Gas vesicles in actinomycetes?

Gas vesicles encoded by gvp genes provide buoyancy in many prokaryotes. In a recent Trends in Microbiology article entitled 'Gas vesicles in actinomycetes: old buoys in novel habitats?' van Keulen et al. documented the occurrence of gvp genes in soil-inhabiting actinomycetes but questioned whether any of them produce gas vesicles. We suggest that the protein encoded by gvpA in actinomycetes might be incompatible with the structure of the standard gas vesicle. Perhaps it has another role associated with the air-water interface.     

Enzymes of ammonia assimilation in hyphae and vesicles of Frankia sp. strain CpI1.

Frankia spp. are filamentous actinomycetes that fix N2 in culture and in actinorhizal root nodules. In combined nitrogen-depleted aerobic environments, nitrogenase is restricted to thick-walled spherical structures, Frankia vesicles, that are formed on short stalks along the vegetative hyphae. The activities of the NH4(+)-assimilating enzymes (glutamine synthetase [GS], glutamate synthase, glutamate dehydrogenase, and alanine dehydrogenase) were determined in cells grown on NH4+ and N2 and in vesicles and hyphae from N2-fixing cultures separated on sucrose gradients. The two frankial GSs, GSI and GSII, were present in vesicles at levels similar to those detected in vegetative hyphae from N2-fixing cultures as shown by enzyme assay and two-dimensional polyacrylamide gel electrophoresis. Glutamate synthase, glutamate dehydrogenase, and alanine dehydrogenase activities were restricted to the vegetative hyphae. Vesicles apparently lack a complete pathway for assimilating ammonia beyond the glutamine stage.     

Biology of Frankia strains, actinomycete symbionts of actinorhizal plants.

Frankia strains are N2-fixing actinomycetes whose isolation and cultivation were first reported in 1978. They induce N2-fixing root nodules on diverse nonleguminous (actinorhizal) plants that are important in ecological successions and in land reclamation and remediation. The genus Frankia encompasses a diverse group of soil actinomycetes that have in common the formation of multilocular sporangia, filamentous growth, and nitrogenase-containing vesicles enveloped in multilaminated lipid envelopes. The relatively constant morphology of vesicles in culture is modified by plant interactions in symbiosis to give a diverse array of vesicles shapes. Recent studies of the genetics and molecular genetics of these organisms have begun to provide new insights into higher-plant-bacterium interactions that lead to productive N2-fixing symbioses. Sufficient information about the relationship of Frankia strains to other bacteria, and to each other, is now available to warrant the creation of some species based on phenotypic and genetic criteria.     

Degradation of [8,9,-14C]endosulfan by soil microorganisms.

Twenty-eight soil fungi, 49 soil bacteria, and 10 actinomycetes were tested as to their ability to degrade the insecticide endosulfan. Using 14C-labeled material, the qualitative as well as the quantitative formation of metabolities, as well as of 14CO2, could be followed. Sixteen fungi, 15 bacteria, and 3 actinomycetes were found capable of metabolizing more than 30% of the applied endosulfan. The major metabolities detected were endosulfate, formed by oxidation of the sulfite group, and endodiol, formed by hydrolysis of the ester bond. The majority of highly active fungi formed endosulfate as the major metabolite, whereas the majority of active bacteria formed endodiol. In addition to endosulfate and endodiol, individual cultures contained small quantities of endohydroxyether and two unidentified products. The very small quantities of 14CO2 evolved from cultures indicated that an extensive mineralization of the carbon skeleton of endosulfan did not occur.     

Degradation of [8,9,-14C]endosulfan by soil microorganisms.

Twenty-eight soil fungi, 49 soil bacteria, and 10 actinomycetes were tested as to their ability to degrade the insecticide endosulfan. Using 14C-labeled material, the qualitative as well as the quantitative formation of metabolities, as well as of 14CO2, could be followed. Sixteen fungi, 15 bacteria, and 3 actinomycetes were found capable of metabolizing more than 30% of the applied endosulfan. The major metabolities detected were endosulfate, formed by oxidation of the sulfite group, and endodiol, formed by hydrolysis of the ester bond. The majority of highly active fungi formed endosulfate as the major metabolite, whereas the majority of active bacteria formed endodiol. In addition to endosulfate and endodiol, individual cultures contained small quantities of endohydroxyether and two unidentified products. The very small quantities of 14CO2 evolved from cultures indicated that an extensive mineralization of the carbon skeleton of endosulfan did not occur.     

