Formation of volutin granules in Corynebacterium glutamicum

Volutin granules are intracellular storages of complexed inorganic polyphosphate (poly P). Histochemical staining procedures differentiate between pathogenic corynebacteria such as Corynebacterum diphtheriae (containing volutin) and non-pathogenic species, such as C. glutamicum. Here we report that strains ATCC13032 and MH20-22B of the non-pathogenic C. glutamicum also formed subcellular entities (18-37% of the total cell volume) that had the typical characteristics of volutin granules: (i) volutin staining, (ii) green UV fluorescence when stained with 4',6-diamidino-2-phenylindole, (iii) electron-dense and rich in phosphorus when determined with transmission electron microscopy and X-ray microanalysis, and (iv) 31P NMR poly P resonances of isolated granules dissolved in EDTA. MgCl2 addition to the growth medium stimulated granule formation but did not effect expression of genes involved in poly P metabolism. Granular volutin fractions from lysed cells contained polyphosphate glucokinase as detected by SDS-PAGE/MALDI-TOF, indicating that this poly P metabolizing enzyme is present also in intact poly P granules. The results suggest that formation of volutin is a more widespread phenomenon than generally accepted.     

Capacity for polyphosphate accumulation of predominant bacteria in activated sludge showing enhanced phosphate removal

The taxonomic identity of predominant bacteria in laboratory-scale anaerobic-aerobic activated sludge systems and their capacity for accumulating polyphosphate (polyP) were investigated. All bacterial isolates accumulated appreciable amounts of polyP under tested conditions, although only 7% of them were positive for the volutin stain. The isolates showing the most pronounced polyP accumulation and volutin formation were the unidentified Gram-positive bacteria containing menaquinone—10. Some other strains of Gram-positive bacteria and menaquinone-containing Gram-negative bacteria also formed volutin granules.     

Identification of organelles in bacteria similar to acidocalcisomes of unicellular eukaryotes

Acidocalcisomes are acidic calcium storage compartments described in several unicellular eukaryotes, including trypanosomatid and apicomplexan parasites, algae, and slime molds. In this work, we report that the volutin granules of Agrobacterium tumefaciens possess properties similar to the acidocalcisomes. Transmission electron microscopy revealed that each intracellular granule was surrounded by a membrane. X-ray microanalysis of the volutin granules showed large amounts of phosphorus, magnesium, potassium, and calcium. Calcium in the volutin granules increased when the bacteria were incubated at high extracellular calcium concentration. Immunofluorescence and immunoelectron microscopy, using antisera raised against peptide sequences conserved in the A. tumefaciens proton pyrophosphatase, indicated localization in intracellular vacuoles. Purification of the volutin granules using iodixanol density gradients indicated a preferential localization of the pyrophosphatase activity in addition to high concentrations of phosphate, pyrophosphate, short- and long-chain polyphosphate, but lack of markers of the plasma membrane. The pyrophosphatase activity was potassium-insensitive and inhibited by the pyrophosphate analogs, amynomethylenediphosphonate and imidodiphosphate, by dicyclohexylcarbodiimide, and by the thiol reagent N-ethylmaleimide. Polyphosphate was also localized to the volutin granules by 4',6'-diamino-2-phenylindole staining. The organelles were acidic, as demonstrated by staining with LysoSensor blue DND-167, a dye especially used to detect very acidic compartments in cells, and cycloprodigiosin, a compound isolated from a marine bacterium that has been shown to uncouple proton pyrophosphatase activity acting as a chloride/proton symport. The results suggest that acidocalcisomes arose before the prokaryotic and eukaryotic lineages diverged.     

Ultrastructural investigation of acidocalcisomes and ATPase activity in yeast Candida guilliermondii NP-4 as ‘complementary’ stress-targets

As a result of electron microscopic studies of morphogenesis in yeast Candida guilliermondii NP-4, the formation of new structures of volutin acidocalcisomes has been established within the cell cytoplasm. Under influence of X-irradiation, the changes in morphometric and electron-dense properties of yeast cells were identified: in yeast cytoplasm, the electron-dense volutin granules were increased up to 400 nm in size. After 24-h post-irradiation incubation of yeasts, the large volutin pellets are fragmented into smaller number particles in size up to 25–150 nm. The ATPase activity in yeast mitochondria was changed under X-irradiation. In latent phase of growth, ATPase activity was decreased 1·35-fold in comparison with non-irradiated yeasts. In logarithmic phase of growth, ATPase activity was three times higher than in latent phase, and in stationary phase of growth it has a value similar to the latent phase. Probably, the cells receive the necessary energy from alternative energy sources, such as volutin. Electron microscopy of volutin granule changes might serve as convenient method for evaluation of damages and repair processes in cells under influence of different environmental stress-factors.     

