Polyethylene Glycol-induced Uptake of Bacteria into Yeast Protoplasts

Cells of the nitrogen-fixing bacterium Azotobacter vinelandii and the unicellular cyanobacterium Anacystis nidulans were introduced into protoplasts of Saccharomyces cerevisiae by the polyethylene glycol (PEG) method. Factors influencing the uptake frequency were examined, and experimental conditions were established for maximizing the uptake frequency. Under optimal conditions, each protoplast took-up a few bacterial cells. Electron-microscopic studies showed the localization of integrated bacterial cells in membrane-bound vesicles of the cytoplasm or large vacuoles. The protoplasts at the intermediate stages of uptake revealed two major mechanisms of uptake: (a) “endocytosis” by a single protoplast and (b) “cell fusion” between two or more protoplasts. Some bacterial cells disintegrated during the subsequent incubation period through a heterophagy-like process.     

Disproving the hypothesis of a common ancestry for the Ochromonas danica chrysoplast and Heliobacterium chlorum

The phylogenetic position of the golden-yellow alga Ochromonas danica chrysoplast was investigated by comparison of the 16S rRNA catalogue and two long 16S rRNA stretches (804 and 454 bases) with catalogues from eubacteria and chloroplasts and with homologoes 16S rRNA regions from Escherichia coli, Bacillus subtilis, Heliobacterium chlorum, Anacystis nidulans and chloroplasts from Zea mays, Nicotiana tabacum, Euglena gracilis and Chlamydomonas reinhardii, respectively. Both approaches indicate a closer relationship of the chrysoplast to chloroplasts and cyanobacteria than to the brownish photoheterotrophic Heliobacterium chlorum for which a common ancestry has recently been hypothesized.     

Selectivity of Ingestion and Digestion in the Chrysomonad Flagellate Ochromonas malhamensis

Ochromonas malhamensis cells were allowed to feed on autoclaved Aerobacter aerogenes, biochemically inert polystyrene latex particles of comparable size, shape and density, or a combination of these to determine if this protozoan is capable of phagotrophic selectivity under defined experimental conditions. Electron microscopic examination of Gomoristained food vacuoles indicated that the type of particle ingested was determined entirely by the type available. Apparently the biochemical or physical configuration of the particle surface is not decisive in particle selection. Gomori acid-phosphatase reaction product, which indicates digestive enzyme activity, was present in all particle-containing vacuoles, suggesting that digestive activity in O. malhamensis is due to the presence of particulate material in the vacuole, but not necessarily to the nature of the particle.     

Amplified expression of ribulose bisphosphate carboxylase/oxygenase in pBR322-transformants of Anacystis nidulans

Prior research suggested that the genes for large (L) and small (S) subunits of ribulose bisphosphate carboxylase/oxygenase (RuBisCO) are amplified in ampicillin-resistant pBR322-transformants of Anacystis nidulans 6301. We now report that chromosomal DNA from either untransformed or transformed A. nidulans cells hybridizes with nick-translated [³²P]-pBR322 at moderately high stringency. Moreover, nick-translated [³²-P]-pCS75, which is a pUC9 derivative containing a PstI insert with L and S subunit genes (for RuBisCO) from A. nidulans, hybridizes at very high stringency with restriction fragments from chromosomal DNA of untransformed and transformed cells as does the ³²P-labeled PstI fragment itself. The hybridization patterns suggest the creation of two EcoRI sites in the transformant chromosome by recombination. In pBR322-transformants the RuBisCO activity is elevated 6- to 12-fold in comparison with that of untransformed cells. In spite of the difference in RuBisCO activity, pBR322-transformants grow in the presence of ampicillin at a similar initial rate to that for wild-type cells. Growth characteristics and RuBisCO content during culture in the presence or absence of ampicillin suggest that pBR322-transformants of A. nidulans 6301 are stable. The data also collectively suggest that a given plasmid in the transformed population replicates via a pathway involving recombination between the plasmid and the chromosome.     