Electron microscopy of the endophyte ofAlnus glutinosa

Earlier light microscopic investigations have revealed that the endophyte ofAlnus glutinosa presents itself in three different forms. In the present study this is confirmed by electron microscopy; also, new data on the cytology of the endophyte have been obtained.The host cells are primarily infected by the hyphal form of the endophyte. A plant cell nucleus and mitochondria can be found in the infected host cells.In the majority of the infected cells, so-called vesicles develop at the tips of the hyphae. Electron micrographs show that these vesicles, as well as the hyphae, are surrounded by the host-cell cytoplasmic membrane. The endophyte cytoplasm inside the vesicles is divided in all directions by cross walls, many of which are incomplete. Plasmalemmosomes are conspicuous. Some vesicles look vigorous but others shrunken or nearly devoid of cytoplasm as if being digested.A minority of host cells situated between the vesicle-containing ones are completely filled by bacteria-like cells. These host cells, in contrast to the other ones, do not contain a nucleus nor mitochondria, nor are the endophyte cells in them enveloped by a host cell cytoplasmic membrane: these host cells are dead. Vesicles are not found in these cells.It is inferred that a living host cell exerts a stimulus on the endophyte to which the latter responds by forming vesicles at the tips of the hyphae. At a later stage the host cells digest the vesicles and the hyphae. On the other hand, if a host cell does not survive the infection, the hyphae divide into bacteria-like cells, which are not digested owing to the absence of host cytoplasm.According to the cytology of the hyphae, the endophyte is an actinomycete.The cytology of the endophyte needs further elucidation. Its plasmalemmosomes, or membranous bodies connected with the cytoplasmic membrane, are beautifully developed. The striated bodies described on p. 359 under 4) may be a new feature, which may turn up in other actinomycetes or bacteria.     

Transport-Specific isolation of large channels reconstituted into lipid vesicles

Summary To develop a technique for purifying and identifying pore-forming membrane proteins, we used a transport-specific increase in buoyant density to select for lipid vesicles containing voltage-dependent anion channels (VDAC). Monodisperse, single-walled vesicles were formed by gel filtration from a detergent-solubilized mixture of lipid and protein in a urea buffer. The vesicles were layered on a linear iso-osmolar density gradient formed of urea and sucrose buffers. Since VDAC is open at zerotrans-membrane voltage and is permeable to urea and sucrose, vesicles containing functional VDAC should become more dense as sucrose enters and urea leaves, while those lacking open channels should maintain their original density. Vesicles formed in the absence of VDAC migrated to a characteristic density, while vesicles formed in the presence of VDAC fractionated into two populations in the gradients, one migrating to the same density as the vesicles formed without VDAC, and one at a significantly greater density. In contrast to the lower density vesicles, the higher density vesicles showed a high permeability to calcein, and contained functional VDAC channels (shown by electrophysiological recordings following fusion with a planar bilayer). Thus, vesicles containing open channels were separable from those that did not by a transport-specific shift in density. This technique may be useful for the enrichment of channels of known permeability properties from impure, material.     

Phosphoinositide-specific phospholipase C-delta 1 binds with high affinity to phospholipid vesicles containing phosphatidylinositol 4,5-bisphosphate.

We studied the binding of phosphoinositide-specific phospholipase C-delta 1 (PLC-delta) to vesicles containing the negatively charged phospholipids phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylserine (PS). PLC-delta did not bind significantly to large unilamellar vesicles formed from the zwitterionic lipid phosphatidylcholine (PC) but bound strongly to vesicles formed from mixtures of PC and PIP2. The apparent association constant for the putative 1:1 complex formed between PLC-delta and PIP2 was Ka congruent to 10(5) M-1. The binding strength increased further (Ka congruent to 10(6) M-1) when the vesicles also contained 30% PS. High-affinity binding of PLC-delta to PIP2 did not require Ca2+. PLC-delta bound only weakly to vesicles formed from mixtures of PC and either PS or phosphatidylinositol (PI); binding increased as the mole fraction of acidic lipid in the vesicles increased. We also studied the membrane binding of a small basic peptide that corresponds to a conserved region of PLC. Like PLC-delta, the peptide bound weakly to vesicles containing monovalent negatively charged lipids; unlike PLC-delta, it did not bind strongly to vesicles containing PIP2. Our data suggest that a significant fraction of the PLC-delta in a cell could be bound to PIP2 on the cytoplasmic surface of the plasma membrane.     

Screening of glucose isomerase-producing microorganisms

The ability to convert D-glucose into D-fructose was found in 14 out of 74 species of actinomycetes and bacteria tested. High intracellular glucose isomerase activity was displayed by Arthrobacter sp. and actinomycetes Streptomyces viridobrunneus, Streptomyces sp. 1 and Streptomyces sp. 32. The first showed maximal glucose-converting potential when cultured in both glucose and xylose media, while glucose isomerase activity of Streptomyces species could be found solely in medium supplemented with xylose. The ketose enzymatically formed from D-glucose was identified as D-fructose.     

