid components. Collectively, these studies demonstrate the importance of sphingomyelin for PMT binding to membranes and suggest the involvement of a protein co-receptor.     

Fusogenic properties of reconstituted hybrid vesicles containing Sendai and influenza envelope glycoproteins: fluorescence dequenching and fluorescence microscopy studies

Co-reconstitution of influenza and Sendai virus phospholipids and glycoproteins resulted in the formation of membrane vesicles containing the envelope glycoproteins from both viruses within the same membrane. Reconstituted influenza-Sendai hybrids (RISH) were able to lyse human erythrocytes and fuse with their membranes or with living cultured cells at pH 5.0 as well as at pH 7.4, thus exhibiting the fusogenic properties of both viruses. This was also inferred from experiments showing that the fusogenic activity of RISH was inhibited by anti-influenza as well as by anti-Sendai virus antibodies. Fusion of FISH and of reconstituted influenza (RIVE) or reconstituted Sendai virus envelopes (RSVE) with recipient membranes was determined by the use of fluorescently labeled envelopes and fluorescence dequenching methods. Observations with the fluorescence microscope were used to study localization of fused reconstituted envelopes within living cells. Incubation of RISH and RSVE with living cells at pH 7.4 resulted in the appearance of fluorescence rings around the cell plasma membranes and of intracellular distinct fluorescent spots indicating fusion with cell plasma membranes and with membranes of endocytic vesicles, respectively. The fluorescence microscopy observations clearly showed that RIVE failed to fuse, at pH 7.4, with cultured cell plasma membranes, but fused with membranes of endocytic vesicles.     

Fusogenic properties of reconstituted hybrid vesicles containing Sendai and influenza envelope glycoproteins: fluorescence dequenching and fluorescence microscopy studies

Co-reconstitution of influenza and Sendai virus phospholipids and glycoproteins resulted in the formation of membrane vesicles containing the envelope glycoproteins from both viruses within the same membrane. Reconstituted influenza-Sendai hybrids (RISH) were able to lyse human erythrocytes and fuse with their membranes or with living cultured cells at pH 5.0 as well as at pH 7.4, thus exhibiting the fusogenic properties of both viruses. This was also inferred from experiments showing that the fusogenic activity of RISH was inhibited by anti-influenza as well as by anti-Sendai virus antibodies. Fusion of FISH and of reconstituted influenza (RIVE) or reconstituted Sendai virus envelopes (RSVE) with recipient membranes was determined by the use of fluorescently labeled envelopes and fluorescence dequenching methods. Observations with the fluorescence microscope were used to study localization of fused reconstituted envelopes within living cells. Incubation of RISH and RSVE with living cells at pH 7.4 resulted in the appearance of fluorescence rings around the cell plasma membranes and of intracellular distinct fluorescent spots indicating fusion with cell plasma membranes and with membranes of endocytic vesicles, respectively. The fluorescence microscopy observations clearly showed that RIVE failed to fuse, at pH 7.4, with cultured cell plasma membranes, but fused with membranes of endocytic vesicles.