Staining Streptomyces Scabies in Lesions of Common Scab of Potato

Toluidine blue can be used to stain Streptotnyces scabies distinctively in slide cultures or in the lesions of common potato scab. This staining method is based on the metachromaticism of volutin, a constant constituent of the spores and mycelium of S. scabies. Either sections or smears are fixed in FPA (formalin, 5 parts; propionic acid, 7.5; 50% alcohol, 87.5), stained in a 1:100 dilution of saturated aqueous toluidine blue from 20 minutes to 24 hours, dehydrated in an acetone-xylene series and mounted. Cellular constituents of the potato tuber stain blue or are colorless whereas the mycelium of Streptomyces appears as a series of red volutin spheres in the blue stained cytoplasm. The criteria of volutin and cytoplasmic staining along with the 1 µ diameter of the mycelium make it possible to distinguish Streptomyces from the other microorganisms and cells in the lesion region.     

Volutin Granules in Zoogloea ramigera

Zoogloea ramigera, a gram-negative bacterium found in activated sludge, formed volutin granules when excess orthophosphate was added to a phosphate-starved culture. These volutin granules were stainable by hydrogen sulfide after lead acetate treatment and extractable by N-perchloric acid but were not adsorbed by activated charcoal. They appeared to consist of inorganic polyphosphate. Optimum granule formation in the arginine broth required 10 g of glucose, 3 mg of phosphate, and 1 to 20 mg of magnesium per liter of medium. At an Mg(2+) concentration of 1 mg/liter, very large granules appeared which often appeared to fill the cell. An excess of glucose, orthophosphate, or magnesium reduced granule formation. In the absence of sulfate, moderate granulation occurred in arginine broth before the addition of excess orthophosphate; granulation did not increase after the addition of phosphate.     

Volutin as a Substrate of the Gram Reaction

A modified Gram procedure, with the use of an extremely diluted or acidified crystal violet solution, stained only volutin in contrast with nonstaining of the rest of cell in Gram-positive bacteria. The substrate of the Gram reaction is not only a ribonucleic acid-magnesium-protein complex in cytoplasm (Henry and Stacey 1946), but also a metaphosphate-ribonucleic acid complex in volutin and deoxyribonucleic acid in nuclei in Gram-positive cells. The isoelectric-point theory and permeability theory of the Gram stain are unsupported by the experiments.     

In vivo Observations of Plastid and Cell Division in Raphid Diatoms and Their Relevance to Diatom Systematics

Premitotic rearrangements of the protoplast, cytokinesis andpostmitotic rearrangements were followed in vivo in Naviculapupula Küitz. and N. bacillum Ehrenb. The plastids andnuclcus perform translational movements before cytokinesis,taking up well-defined positions on opposite sides of the cell.Following this the plastid divides by constriction and the cellcleaves in two. Cytokinesis takes 5–8 min and is effectedby a contractile ring. This is circular, except where constrainedby the cell wall. Parts of the ring appear to be functionalbefore cleavage begins. The two volutin granules persist duringcell division and are segregated one to each daughter cell.The granules are associated with the tonoplast and contractilering until late in cleavage, when they are released into thevacuoles. After value formation, the plastid, which has beenchanging in shape since before mitosis, rotates through 90°.A new volutin granule is formed in each daughter cell. The segregationof the granules, the tilt of the dividing nucleus and the rotationof the plaslid are chiral. The positions and shapes taken bythe organelles during the cell cycle suggest the presence ofintracellular recognition and attachment sites, which existfor specific periods. The taxonomic value of cell cycle eventsis discussed. Navicula, cell cycle, cell division, diatom systematics, plastid division, plastid rotation, volutin granules     

Isolation of intact chains of polyphosphate from "Propionibacterium shermanii" grown on glucose or lactate