Screening of glucose isomerase-producing microorganisms

The ability to convert D-glucose into D-fructose was found in 14 out of 74 species of actinomycetes and bacteria tested. High intracellular glucose isomerase activity was displayed by Arthrobacter sp. and actinomycetes Streptomyces viridobrunneus, Streptomyces sp. 1 and Streptomyces sp. 32. The first showed maximal glucose-converting potential when cultured in both glucose and xylose media, while glucose isomerase activity of Streptomyces species could be found solely in medium supplemented with xylose. The ketose enzymatically formed from D-glucose was identified as D-fructose.     

Gas vesicles in actinomycetes: old buoys in novel habitats?

Gas vesicles are gas-filled prokaryotic organelles that function as flotation devices. This enables planktonic cyanobacteria and halophilic archaea to position themselves within the water column to make optimal use of light and nutrients. Few terrestrial microbes are known to contain gas vesicles. Genome sequences that have become available recently for many bacteria from non-planktonic habitats reveal gas vesicle gene clusters in members of the actinomycete genera Streptomyces, Frankia and Rhodococcus, which typically live in soils and sediments. Remarkably, there is an additional level of complexity in cluster number and gene content. Here, we discuss whether putative gas vesicle proteins in these actinomycetes might actually be involved in flotation or whether they might fulfil other cellular functions.     

Interaction of apoliprotein C-III with phosphatidylcholine vesicles. Dependence of aproprotein-phospholipid complex formation on vesicle structure.

We have studied the interaction of an apolipoprotein from human very low density lipoproteins (apoC-III) with egg yolk phosphatidylcholine in the form of single- and multi-bilayer vesicles. The reactivity of single-bilayer vesicles with apoC-III appears to be greater than that of the multi-bilayer vesicles according to several thermodynamic and spectrosconic criteria. In the complexes formed by the association of apoC-III with single-bilayer vesicles, the alpha-helical content of the peptide backbone and the apolarity of the environment around the tryptophan residues are greater than that observed in the complexes formed with the multibilayer vesicles. A higher yield and more homogeneous density distribution of lipid-apoprotein complexes results from the interaction of apoC-III with the single-bilayer vesicles relative to those obtained with the multi-bilayer vesicles. The enthalpy of association of apoC-III with phospholipid was greater for the single-shelled vesicles (25 kcal/mol apoC-III) than for the multi-shelled ones (18 kcal/mol apoC-III). The difference in reactivity of these two types of liposomes is not due to a difference in their fluidities since their fatty acid compositions are identical, but may be due to a difference in their areas of sterically accessible phospholipid, their permeabilities to the apoprotein, their radii of curvation, or a combination of these factors.     

Origin of the brushborder in the differentiating midgut of Melasoma saliceti (Chrysomelidae, Coleoptera) embryos

The embryonic development of Melasoma saliceti takes eight days at room temperature. At the beginning of the 5th day the endoderm cells have already formed a unilayered epithelium of the midgut primordium. The midgut epithelium is formed by flat cells that are not connected by specialized intercellular junctions. Large vesicles can be seen in dilated intercellular spaces of the epithelium. Cytoplasmic projections, similar to microvilli, appear in the vesicles. During the 5th day ofdevelopment, the vesicles grow and become enclosed by the intercellular junctions of a zonula adherens type. During the 6th day of development the cell junctions surrounding the vesicles become transformed into a septate type. On the 8th day of development the vesicles come close to the apical sides of the midgut cells and open towards the yolk. At the same time the microvilli spread over the apical surface of the midgut primordium to form the regular brushborder of the larval midgut. In the species studied the vesicles appear to "prefabricate" the apical surfaces of the future midgut epithelium.     

