Morphology and Round Body Formation in Vibrio marinus

The morphology of Vibrio marinus MP-1 was studied by phase and electron microscopy. The ultrastructure of the vibrio form of V. marinus was found to be typically gram-negative with a trilaminar plasma membrane and cell wall. The coccoid or round bodies noted in otherwise pure cultures of V. marinus were frequently found in early and late stationary phase of growth. The round bodies in ultrathin section were found to contain at least one, and often three or four, cell units. Three types of round bodies were observed in ultrathin section, each differing in size and behavior: "spherules," "spheres" or the "round body," and "giant cells" or "macrospheres." The round bodies appeared to be associated with, or to result from, the constrictive cell division of V. marinus.     

New view of the surface projections of Chlamydia trachomatis.

Two kinds of surface specializations of chlamydiae have been described: hemispheric projections and spikelike rods. We undertook the present studies to demonstrate chlamydial ultrastructure in greater detail in conventional thin-sectioned specimens. Chlamydia trachomatis (LGV strain L2/434/Bu), cultured for 40 h in L929 mouse fibroblasts, was fixed in glutaraldehyde-acrolein, p-formaldehyde-glutaraldehyde, or glutaraldehyde-osmium tetroxide mixtures, postfixed in osmium tetroxide, stained in uranyl acetate, dehydrated in ethanols, and embedded in Epon. By the use of fixatives that penetrate and fix rapidly, chlamydial outer and plasma membranes were clearly revealed. Our results indicate that the hemispheric projections are specializations of the plasma membrane of elementary bodies. The spikelike projections are found in intermediate forms, originate beneath depressions of the plasma membrane, and extend through the periplasmic space and outer membrane to end with pointed tips. Improved preservation of chlamydiae provides a new, informative view of their complex structure. Significant interactions between chlamydiae and host cells might be influenced by the surface structures shown in this study.     

Thermolability of Malic Dehydrogenase from the Obligate Psychrophile Vibrio marinus

Langridge, Patricia (Oregon State University, Corvallis), and Richard Y. Morita. Thermolability of malic dehydrogenase from the obligate psychrophile Vibrio marinus. J. Bacteriol. 92:418-423. 1966.-The thermolability of malic dehydrogenase in whole cells of Vibrio marinus MP-1 grown at 15 C was compared with that of cell-free extracts and partially purified fractions. The intracellular enzyme was found to be stable between 0 C, and the organism's optimal growth temperature, 15 C. In cell-free extracts, considerable lability was noted even at 0 C, and this lability did not increase further until the enzyme was exposed to temperatures above the organism's maximal growth temperature (20 C). Twenty-fold purified enzyme was stable between 15 and 20 C, but both above and below this there was considerable inactivation. A 5-min exposure of both cold- and heat-inactivated enzyme to 15 C allowed reactivation, although to a different extent. Ammonium sulfate was found both to stimulate enzyme activity and to reactivate temperature-inactivated enzyme.     

Cloning and sequencing of clustered genes involved in fatty acid biosynthesis from the docosahexaenoic acid-producing bacterium, Vibrio marinus strain MP-1

A cluster of genes involved in fatty acid biosynthesis (fab) was isolated from docosahexaenoic acid (DHA)-producing Vibrio marinus strain MP-1. This fab gene cluster included five genes highly homologous to the Escherichia coli counterparts, and their order in the cluster was the same with that of the E. coli fab gene cluster except that the latter included the additional fabH gene. These fab genes should be involved in early steps of DHA biosynthesis in V. marinus strain MP-1.     

Periplasmic Structure of Frozen-Etched and Negatively Stained Cells of Bacillus licheniformis as Correlated with Penicillinase Formation

