Study of Molecular Karyotypes in Amoeboaphelidium protococcarum,the Endotrophic Parasite of Chlorophycean alga Scenedesmus

On the basis of the pulsed field gel electrophoresis (PFGE) technique, molecular karyotypes in Amoeboaphelidium protococcarum (the endotrophic parasite of chlorophycean algae, which combines protozoon- and fungus-type characters) were determined. Molecular karyotypes in the strains X1, X5, and X31, which differ in the host range and originate from Western, Central, and Eastern Euro-Asia respectively, demonstrate the intrageneric polymorphism—from 7 to 13 chDNA bands—with the estimated molecular size of 0.3–2.2 Mb were resolved, and the genome size was determined to be between 7.4 and 10.2 Mb. The molecular karyotype pattern in A. protococcarum is different from that in Protozoa. The low-grade distinction in molecular karyotypes between X1 and X5, on one hand, and high-grade distinction between X1/X5 and X31, on the other hand, makes it possible to place X31 in a separate taxon. Received: 24 June 1996 / Accepted: 1 August 1996     

两株筛自大规模生产跑道池的节旋藻性能比较研究

温度是影响节旋藻大规模培养中生物质产率的重要因素。筛选高产和耐受温度胁迫的优良藻株对提高节旋藻产量具有重要意义。从生产规模跑道池中分离到形态特征差异显著而亲缘关系很近的两株节旋藻Arthrospira sp.DICP-D(D)和Arthrospira sp.DICP-F(F),对其生物质积累性能和对低温、高温胁迫的耐受能力进行了评估。结果显示,尽管藻株D和藻株F在正常条件下拥有相似的生物质组成,在N胁迫和低温胁迫下拥有相同的碳水化合物积累能力,然而藻株D的生物质产率比相同条件下的藻株F高33%~230%。藻株D对高温胁迫的耐受能力是藻株F的2.1倍。藻株D在41℃和15℃下的生物质产率分别维持在正常温度下的73%和61%,显示其对温度胁迫的良好耐受能力。藻株D比藻株F拥有更有效的光保护机制,使其能够更好地适应胁迫环境。藻株D在生物质产率和胁迫耐受能力上的优良性状使其在户外大规模培养中具有良好的应用前景。     

Morphological,Molecular, and Phylogenetic Characterization of Nosema ceranae,a Microsporidian Parasite Isolated from the European Honey Bee,Apis mellifera1

ABSTRACT. Nosema ceranae, a microsporidian parasite originally described from Apis cerana, has been found to infect Apis melllifera and is highly pathogenic to its new host. In the present study, data on the ultrastructure of N. ceranae, presence of N. ceranae-specific nucleic acid in host tissues, and phylogenetic relationships with other microsporidia species are described. The ultrastructural features indicate that N. ceranae possesses all of the characteristics of the genus Nosema. Spores of N. ceranae measured approximately 4.4 × 2.2 μm on fresh smears. The number of coils of the polar filament inside spores was 18–21. Polymerase chain reaction (PCR) signals specific for N. ceranae were detected not only in the primary infection site, the midgut, but also in the tissues of hypopharyngeal glands, salivary glands, Malpighian tubules, and fat body. The detection rate and intensity of PCR signals in the fat body were relatively low compared with other examined tissues. Maximum parsimony analysis of the small subunit rRNA gene sequences showed that N. ceranae appeared to be more closely related to the wasp parasite, Nosema vespula, than to N. apis, a parasite infecting the same host.     

Vernalophrys algivore gen. nov., sp. nov. (Rhizaria: Cercozoa: Vampyrellida), a New Algal Predator Isolated from Outdoor Mass Culture of Scenedesmus dimorphus

Microbial contamination is the main cause of loss of biomass yield in microalgal cultures, especially under outdoor environmental conditions. Little is known about the identities of microbial contaminants in outdoor mass algal cultures. In this study, a new genus and species of vampyrellid amoeba, Vernalophrys algivore, is described from cultures of Scenedesmus dimorphus in open raceway ponds and outdoor flat-panel photobioreactors. This vampyrellid amoeba was a significant grazer of Scenedesmus and was frequently associated with a very rapid decline in algal numbers. We report on the morphology, subcellular structure, feeding behavior, molecular phylogeny, and life cycle. The new amoeba resembles Leptophrys in the shape of trophozoites and pseudopodia and in the mechanism of feeding (mainly by engulfment). It possesses two distinctive regions in helix E10_1 (nucleotides 117 to 119, CAA) and E23_1 (nucleotides 522 and 523, AG) of the 18S rRNA gene. It did not form a monophyletic group with Leptophrys in molecular phylogenetic trees. We establish a new genus, Vernalophrys, with the type species Vernalophrys algivore. The occurrence, impact of the amoeba on mass culture of S. dimorphus, and means to reduce vampyrellid amoeba contamination in Scenedesmus cultures are addressed. The information obtained from this study will be useful for developing an early warning system and control measures for preventing or treating this contaminant in microalgal mass cultures.     