Glucose utilization by enzymatically-formed trophoblastic vesicles and Day-14 porcine blastocysts

The total glucose metabolism of 48-h spherical trophoblastic vesicles, Day-60 trophoblastic vesicles sections and Day-14 porcine blastocyst sections was measured by the method of O'Fallon and Wright (1). Trophoblastic vesicles were formed by enzyme dispersal in Day-14 porcine blastocysts. Glucose was based on DNA content of the tissue measured by diamino benzoic acid reaction with DNA (2). Slope of the lines (PMoles glucose utilized/4 h x DNA content) was different between Day-14 blastocyst sections and 48 h trophoblastic vesicles (P /= 0.05). Slopes of the lines were identical between 48-h trophoblastic vesicles and Day-60 trophoblastic vesicles sections (P >/= 0.87). Average glucose utilization on a per ng DNA basis was calculated. Day-14 blastocyst sections utilized 0.67 Pmoles glucose/4 h per ng DNA, Day-60 trophoblastic vesicles sections; 0.57; and 48-h sperical trophoblastic vesicles used 0.29. It is hypothesized that the change in glucose utilization between the Day-14 porcine blastocyst and enzymatically formed trophoblastic vesicles may be due to a decrease in metabolism as a consequence of in vitro culture. Further, it is theorized that Day-60 trophoblastic vesicles sections used higher quantities of glucose than 48-h sperical trophoblastic vesicles on a per ng DNA basis due to the increased availability of glucose to the cells of the inner layers, caused by the sectioning of the tissue. The results of this study identify changes in glucose metabolism of enzymatically formed porcine trophoblastic vesicles during culture. It is proposed that enzymatically-formed trophoblastic vesicles be used as a model system for the study of embyro metabolism.     

Compound exocytosis of casein micelles in mammary epithelial cells

Ultrastructure of lactating bovine and rat mammary epithelial cells was studied with emphasis on secretory vesicle interactions. In the apical zone of the cell, adjacent secretory vesicles formed ball and socket configurations at their points of apposition. Similar configurations were formed between plasma membrane and secretory vesicle membrane. These structures may be formed by the diffusion of water between vesicles with different osmotic potentials. Frequently, vesicular chains consisting of 10 or more linked secretory vesicles were observed. Prior to the exocytotic release of casein micelles, adjacent vesicles fused through fragmentation of the ball and socket membrane. These membrane fragments and the casein micelles appeared to be secreted into the alveolar lumen after passing from one vesicle into another and finally through a pore in the apical plasma membrane. Emptied vesicular chains appeared to collapse and fragmentation of their membrane was observed. Based on these observations, we suggest that most vesicular membrane does not directly contact or become incorporated into the plasma membrane during secretion of the nonfat phase of milk.     

Asymmetric disposition of detergents within vesicle bilayer and its effect on ion permeation through the membrane

Phospholipid vesicles were prepared by detergent removal using hydrophobic porous beads, Amberlite XAD-2, or dialysis from detergent-phospholipid mixed micelles. The liposomes formed were found to be mostly unilammellar vesicles. The vesicle diameter was estimated, by both quasi-elastic light-scattering and gel-exclusion chromatography on Sephacryl S-1000, to be 80 nm for the vesicles formed by removal of octaethylene glycol monododecyl ether by the bead method. The effect of detergents within a bilayer on ion permeation was demonstrated. When the content of octaethylene glycol monododecyl ether reached a molar ratio of 0.2, the intrinsic ion selectivity of the phospholipid membrane between anion and cation was diminished. The ion permeability measured for vesicles with detergent incorporated into initially detergent-free vesicles was about 10-times greater than that for vesicles with detergent remaining following the process of detergent removal. This observation was explained by the different disposition of the detergent in the bilayer, that is, when vesicles were formed by the removal of detergent from mixed micelles, the residual detergent became distributed in both the outer and inner leaflets, and when the detergent was incorporated into initially detergent-free vesicles, the detergent became distributed only in the outer leaflet within the experimental time limits. This idea was supported by the NMR studies. It was also found that, as a detergent, octaethylene glycol monododecyl ether has a stronger effect on ion permeation than octyl glucoside.     

Uptake of ferritin by the mosaic egg surface of Brachydanio

The internalization of membrane from the mosaic egg surface of the zebra fish, Brachydanio, was investigated using anionic ferritin and transmission electron microscopy. The cortical cytoplasm of the 5-min activated egg showed numerous membrane-bound vesicles not found in the unactivated egg cortex. Two types of vesicles were identified: uncoated (smooth) and coated. Coated vesicles measured about 0.7 to 0.9 micrometer in diameter. Coated pits, considered to be precursors to the formation of coated vesicles, were frequently observed at the base of membrane-lined cortical granule crypts. Anionic ferritin was localized over coated pits and in both smooth and coated vesicles. The absence of any morphological evidence of a surface origin for smooth vesicles suggested these ferritin-labeled organelles might be formed by coated vesicle fusion. Our results indicate that the plasma membrane redundancy created by the exocytosis of cortical granules in Brachydanio appears to be resolved in part by the internalization of membrane through endocytosis.     