Sequence studies on 16S ribosomal RNA from a blue-green alga

Summary The 16S ribosomal RNA of the blue green algaAnacystis nidulans has been characterized in terms of the oligomers generated by digestion with T1 ribonuclease.A. nidulans by this criterion is definitely a procaryote; being no more distant from Bacilli or Enterics than the latter two are from one another.A. nidulans appears to be somewhat more closely related to the Bacilli than to the Enterics.This is contribution III in a series entitled Procaryote phylogeny.     

Confusing Selective Feeding with Differential Digestion in Bacterivorous Nanoflagellates

Food selectivity and the mechanisms of food selection were analyzed by video microscopy for three species (Spumella, Ochromonas, Cafeteria) of interception-feeding heterotrophic nanoflagellates. The fate of individual prey particles, either live bacteria and/or inert particles, was recorded during the different stages of the particle-flagellate-interaction, which included capture, ingestion, digestion, and egestion. The experiments revealed species-specific differences and new insights into the underlying mechanisms of particle selection by bacterivorous flagellates. When beads and bacteria were offered simultaneously, both particles were ingested unselectively at similar rates. However, the chrysomonads Spumella and Ochromonas egested the inert beads after a vacuole passage time of only 2-3 min, which resulted in an increasing proportion of bacteria in the food vacuoles. Vacuole passage time for starved flagellates was significantly longer compared to that of exponential-phase flagellates for Spumella and Ochromonas. The bicosoecid Cafeteria stored all ingested particles, beads as well as bacteria, in food vacuoles for more then 30 min. Therefore "selective digestion" is one main mechanism responsible for differential processing of prey particles. This selection mechanism may explain some discrepancies of former experiments using inert particles as bacterial surrogates for measuring bacterivory.     

Evidence for an arginine-dependent route for the synthesis of NO in the model filamentous fungus Aspergillus nidulans

Nitric oxide (NO) is a signalling molecule in eukaryotic and prokaryotic organisms. NO levels transiently boost upon induction of conidiation in Aspergillus nidulans. Only one pathway for NO synthesis involving nitrate reductase has been reported in filamentous fungi so far, but this does not satisfy all the NO produced in fungal cells. Here we provide evidence for at least one additional biosynthetic pathway in A. nidulans involving l -arginine or an intermediate metabolite as a substrate. Under certain growth conditions, the addition of l -arginine to liquid media elicited a burst of NO that was not dependent on any of the urea cycle genes. The NO levels were controlled by the metabolically available arginine, which was regulated by mobilization from the vacuoles and during development. In vitro assays with protein extracts and amino acid profiling strongly suggested the existence of an arginine-dependent NO pathway analogous to the mammalian NO synthase. Addition of polyamines induced NO synthesis, and mutations in the polyamine synthesis genes puA and spdA reduced the production of NO. In conclusion, here we report an additional pathway for the synthesis of NO in A. nidulans using urea cycle intermediates.     

Growth and macromolecular composition of Anacystis nidulans at different temperatures in Mg2+- and K+-limited chemostats

Alterations in dry weight, macromolecular composition and cell volume with temperature, were examined for Mg²⁺- and K⁺-limited Anacystis nidulans. The experiments were performed in chemostats with constant dilution rate. Increasing the temperature from 30–40°C resulted in a 2.1 times increase in yield (g dry weight/g ion) for the Mg²⁺-limited culture, while it increased 1.3 times in the K⁺-limited culture. This difference in yield increase with temperature was caused by a large accumulation of carbohydrate in the Mg²⁺-limited cells. The relation between RNA and protein was found to be independent of temperature in both cultures. This indicated that A. nidulans contained extra inactive RNA under the growth conditions used. These results are discussed to indicate that A. nidulans regulates the rate of protein synthesis by activating/inactivating RNA in protein synthesis. The filament size and cellular DNA content both increased 1.6 times in the Mg²⁺-limited cells when decreasing the temperature from 40 to 30°C. The chlorophyll content of A. nidulans was found to be independent of temperature in both cultures.     