A procedure is presented for the isolation of intact polyphosphate (poly P) from "Propionibacterium shermanii." It is demonstrated, by including [32P]poly P during the extraction, that this procedure does not hydrolyze the poly P, and it is shown that two other widely used procedures do cause breakdown of the poly P. The procedure presented allows isolation of three fractions, short-chain poly P which is soluble in trichloroacetic acid, long-chain poly P which is soluble at neutral pH, and long-chain poly P which is present in volutin granules. Cells which had been grown on lactate did not contain short-chain poly P but did contain a high amount of long-chain poly P, which accumulated to 3% of the cell dry weight. At least 70% of this poly P was present in volutin granules. The poly P ranged in length from 250 to 725 phosphate residues and was the same average size as that synthesized in vitro by the poly P kinase from "P. shermanii". This indicates that the poly P kinase is responsible for catalyzing the synthesis of the poly P. In contrast to cells grown on lactate, those which had been grown on glucose did not contain volutin granules, did contain short-chain poly P and had 100-fold less long-chain poly P than lactate-grown cells. We propose that during the fermentation of glucose, the amount of poly P is lower than during growth on lactate because it is continuously utilized as a substrate in the phosphorylation of glucose.     

On the nature of the granules of the genus spirillum

Summary Chemical analyses of centrifugal fractions of cell-free extracts of three spirilla and Sacch. cerevisiae showed that the granular inclusions accumulated by these organisms do not contain significant amounts (if any) of polyphosphates. Further, no trichloroacetic acid extractable-barium precipitable polyphosphates could be isolated from the spirilla. Thus, the granules of spirilla, heretofore called volutin, are not identifiable with polyphosphates.The phase opaque bodies present in members of this genus are composed primarily of poly--hydroxybutyrate. It is recommended that the term volutin no longer be used to describe granules showing a metachromatic reaction.     

Alterations Induced by the Antifungal Compounds Ketoconazole and Terbinafine in Leishmania

ABSTRACT. The antiproliferative effects and ultrastructural alterations induced in vitro by two antifungal compounds, the azole ketoconazole and the allylamine terbinafine on Leishmania amazonensis are reported. Promastigotes treatment with ketoconazole and terbinafine induced growth arrest and cell lysis in 72 hours. Combination of the two agents produced additive effects on promastigote axenic growth and synergistic effects on intracellular amastigote proliferation. The amastigotes, either axenically grown or infecting murine macrophages, were about 100-fold more sensitive to the drugs. These compounds induced the appearance of large multivesicular bodies, especially after ketoconazole treatment, increased amount of lipid inclusions as well as numerous, polymorphic volutin granules, particularly in terbinafine-treated cells. Multivesicular bodies were observed in close apposition with organelles such as mitochondria, which also showed alterations in the distribution and appearance of cristae, and the formation of paracrystalline arrays within the matrix. Some cells presented large portions of cytoplasm wrapped by endoplasmic reticulum and many parasites also presented myelin-like endoplasmic reticulum profiles. Such alterations together with the strong acid phosphatase activity observed in the multivesicular bodies and volutin granules may indicate the existence of an unusual autophagic process in cells treated with ergosterol biosynthesis inhibitors.     

Ultrastructural organization of marine luminescent bacteria and their mutants

The aim of this work was to study the submicroscopic organization of luminescent bacteria belonging to the genera Photobacterium and Lucibacterium as well as that of their "dark" mutants incapable of luminescence. The ultrastructural organization of all studied bacteria is typical of gram-negative species. The luminescent bacteria are characterized by the presence, in their cytoplasm, of osmophilic formations 22--110 nm in size. The cells of "dark" mutants accumulate volutin and contain complex membrane systems which are related to decelerated growth of the cultures.     

Haemoproteus gabaldoni n. sp. (Apicomplexa: Haemoproteidae) from the Muscovy duck Cairina moschata (Aves: Anatidae)

Haemoproteus gabaldoni n. sp. is described from the Muscovy duck Cairina moschata from Caracas, Venezuela and is compared to H. greineri and H. nettionis which have been described previously from the Anatidae. The highly amoeboid outline, volutin granules and small number of pigment granules of H. gabaldoni serve to readily separate this species from the other two.     

Neutral Red as a Lipid Fluorochrome

Aqueous 0.1% neutral red adjusted to pH 6.5, or mixed 1:1 with seawater, produced conspicuous fluorescence in plant and animal fats while acting as a conventional stain for acidic groups including volutin. The water-soluble, nonfluorescent dye predominating in acid solutions became deprotonized, lipophilic and fluorescent under neutral and alkaline conditions. Neutral dye crystals fluoresced dull red, whereas the fluorescence was either bright lemon yellow or blue-green in lipids, apparently dependent upon their composition.     