Bacillus licheniformis strain 749/C (constitutive for penicillinase formation) and uninduced cells of strain 749 (penicillinase-inducible) were examined after freezeetching. In the early stationary phase, strain 749/C organisms had clusters of vesicles (30 to 40 nm in diameter) on the outer surface of the plasma membrane. These are randomly distributed on the membrane, including the region of septum formation. The vesicles are not intimately associated with the plasma membrane, and their inner and outer surfaces are devoid of particles. Periplasmic vesicles were not detected by freeze-etching in strain 749 (uninduced) or in young cells of 749/C; however, the membrane of mid-logarithmic phase 749/C cells had a corrugated appearance. Negatively stained 749/C cells (logarithmic phase) also showed many vesicular and tubular bodies in the periplasm as well as septal and cytoplasmic mesosomes of typical morphology. The periplasmic structures appear to be formed either by evagination of plasma membrane or by migration of vesicular bodies from the membranous pockets of the cytoplasm. Stationary phase cells of 749/C still have many periplasmic vesicular bodies; however, the mesosomes are greatly reduced both in number and size. In sharp contrast, strain 749 organisms have very few structures similar to the periplasmic bodies of strain 749/C. These findings support our previous view that penicillinase-producing cells of 749/C have periplasmic membranous structures that are rare in the uninduced strain 749, though there is some lack of correspondence between freeze-etching, negative staining, and thin section data. These structures may be important for the retention or storage of penicillinase in the cell.     

Conformational change and localization of calpactin I complex involved in exocytosis as revealed by quick-freeze, deep-etch electron microscopy and immunocytochemistry

Calpactin I complex, a calcium-dependent phospholipid-binding protein, promotes aggregation of chromaffin vesicles at physiological micromolar calcium ion levels. Calpactin I complex was found to be a globular molecule with a diameter of 10.7 +/- 1.7 (SD) nm on mica. When liposomes were aggregated by calpactin, quick-freeze, deep-etching revealed fine thin strands (6.5 +/- 1.9 [SD] nm long) cross-linking opposing membranes in addition to the globules on the surface of liposomes. Similar fine strands were also observed between aggregated chromaffin vesicles when they were mixed with calpactin in the presence of Ca2+ ion. In cultured chromaffin cells, similar cross-linking short strands (6-10 nm) were found between chromaffin vesicles and the plasma membrane after stimulation with acetylcholine. Plasma membranes also revealed numerous globular structures approximately 10 nm in diameter on their cytoplasmic surface. Immunoelectron microscopy on frozen ultrathin sections showed that calpactin I was closely associated with the inner face of the plasma membranes and was especially conspicuous between plasma membranes and adjacent vesicles in chromaffin cells. These in vivo and in vitro data strongly suggest that calpactin I complex changes its conformation to cross-link vesicles and the plasma membrane after stimulation of cultured chromaffin cells.     

苏云金芽孢杆菌发酵上清中增效物质生成的相关研究

利用BIOSTAT ^?-CL1 5L全自动发酵罐和 2 0t不锈钢发酵罐 ,对苏云金芽孢杆菌不同菌株 (GC-91 ,MP342 ,HD-1 )发酵上清液中增效物质的生成进行了研究 ,发现增效物质于对数生长期前期开始产生并积累 ,至对数生长期末期达到高峰 ,并保持稳定 ;不同菌株的发酵上清中增效物质生成量不同 ,其中GC-91最强 (增效倍数f=6.0 ) ,MP342次之 (f=3.7) ,HD 1最弱 (f=1.5 ) ;GC-91菌株上清液中增效物质生成曲线与晶体含量 ,效价代     

Isolation of a sodium dodecyl sulfate-insoluble transglutaminase substrate from liver plasma membranes

Rat liver plasma membranes contain transglutaminase activity and a large molecular weight protein complex which serves as a substrate for this enzyme. When plasma membranes were solubilized in sodium dodecyl sulfate and disulfide-reducing agents the transglutaminase substrate was recovered in the detergent-insoluble fraction. The insolubility of the complex suggested that it might be further studied by adsorbing membranes onto glass slides, then extracting with the detergent and reducing agent. After extraction, dark field light microscopy revealed numerous flattened sheets which varied in size from 4 to 12 micrometers. To confirm that these structures were the large molecular weight transglutaminase substrate, the plasma membranes were solubilized in sodium dodecyl sulfate and dithiothreitol and sedimented through a sucrose gradient containing the agent. The large molecular weight substrate was the only material found at the 1.11/1.23 g/cm3 interface. Microscopic examination showed the same structures previously observed on the glass slides. We conclude that the large molecular weight transglutaminase substrate is a sodium dodecyl sulfate-insoluble, morphologically distinct, protein complex. Due to its considerable size, nondissociable nature, and association with the lateral membrane, the sodium dodecyl sulfate-insoluble transglutaminase substrate may serve as a type of skeleton or scaffolding for this plasma membrane domain.     