THE INFLUENCE OF THE MODE OF NUTRITION ON THE DIGESTIVE SYSTEM OF OCHROMONAS MALHAMENSIS

The intracellular distribution and level of acid hydrolases in Ochromonas malhamensis were studied in cells grown osmotrophically in a defined medium, in a carbon-free starvation medium, and during phagotrophy in each of these media. By cytochemical techniques, little enzymic reaction product was observed in the vacuoles of osmotrophic cells grown in the defined medium. Starved cells, however, contained autophagic vacuoles and cannibalized other Ochromonas cells. Dense enzymic reaction product was observed in the digestive vacuoles and in the Golgi cisternae of these starved cells. Moreover, starved cells and cells grown in a nutritionally complete medium ingested Escherichia coli which appeared in digestive vacuoles containing enzymic reaction product. Biochemical assays for lysosomal acid phosphatase (E.C. 3.1.3.2 orthophosphoric monoester phosphohydrolase) and acid ribonuclease (E.C. 2.7.7.16 ribonucleate nucleotido-2'-transferase) were done on Ochromonas cultures in the same experimental treatments and under identical assay conditions as the cytochemical study. During starvation, the acid hydrolase specific activities were consistently twice those found in cells grown in an osmotrophic complete medium. Ochromonas fed E. coli showed no increase in acid hydrolase specific activity as compared to controls not fed E. coli. The latency of lysosomal acid hydrolases in cells fixed with glutaraldehyde was reduced, suggesting that this fixative increases lysosomal membrane permeability and may release enzymes or their reaction products into the cytoplasmic matrix during cytochemical analysis. This could explain the cytoplasmic staining artifact sometimes observed with glutaraldehyde-fixed cells when studied by the Gomori technique. This study confirms that Ochromonas malhamensis, a phytoflagellate, does produce digestive vacuoles and can ingest bacteria, thereby fulfilling its role as a heterotroph in an aquatic food chain. When Ochromonas is grown in a nutritionally complete osmotrophic medium, phagocytosis causes appearance of acid hydrolases in the digestive vacuoles, whereas the total activity of the enzymes remains unchanged. An organic carbon-free medium strongly stimulates acid hydrolaes activity and causes these enzymes to appear in the digestive vacuoles whether phagocytosis occurs or not.     

Evidence for two distinct origins of replication in the large endogenous plasmid of Anacystis nidulans R2

Summary Shuttle cloning vectors, capable of replication in Escherichia coli and in the cyanobacterium Anacystis nidulans R2, have been used to localize a putative origin of replication of the large endogenous plasmid (pANL) of A. nidulans R2. Utilizing the cloning flexiblity of the polylinker containing E. coli plasmid pDPL 13, we have constructed a series of deletion derivatives of a 11.7 kilobase segment of pANL believed to contain a functional origin of replication. Two distinct segments of pANL that are 5.7 and 4.7 kilobases in size carry sufficient information to support transformation of A. nidulans R2. These DNA fragments, designated by us ORI 1 and ORI 2, do not overlap and show no DNA homology by blot hybridization analysis. Additionally, neither of these fragments are homologous to the replication origin of the other endogenous plasmid (pANS) of A. nidulans R2. Intact hybrid plasmids capable of transforming A. nidulans R2 have been re-isolated from transformed cells indicating that these plasmids exist autonomously in both A. nidulans R2 and E. coli.Dedicated to Professor Georg Melchers to celebrate his 50-year association with the journal     

Ultrastructural changes in the gastrodermal phagocytic cells of the planarian Dugesia gonocephala s.l. during food digestion (Plathelminthes)