Differentiation of polyphosphate and poly-β-hydroxybutyrate granules in an Acinetobacter sp. isolated from activated sludge

Cells containing polyphosphate 71 micrograms P (mg protein)-1 and no poly-beta-hydroxybutyrate showed metachromatic granules but no lipid granules; cells containing poly-beta-hydroxybutyrate (15% of dry weight) showed fluorescence lipid granules but no metachromatic granules; whereas cells containing both polyphosphate and poly-beta-hydroxybutyrate showed both types of granules. These observations, together with a critical review of the literature, show a clear distinction between metachromatic (or volutin) granules and lipid granules.     

Polyphosphate metabolism in the blue-green alga Microcystis aeru-ginosa

The uptake of inorganic phosphorus was studied in an axenicstrain of phosphorus-starved cells of the blue-green alga Microcystisaeruginosa, an organism often causing blooms in freshwater bodies.Rates of growth and of cellular polyphosphate content as a functionof initial orthophosphate in the medium indicate the operationof the ‘phosphorus overplus’ phenomenon in M. aeruginosa,accompanied by formation of volutin granules. The granules wereisolated by a non-aqueous centrifugation method, and identifiedas polyphosphate bodies.     

Comparative Ultrastructure of the Thiobacilli

The ultrastructure of seven Thiobacillus species was studied. The structure of their cell envelopes is similar, if not identical, to that found in other gram-negative bacteria. Obvious differences were noted in the middle layer of the cell envelope of the seven cultures. Polyhedral inclusion bodies were apparent in four of the organisms: T. thioparus, T. neapolitanus, T. intermedius, and T. thiooxidans. Lamellar bodies, similar to those present in certain photosynthetic bacteria were found in a few cells of T. thioparus. Structures resembling mesosomes were discovered in T. dinitrificans. A few cells of T. intermedius possessed paracrystalline bodies. Other inclusions, probably volutin and polysaccharide, were present in some of the cultures.     

Volutin granules of Eimeria parasites are acidic compartments and have physiological and structural characteristics similar to acidocalcisomes

The structural organization of parasites has been the subject of investigation by many groups and has lead to the identification of structures and metabolic pathways that may represent targets for anti-parasitic drugs. A specific group of organelles named acidocalcisomes has been identified in a number of organisms, including the apicomplexan parasites such as Toxoplasma and Plasmodium, where they have been shown to be involved in cation homeostasis, polyphosphate metabolism, and osmoregulation. Their structural counterparts in the apicomplexan parasite Eimeria have not been fully characterized. In this work, the ultrastructural and chemical properties of acidocalcisomes in Eimeria were characterized. Electron microscopy analysis of Eimeria parasites showed the dense organelles called volutin granules similar to acidocalcisomes. Immunolocalization of the vacuolar proton pyrophosphatase, considered as a marker for acidocalcisomes, showed labeling in vesicles of size and distribution similar to the dense organelles seen by electron microscopy. Spectrophotometric measurements of the kinetics of proton uptake showed a vacuolar proton pyrophosphatase activity. X-ray mapping revealed significant amounts of Na, Mg, P, K, Ca, and Zn in their matrix. The results suggest that volutin granules of Eimeria parasites are acidic, dense organelles, and possess structural and chemical properties analogous to those of other acidocalcisomes, suggesting a similar functional role in these parasites.     

Ultrastructure of Trypanosoma duttoni.

Trypanosoma duttoni exhibits the typical trypanosome morphology in that it is bounded by a unit membrane which included its anterior flagellum; it is surrounded by a framework of suppellicular fibrils, and it contains a nucleus and the posteriorly located kinetoplast-blepharoplast structures along with cytoplasmic organelles (mitochondria, Golgi, and enoplasmic reticulum). Very prominently displayed in T. duttoni is the contractile vacuole, which has not previously received wide-spread recognition. The kinetoplast is clearly continuous with mitochondria in some cases. Inclusion bodies are categorized into three distinct types, none of which can be conclusively designated as the very popular volutin granules, without cytochemical evidence. The existence of a third tubule among peripheral tubule doublets of the flagellum is observed.     

Morphology of the bifidobacteria by light and electron microscopy and their biological properties]

The morphology and the acid-producing, antagonistic and enzymatic activity of the strains of bifidobacteria isolated from the intestine of adults and children were studied. Bifidobacteria showed a considerable polymorphism, all the strains were antagonistically active. The strains isolated from adults were found to have greater acid-producing activity; the predominant species were B. longum and B. adolescentis, and in children B. bifidum were also isolated. The characteristic feature of B. longum strains was the presence of a slime-like layer and formations resembling bubbles around bacterial cells. The stains with greater physiological activity were found to have an extensive mesosomal complex, as well as a great number of volutin granules.