Detergent-free domain isolated from Xenopus egg plasma membrane with properties similar to those of detergent-resistant membranes

Microdomains known as "rafts" have been isolated from many cell types as detergent-resistant membranes (DRMs) and are enriched in sphingolipids and cholesterol. However, there has been considerable controversy over whether such domains are found in native membranes or are artificially generated by the purification procedure. This controversy is based at least in part on the fact that raft membranes were first detected following detergent extraction in the cold. We isolated two plasma membrane fractions, without detergent treatment, using a discontinuous sucrose density gradient. One fraction was designated "light" and the other "heavy." These fractions were compared with DRMs, which were isolated in the presence of 1% Triton X-100. We found that Xenopus DRMs are enriched with sphingomyelin and cholesterol and exhibit a phase state similar to the liquid-ordered phase. Comparison of DRM complexes with the light and heavy plasma membrane fractions revealed some physical and biochemical similarities between the light fraction of the plasma membrane and the DRM complexes, based on (1) the phosphatidylcholine/sphingomyelin ratio and (2) the protein composition visualized on a two-dimensional gel. These two fractions are also quite similar in their thermotropic phase behavior, and their high levels of ganglioside GM1. We conclude that the light membrane fraction isolated in a detergent-free environment has many of the characteristics normally associated with DRMs.     

Isolation and partial characterization of the plasma membrane of the sea urchin egg

The cell surface complex (Detering et al., 1977, J. Cell Biol. 75, 899-914) of the sea urchin egg consists of two subcellular organelles: the plasma membrane, containing associated peripheral proteins and the vitelline layer, and the cortical vesicles. We have now developed a method of isolating the plasma membrane from this complex and have undertaken its biochemical characterization. Enzymatic assays of the cell surface complex revealed the presence of a plasma membrane marker enzyme, ouabain-sensitive Na+/K+ ATPase, as well as two cortical granule markers, proteoesterase and ovoperoxidase. After separation from the cortical vesicles and purification on a sucrose gradient, the purified plasma membranes are recovered as large sheets devoid of cortical vesicles. The purified plasma membranes are highly enriched in the Na+/K+ ATPase but contain only very low levels of the proteoesterase and ovoperoxidase. Ultrastructurally, the purified plasma membrane is characterized as large sheets containing a "fluffy" proteinaceous layer on the external surface, which probably represent peripheral proteins, including remnants of the vitelline layer. Extraction of these membranes with Kl removes these peripheral proteins and causes the membrane sheets to vesiculate. Polyacrylamide gel electrophoresis of the cell surface complex, plasma membranes, and Kl-extracted membranes indicates that the plasma membrane contains five to six major proteins species, as well as a large number of minor species, that are not extractable with Kl. The vitelline layer and other peripheral membrane components account for a large proportion of the membrane-associated protein and are represented by at least six to seven polypeptide components. The phospholipid composition of the Kl-extracted membranes is unique, being very rich in phosphatidylethanolamine and phosphatidylinositol. Cholesterol was found to be a major component of the plasma membrane. Before Kl extraction, the purified plasma membranes retain the same species-specific sperm binding property that is found in the intact egg. This observation indicates that the sperm receptor mechanisms remain functional in the isolated, cortical vesicle-free membrane preparation.     

THE FINE STRUCTURE OF DIPLOCOCCUS PNEUMONIAE

The fine structure of an unencapsulated strain of Diplococcus pneumoniae is described. A striking feature of these bacteria is an intracytoplasmic membrane system which appears to be an extension of septa of dividing bacteria. The possible function of these structures and their relationship to the plasma membrane and other types of intracytoplasmic membranes found in pneumococcus is discussed.     