Summary In the present report the functional morphology of the planarian gastrodermal phagocytic cells is examined in feeding animals. A functional interpretation of some of the morphological findings is given. The events in the fine-structure modifications of the phagocytic cells in the course of phagocytosis and intracellular digestion of food particles were followed through five post-feeding stages in the planarian Dugesia gonocephala. Light and electron microscopical observations demonstrate that there is preliminary intraluminal digestion of food particles; their phagocytosis takes place quickly.Beef hepatocytes that served as food are found engulfed at first in food vacuoles near the apical border of the phagocytic cells, and are clearly recognizable. The vacuoles increase in number to occupy most of the cytoplasm of these cells. Progressive breakdown and disappearance of phagocytosed hepatocytes occurs. In time the vacuoles move deeper into the cells, their contents lose their identity, and condense to homogeneous or heterogeneous residual bodies. These are returned to the distal surface of the cells, and then voided into the intestinal lumen. At the same time, synthesis and accumulation of numerous lipid droplets occurs, probably as a final product resulting from metabolism of the digested material. When feeding is over, the phagocytic cells are filled with lipid droplets, acquiring their typical appearance.It is suggested that disintegration of phagocytic cells during starvation is balanced by proliferation and differentiation of neoblasts into new phagocytic cells during the feeding-starvation cycle.     

Symbiotic Chlorella sp. of the ciliate Paramecium bursaria do not prevent acidification and lysosomal fusion of host digestive vacuoles during infection

Summary. Each symbiotic Chlorella sp. of the ciliate Paramecium bursaria is enclosed in a perialgal vacuole derived from the host digestive vacuole, and thereby the alga is protected from digestion by lysosomal fusion. Algae-free cells can be reinfected with algae isolated from algae-bearing cells by ingestion into digestive vacuoles. To examine the timing of acidification and lysosomal fusion of the digestive vacuoles and of algal escape from the digestive vacuole, algae-free cells were mixed with isolated algae or yeast cells stained with pH indicator dyes at 25 ± 1 °C for 1.5 min, washed, chased, and fixed at various time points. Acidification of the vacuoles and digestion of Chlorella sp. began at 0.5 and 2 min after mixing, respectively. All single green Chlorella sp. that had been present in the host cytoplasm before 0.5 h after mixing were digested by 0.5 h. At 1 h after mixing, however, single green algae reappeared in the host cytoplasm, arising from those digestive vacuoles containing both nondigested and partially digested algae, and the percentage of such cells increased to about 40% at 3 h. At 48 h, the single green algae began to multiply by cell division, indicating that these algae had succeeded in establishing endosymbiosis. In contrast to previously published studies, our data show that an alga can successfully escape from the host’s digestive vacuole after acidosomal and lysosomal fusion with the vacuole has occurred, in order to produce endosymbiosis. Correspondence and reprints: Biological Institute, Faculty of Science, Yamaguchi University, Yoshida 1677-1, Yamaguchi 753-8512, Japan.     

Photosynthetic activity of diimidoester-modified cells,permeaplasts, and cell-free membrane fragments of the blue-green alga Anacystis nidulans

On treating the blue-green alga Anacystis nidulans with dimethylsuberimidate up to 70% of the free NH₂ of the photosynthetic membrane is amidinated, and presumably inter- and intramolecular cross-links are established in the membrane proteins. Amidination destroys the ability of A. nidulans to photoreduce HCO₃^? but leaves the photochemical activities of Photosystems II and I nearly intact. With added electron acceptors, photosynthetic O₂ evolution can be demonstrated both with permeable cells (permeaplasts) prepared by digestion of the cell wall of dimethylsuberimidate-reacted A. nidulans with lysozyme, as well as with heavy membrane particles (36 000 × g) prepared from dimethylsuberimidate-reacted cells.Permeaplasts prepared from dimethylsuberimidate-reacted cells resist damage in hypoosmotic medium, whereas those prepared from unreacted cells are induced to release C-phycocyanin. On the other hand, the former are inactivated more easily by heat stress than the latter. On this basis, it is concluded that cross-linking with dimethylsuberimidate confers functional instability to photosynthetic membranes.     