Inhibition of hydrolysis of phosphorylated Ca2+,Mg2+-ATPase of the sarcoplasmic reticulum by Ca2+ inside and outside the vesicles

The effects of intra- and extravesicular calcium and magnesium ions on the hydrolysis of the phosphoenzyme (EP) intermediate formed in the reaction of Ca2+,Mg2+-dependent ATPase of the sarcoplasmic reticulum were investigated. The rate constants of EP hydrolysis were measured under conditions that allowed a single turnover of ATP hydrolysis to minimize the increase in calcium concentration inside the vesicles. The EP formed during a single turnover was hydrolyzed biphasically and could be resolved into fast- and slow-decomposing components. When free Mg2+ outside the vesicles was chelated by adding excess EDTA, EP could also be kinetically resolved into two components; EDTA-sensitive EP, which could be quickly decomposed by adding EDTA, and EDTA-insensitive EP, which could be prevented from decomposing by adding EDTA. The amount of EDTA-sensitive EP decreased rapidly during the initial phase of the reaction, while that of EDTA-insensitive EP decreased slowly with the same rate constant as that of the slow-decomposing EP. These results showed that the biphasic time course of EP hydrolysis was caused by the formation of EDTA-sensitive and -insensitive EP during the reaction. The time course of EP hydrolysis could be quantitatively analyzed in terms of the following reaction mechanism. (formula; see text) The decomposition of EDTA-insensitive EP required Mg2+ outside the vesicles and was competitively inhibited by extravesicular Ca2+. The decomposition of EDTA-sensitive EP was inhibited by Ca2+ inside the vesicles but not by external Ca2+. The linear relationships between the inverse of the rate constants of EP decomposition during the initial phase and the intravesicular CaCl2 concentrations suggested that decomposition of EDTA-sensitive EP was inhibited by the binding of 1 mol of intravesicular Ca2+ to 1 mol of EP. Furthermore, Mg2+ inside the vesicles scarcely affected the inhibition of EP hydrolysis by intravesicular Ca2+. These results suggested that magnesium ions are not counter-transported during the active transport of calcium by SR vesicles.     

Synthetic lipid (DOPG) vesicles accumulate in the cell plate region but do not fuse

The cell plate is the new cell wall, with bordering plasma membrane, that is formed between two daughter cells in plants, and it is formed by fusion of vesicles (approximately 60 nm). To start to determine physical properties of cell plate forming vesicles for their transport through the phragmoplast, and fusion with each other, we microinjected fluorescent synthetic lipid vesicles that were made of 1,2-dioleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (DOPG) into Tradescantia virginiana stamen hair cells. During interphase, the 60-nm wide DOPG vesicles moved inside the cytoplasm comparably to organelles. During cytokinesis, they were transported through the phragmoplast and accumulated in the cell plate region together with the endogenous vesicles, even inside the central cell plate region. Because at this stage microtubules are virtually absent from that region, while actin filaments are present, actin filaments may have a role in the transport of vesicles toward the cell plate. Unlike the endogenous vesicles, the synthetic DOPG vesicles did not fuse with the developing cell plate. Instead, they redistributed into the cytoplasm of the daughter cells upon completion of cytokinesis. Because the redistribution of the vesicles occurs when actin filaments disappear from the phragmoplast, actin filaments may be involved in keeping the vesicles inside the developing cell plate region.     

7株放线菌在辣椒根部定殖及对辣椒叶片PAL与PPO活性的影响

采用盆栽接种试验、平皿涂抹法测数及常规酶活测定法研究了7株拮抗性放线菌在辣椒根部的定殖能力及接种24d对辣椒叶片苯丙氨酸解氨酶（PAl。）与多酚氧化酶活性（PPO）的诱导效应。结果表明：（1）供试7株放线菌单独接种均不能在辣椒根内定殖,但与辣椒疫霉P3混合接种时有5株可定殖;供试放线菌在辣椒根部的定殖能力与其体外平皿试验中产生的的拈抗圈大小基本无关;可定殖放线菌的定殖密度随时间延长而降低,至40d时均无活菌检出。（2）在放线菌单接处理中,5株菌接种后可诱导辣椒叶片PAL,活性提高,全部供试菌均能诱导PPO活性提高,其中可使两种酶同步提高的有5株菌;在放线菌＋P3混接处理中,有6株接种后可诱导PAL,活性提高,5株菌能诱导PPO活性提高,其中可使两种酶同步提高的有4株菌;在接入放线菌时同时混接辣椒疫霉,能增强2株供试放线菌对辣椒叶片PAL活性及6株供试放线菌对辣椒叶片PPO活性的诱导作用;供试放线菌的定殖能力与辣椒叶片PAL及PPO活性变化无明显规律性关系。