Electron Microscopy of Peripheral Nerves and Neuromuscular Junctions in the Wasp Leg

The peripheral nerve branch innervating the femoral muscles of the common yellow jacket (Vespula carolina) has been found to possess a thick lemnoblast basement membrane and a complex mesaxon. The term "tunicated nerve" is proposed to designate the type of peripheral nerve in which one or several axons are loosely mantled by meandering, cytoplasm-enclosing membranes of the lemnoblast. The peripheral axon courses longitudinally in a groove in the muscle fiber between the plasma membrane of the muscle fiber and a cap formed by lemnoblast and tracheoblast. The junction is characterized by apposition of plasma membranes of axon and muscle fiber, abundant mitochondria, and synaptic vesicles in the axon, and aggregates of "aposynaptic granules" plus mitochondria and endoplasmic reticulum on the muscle side of the synapse. Unlike the vertebrate striated muscle fiber, no complex infolding of the synapsing plasma membrane of the muscle fiber occurs. The "connecting tissue" of the insect is formed by tracheoblasts, their basement membranes, and the basement membranes of other cells. Further mechanical support is given by the ramifying tracheoles. The physiologic roles of the specialized structures are considered.     

Ultrastructure of L forms. IV. L forms of nonagglutinating vibrios]

A study was made of the ultrastructure of stable L-forms of Nag vibrios aged 24 hours. Cells of all types of the L-forms had cytoplasmic membranes, and a three-layered structure, which was found not everywhere. Externally of the cytoplasmic membrane, in some areas of the individual cells there were revealed a plastic layer of cell wall and a basal membrane. However, in difference to bacterial forms of the vibryos, rigidity of the cell wall was disturbed, and the links between the cell wall and the cytoplasmic membrane were indetectable. There were regularly revealed lamellar of myelin-like membranous structures in the cytoplasm, which did not occur in bacterial forms, and also lamellar mesosomes. The latter were found in the sites of cell division. Viability of small bodies as the minimal reproductive forms of the L-cultures is confirmed by the presence in them of a nucleoid and of the binary division.     

The oxygen-evolving complex of chloroplast membranes

The oxygen-evolving complex (OEC) of plants is the main energy-transforming structure of chloroplast membranes, in which light energy is used for photosynthetic oxidation of intracellular water and oxygen formation. The conducted research has resulted in isolation of functionally active OEC of higher plants and elucidation of its molecular composition, photochemical properties and structural organization. The OEC has been revealed to represent the dimer of the pigment-lipoprotein complexes of photosystem 2 (PLPC PS-2) associated in a chloroplast membrane according to the mirror symmetry rule into an integrate structure based on hydrophobic bonds. The model has been developed for the structure of the dimeric complex of PS-2 that has the function of oxygen formation. This model was confirmed by the X-ray analysis of crystals of the dimeric complex of PS-2. The concept about the fact that the “hydrophobic boiler” determining the formation of the water-oxidizing center of the OEC is formed in the area of association of the reaction centers of monomeric PLPCs PS-2 was advanced based on the regularities of change in the functional activity of the OEC under the action of stress-factors. The new scheme has been advanced for the two-anode organization of the water-oxidizing center as the main condition for realizing the process of molecular oxygen formation. The mechanism of the process of photosynthetic water oxidation and molecular oxygen formation has been developed based on the experimental data about the structural organization of the OEC and its water-oxidizing center. The quantum-chemical modeling of the process showed that its course corresponds to the mechanism suggested.     

Morphology and Ultrastructure of Mycoplasma pneumoniae Spherules

During growth in fluid medium, most strains of Mycoplasma pneumoniae produce free-floating granules which become larger with time. We have called these granules "spherules." This study describes the morphological and ultrastructural features of M. pneumoniae strain AP-164 spherules, both free and in association with HeLa cells in cell culture. In thin section, spherules were composed of lobulated cells, connected together by membranes, and ring-shaped cells. The two-dimensional morphology observed varied according to the plane of section and to the age of the culture. In HeLa cell cultures, mycoplasmata were found attached to plasma membranes of cells; in sections, individual mycoplasmata were often aligned in radial apposition to the membranes. Mycoplasmata were not found intracellularly. The three-dimensional morphology of spherules was examined by the critical point method and by scanning electron microscopy. Both methods revealed lobulated forms, ring-shaped forms, and star-shaped forms. Treatment of the spherules with crude porcine pancreatic lipase effectively released large numbers of free organisms. Phosphotungstic acid preparations of these uncentrifuged forms revealed a morphology in agreement with the other methods used. Lobulated ring forms with "beaded" filaments were prominent. In respect to morphology, M. pneumoniae under our conditions resembles that of the type species M. mycoides.     