Light and Electron Microsopic Observations on the Pathogenesis of Naegleria fowleri in Mouse Brain and Tissue Culture

Two strains of pathogenic Naegleria were employed to infect mice and monkey kidney (Vero line) cell cultures. Mice were infected intranasally. Moribund mice were sacrificed and their brains processed for light and electron microscopy. The normal architecture of the infected brain was completely destroyed; the olfactory lobes and the cerebral cortex showed the heaviest damage. The inflammatory response was mainly in the form of neutrophil polymorphs (PMN) and was confined to the olfactory lobes and the superficial regions of cerebral cortex. Numerous amebas were seen interspersed with the degenerating neurons, glial processes, and PMN. Most conspicuous were the food vacuoles which contained host tissue in various stages of digestion. Amebas in the brain tissue also produced many micropinocytotic vesicles from the surface of the plasma membrane. These vesicles are interpreted as vehicles of transport of nutritive materials from the host tissue. The infected cell culture showed the characteristic cytopathic effect (CPE). The CPE was chiefly in the form of cell shrinkage, nuclear pycnosis and discontinuity of cell sheet. Amebas were often seen in an intracellular location. The Vero cells produced many fuzzy pinocytotic vesicles at these loci where the ameba plasma membrane and Vero cell membrane were in close apposition; the probable significance of this is discussed. Most impressive, however, were the pseudopodial formation and capturing of the host material which indicated the great phagocytic activity of the amebas. This was confirmed further by the presence of large numbers of food vacuoles containing host material in various stages of digestion. These observations show that the amebas invade and destroy the brain tissue by active phagocytosis.     

Characterization of Aspergillus nidulans peroxisomes by immunoelectron microscopy

In previous work, we have demonstrated that oleate induces a massive proliferation of microbodies (peroxisomes) in Aspergillus nidulans. Although at a lower level, proliferation of peroxisomes also occurrs in cells growing under conditions that induce penicillin biosynthesis. Here, microbodies in oleate-grown A. nidulans cells were characterized by using several antibodies that recognize peroxisomal enzymes and peroxins in a broad spectrum of eukaryotic organisms such as yeast, and plant, and mammalian cells. Peroxisomes were immunolabeled by anti-SKL and anti-thiolase antibodies, which suggests that A. nidulans conserves both PTS1 and PTS2 import mechanisms. Isocitrate lyase and malate synthase, the two key enzymes of the glyoxylate cycle, were also localized in these organelles. In contrast to reports of Neurospora crassa, our results demonstrate that A. nidulans contains only one type of microbody (peroxisomes) that carry out the glyoxylate cycle and contain 3-ketoacyl-CoA thiolase and proteins with the C-terminal SKL tripeptide. Received: 4 March 1998 / Accepted: 2 July 1998     

Proteomic analysis of Aspergillus nidulans cultured under hypoxic conditions

The fungus Aspergillus nidulans reduces nitrate to ammonium and simultaneously oxidizes ethanol to acetate to generate ATP under hypoxic conditions in a mechanism called ammonia fermentation (Takasaki, K. et al.. J. Biol. Chem. 2004, 279, 12414–12420). To elucidate the mechanism, the fungus was cultured under normoxic and hypoxic (ammonia fermenting) conditions, intracellular proteins were resolved by 2‐DE, and 332 protein spots were identified using MALDI MS after tryptic digestion. Alcohol and aldehyde dehydrogenases that play key roles in oxidizing ethanol were produced at the basal level under hypoxic conditions but were obviously provoked by ethanol under normoxic conditions. Enzymes involved in gluconeogenesis, as well as the tricarboxylic and glyoxylate cycles, were downregulated. These results indicate that the mechanism of fungal energy conservation is altered under hypoxic conditions. The results also showed that proteins in the pentose phosphate pathway as well as the metabolism of both nucleotide and thiamine were upregulated under hypoxic conditions. Levels of xanthine and hypoxanthine, deamination products of guanine and adenine were increased in DNA from hypoxic cells, indicating an association between hypoxia and intracellular DNA base damage. This study is the first proteomic comparison of the hypoxic responses of A. nidulans.     