Dimerisation and an increase in active site aromatic groups as adaptations to high temperatures: X-ray solution scattering and substrate-bound crystal structures of Rhodothermus marinus endoglucanase Cel12A

Cellulose, a polysaccharide consisting of beta-1,4-linked glucose, is the major component of plant cell walls and consequently one of the most abundant biopolymers on earth. Carbohydrate polymers such as cellulose are molecules with vast diversity in structure and function, and a multiplicity of hydrolases operating in concert are required for depolymerisation. The bacterium Rhodothermus marinus, isolated from shallow water marine hot springs, produces a number of carbohydrate-degrading enzymes including a family 12 cellulase Cel12A. The structure of R.marinus Cel12A in the ligand-free form (at 1.54 angstroms) and structures of RmCel12A after crystals were soaked in cellopentaose for two different lengths of time, have been determined. The shorter soaked complex revealed the conformation of unhydrolysed cellotetraose, while cellopentaose had been degraded more completely during the longer soak. Comparison of these structures with those of mesophilic family 12 cellulases in complex with inhibitors and substrate revealed that RmCel12A has a more extensive aromatic network in the active site cleft which ejects products after hydrolysis. The substrate structure confirms that during hydrolysis by family 12 cellulases glucose does not pass through a (2,5)B conformation. Small-angle X-ray scattering analysis of RmCel12A showed that the enzyme forms a loosely associated antiparallel dimer in solution, which may target the enzyme to the antiparallel polymer strands in cellulose.     

Morphology of Halprowia (Chlamydia) isolated in Reiter's syndrome]

Morphology and ultrastructure of Halprowia arthritidis, strain SR-1 (HSR), isolated from the synovial fluid of a patient with Reiter's syndrome, was studied in the membranes of the yolk sacs of the developing chick embryos and the L-cell culture. In acridine orange staining for light and fluorescent microscopy there was revealed intracellular cytoplasmic inclusions containing HSR structures at various stages of its reproduction characteristic of halprowia (chlamydia). The direct immunofluorescent method demonstrated the presence of a characteristic HSR antigen not only in the developed inclusions, but also at the early stages of infection, when the morphological HSR structures could not be found by light microscopy. The ultrastructure of the HSR inclusions and forms in the cycle of development (of the initial and elementary bodies) of the SR-1 strain was typical of other halprowia. A peculiar structure of a complex of cell wall and cytoplasmic membrane of the elementary body was described. Taking into consideration the biological characteristics of HSR revealed earlier it can be considered to be a typical representative of Halprowiales s. Chlamydiales. The data obtained on other halprowia, pointing out the fact that criteria of compactness and diffuseness of inclusions, the presence of absence of glycogen in the inclusions could not serve as taxonomic signs in classification of halrpowia, were confirmed on a model of the SR-1 studied.     

Spin-label studies on rat liver and heart plasma membranes: do probe-probe interactions interfere with the measurement of membrane properties?

The structures of purified rat liver and heart plasma membranes were studied with the 5-nitroxide stearic acid spin probe, I(12,3). ESR spectra were recorded with a 50 gauss field sweep, and also with a new technique which "expands" the spectrum by (1) recording pairs of adjoining peaks with a smaller field sweep and (2) superposing the common peaks. The hyperfine splittings measured from the "expanded" spectra were significantly more precise than those obtained from the "unexpanded" spectra. Both procedures were used to study the effects of various I(12,3) probe concentrations on the spectra of liver and heart membranes, as well as the effects of temperature and CaCl2 additions on the spectra of liver membranes, and revealed the following: The polarity-corrected order parameters of liver (31 degrees) and heart (22 degrees) membranes were found to be independent of the probe concentration, if experimentally-determined low I(12,3)/lipid ratios were employed. The absence of obvious radical-interaction broadening in the unexpanded spectra indicated that "intrinsic" membrane properties may be measured at these low probe/lipid ratios. Here, "intrinsic" properties are defined as those which are measured when probe-probe interactions are negligible, and do not refer to membrane behavior in the absence of a perturbing spin label. At higher I(12,3)/lipid ratios, the order parameters of liver and heart membranes were found to substantially decrease with increasing probe concentration. The increase in the "apparent" fluidity of both membrane systems is attributed to enhanced radical interactions; however, an examination of these spectra (without reference to "low" probe concentration spectra) might incorrectly suggest that radical interactions were absent. For the membrane concentrations employed in these studies, the presence of "liquid-lines" (or "fluid components") in the unexpanded ESR spectra was a convenient marker of high probe concentrations. A thermotropic phase separation was observed in liver membranes between 19 degrees and 28 degrees. Addition of CaCl2 to liver plasma membrane [labelled with "low" I(12,3) concentrations] increased the rigidity of the membrane at 31 degrees and 37 degrees, without inducing a segregation of the probe in the bilayer. Previously reported data are discussed in relation to these results, and suggested minimal criteria for performing membrane spin label studies are included.     