Cryopreservation of Ochromonas in Liquid Nitrogen with Reproducibility of Thiamin Assay

The first successful cryopreservation of Ochromonas danica and Ochromonas malhamensis is reported. The freezing method was consistently reproducible for the former, but not for the latter. Ochromonas danica cultures established from frozen material still could be used as test organisms for assay of thiamin. This is the first report of a protozoon retaining its assay property after being frozen to -196 C.     

The buoyancy-providing role of gas vacuoles in an aerobic bacterium

The heterotrophic, freshwater bacterium Prosthecomicrobium pneumaticum Staley possesses sufficient gas vacuoles to render it buoyant at all stages of growth. Although the cells have a turgor pressure of about 300 kPa, there is no evidence that this pressure is important in causing collapse of the constituent gas vesicles. A mutant of the bacterium, which produced only 0.2% of the amount of gas vacuoles produced by the wild type, was isolated. It always sank in liquid culture. Wild type and mutant bacteria grew at the same rate in shaken culture, but in static culture the wild type, which floated to the liquid surface grew more quickly than the mutant, which sank. Other competition experiments suggested that the advantage gained in floating at the surface was simply that oxygen was more readily available there to this obligate aerobe. Similar advantages may benefit gas vacuolate forms in natural habitats.A second mutant was isolated which produced about 40% fewer gas vacuoles than the wild type in corresponding stages of growth, insufficient to provide buoyancy, and unlikely to be of selective value. The occurrence of this mutant suggests there may be duplication of the gas vacuole gene.Abbreviations T turbidity - PST pressure sensitive turbidity - kPa kilo-Pascals (100 kPa=1 bar)     

Ultrastructure and histochemistry,of the stomach of Asplanchna sieboldi

Stomach cells of female Asplanchna sieboldi are specialized for absorption and intracellular digestion of nutrients. Evidence is presented to show that electron-opaque colloidal substances, present in the medium and within digestive vacuoles of the prey (Paramecium), are taken up by the stomach cells at the apical cell membrane and sequestered within food vacuoles which contain hydrolases working in both the acid and alkaline pH range. The stomach cells are also implicated in the absorption of molecules below the resolving power of the electron microscope. In rotifers possessing a complete digestive tract, this task is presumed to be handled by the intestine.     

CHLOROPHYLL,NITROGEN, AND PHOTOSYNTHETIC PATTERNS DURING GROWTH AND SENESCENCE OF TWO BLUE-GREEN ALGAE1,2

A standardized, multiflask, batch culture system was developed to study the processes of algal senescence in Anacystis nidulans and Phormidium molle Gom, var. tenuior W. et G. West. Growth data over a 3-year period gave reproducible and comparable time-course curves. Although A. nidulans is unicellular and P. molle filamentous, the patterns of change with age were similar. Mean logarithmic doubling times and carbon yields were, respectively, 6.9 hr and 390 mg C/liter for A. nidulans and 7.2 hr and 710 mg C/liter for P. molle. Chlorophyll concentration and photo-synthetic capacity per unit carbon rose rapidly during the logarithmic phase to maximum levels in either late log phase (P. molle) or early linear phase (A. nidulans), then fell throughout the declining growth phase to low levels in the stationary phase. Nitrate was rapidly exhausted from the medium during the period of logarithmic growth and stoichiometrically converted to particulate organic form; very little subsequent fixation of molecular nitrogen occurred. The phycocyanins were rapidly destroyed during the logarithmic phase while the carotenoids remained relatively constant throughout the whole growth period and then slowly declined. Preliminary electron micrographs showed a progressive deterioration in cellular ultrastructure, especially a reduction in the number of photosynthetic thylakoids, commenting in the linear growth phase. Analysis of the results suggests that occurrence of linear growth kinetics and termination of culture growth were caused by exhaustion of nitrate. The observed decreases in chlorophylls, phycocyanins, and photosynthetic capacity during active culture growth show that senescence effects may not be, as assumed, restricted to the stationary phase of growth.