Electron tomography of the Maurer's cleft organelles of Plasmodium falciparum-infected erythrocytes reveals novel structural features

During intraerythrocytic development, the human malaria parasite, Plasmodium falciparum, establishes membrane-bound compartments, known as Maurer's clefts, outside the confines of its own plasma membrane. The Maurer's compartments are thought to be a crucial component of the machinery for protein sorting and trafficking; however, their ultrastructure is only partly defined. We have used electron tomography to image Maurer's clefts of 3D7 strain parasites. The compartments are revealed as flattened structures with a translucent lumen and a more electron-dense coat. They display a complex and convoluted morphology, and some regions are modified with surface nodules, each with a circular cross-section of approximately 25 nm. Individual 25 nm vesicle-like structures are also seen in the erythrocyte cytoplasm and associated with the red blood cell membrane. The Maurer's clefts are connected to the red blood cell membrane by regions with extended stalk-like profiles. Immunogold labelling with specific antibodies confirms differential labelling of the Maurer's clefts and the parasitophorous vacuole and erythrocyte membranes. Spot fluorescence photobleaching was used to demonstrate the absence of a lipid continuum between the Maurer's clefts and parasite membranes and the host plasma membrane.     

The ultrastructure of plasma and thylakoid membrane vesicles from the fresh water cyanobacteriumAnacystis nidulans adapted to salinity

Summary Photoautotrophically growing cultures of the fresh water cyanobacteriumAnacystis nidulans adapted to the presence of 0.4–0.5 M NaCl (about sea water level) with a lag phase of two days after which time the growth rate reassumed 80–90% of the control. Plasma and thylakoid membranes were separated from cell-free extracts of French pressure cell treatedAnacystis nidulans by discontinuous sucrose density gradient centrifugation and purified by repeated recentrifugation on fresh gradients. Identity of the plasma and thylakoid membrane fractions was confirmed by labeling of intact cells with impermeant protein markers prior to breakage and membrane isolation. Electron microscopy revealed that each type of membrane was obtained in the form of closed and perfectly spherical vesicles. Major changes in structure and function of the plasma membranes (and, to a much lesser extent, of the thylakoid membranes) were found to accompany the adaptation process. On the average, diameters of plasma membrane vesicles from salt adapted cells were only one-third of the diameters of corresponding vesicles from control cells. By contrast, the diameters of thylakoid membrane vesicles were the same in both cases.Freeze-etching the cells and counting the number of membrane-intercalating particles on both protoplasmic and exoplasmic fracture faces of plasma and thylakoid membranes indicated a roughly 50% increase of the particle density in plasma membranes during the adaptation process while that in thylakoid membranes was unaffected. Comparison between particle densities on isolated membranes and those on corresponding whole cell membranes permitted an estimate as to the percentage of inside-out and right-side-out vesicles. Stereometric measurement of particle sizes suggested that two distinct sub-populations of the particles in the plasma membranes increased during the adaptation process, tentatively correlated to the cytochrome oxidase and sodium-proton antiporter, respectively. The effects of salt adaptation described in this paper were fully reversed upon withdrawal of the additional NaCl from the growth medium (deadaptation). Moreover, they were not observed when the NaCl was replaced by KCl.Abbreviations CM cytoplasmic or plasma membrane - ICM intracytoplasmic or thylakoid membrane - EF exoplasmic fracture face - PF protoplasmic fracture face - DABS diazobenzosulfonate; Hepes N-2-hydroxyethylpiperazine-N-2-ethane-sulfonate - PMSF phenylmethylsulfonylfluoride Dedicated to the memory of Professor Oswald Kiermayer