In Vitro Characterization of a Recombinant AHL-Lactonase from Bacillus cereus Isolated from a Striped Catfish (Pangasianodon hypophthalmus) Pond

aiiA gene encoding AHL-lactonase was isolated from Bacillus cereus strain N26.2, originating from a striped catfish (Pangasianodon hypophthalmus) pond in Vietnam. This gene, abbreviated as aiiA(N26.2), was cloned and expressed in a competent Escherichia coli strain BL21(DE3)pLysS. The resulting protein, abbreviated as AiiA_N26.2, was highly active in the pH range of 6–8 and could retain 80 % of the maximum activity under storage for 5 days at 4 °C or for 3 days at 20 °C. These properties of AiiA_N26.2 protein confers its future application via feed supplementation, with the purpose of controlling aquaculture pathogens which regulate the virulence via a quorum sensing system.     

Isolation,Characterization and Synthesis of DNA from a Malaria Parasite*

SYNOPSIS. Methods for the isolation and purification of DNA from the intraerythrocytic stages of the avian malaria parasite Plasmodium lophurae are described. The DNA of P. lophurae was found to have a 19 mole-percent guanine plus cytosine (G+C) composition as determined by melting temperature and density measurements in CsCl gradients, whereas that of the host cell nucleus was 35 molepercent G+C. Synthesis of DNA by P. lophurae was studied by following the incorporation of P³² during the in vitro growth of plasmodia in infected cell suspension cultures. DNA synthesis was linear during the multiple nuclear divisions of a single growth cycle in highly synchronous cultures. Separation of P³² labeled parasite DNA from the DNA of the duck erythrocyte on CsCl gradients showed that only parasite DNA was synthesized.     

Production and Characterization of an Exopolysaccharide Excreted by a Deep-Sea Hydrothermal Vent Bacterium Isolated from the Polychaete Annelid Alvinella pompejana

The heterotrophic and mesophilic marine bacterium HYD-1545 was isolated on a metal-amended medium from the dorsal integument of the hydrothermal vent polychaete Alvinella pompejana. This strain, which can be assigned to the genus Alteromonas on the basis of its G+C content and phenotypical features, produced large amounts of an acidic polysaccharide in batch cultures. The polysaccharide was excreted during the stationary phase of growth and contained glucose, galactose, glucuronic acid, galacturonic acid, and 4,6-O-(1-carboxyethilidene)-galactose as major components. This polysaccharide was a polyelectrolyte, and the viscosity of its solutions depended on the ionic strength. The decrease in viscosity with increasing NaCl concentrations and the effect of Ca²⁺ in decreasing the viscosity at low Ca²⁺ concentrations support a model in which the polysaccharide carries anionic groups. However, an unusual behavior was observed at higher concentrations and could be related to intermolecular interactions involving Ca²⁺ ions.     

Enzymatic Characterization of an Amine Oxidase from Arthrobacter sp. Used to Measure Phosphatidylethanolamine

Ethanolamine oxidase was screened with the aim of using it to establish a novel enzymatic phosphatidylethanolamine assay. Ethanolamine oxidase activity was detected in the crude extract of Arthrobacter sp., and the enzyme was purified more than 15-fold in three steps with a 54% yield. SDS–PAGE revealed the presence of only one band, which migrated, with an apparent molecular mass of 70 kDa. Biochemical characterization of the enzyme showed phenylethylamine to be the preferred substrate, with the highest kcat/Km value. The primary structure, determined by sequencing the cloned gene, showed a high degree of identity to Cu-containing phenylethylamine oxidase (64%). When heterologously overexpressed in Escherichia coli, the enzyme exhibited only a trace of amine oxidase activity, but high levels of activity emerged after exposure to Cu²⁺, as is typical of recombinant copper amine oxidases. Preliminary application of this enzyme coupled with phospholipase D for determination of phosphatidylethanolamine is also described. This is the first enzymatic method for the measurement of phosphatidylethanolamine.     

Selection of a Photosynthetic Bacterium Suitable for Hydrogen Production in Outdoor Cultures among Strains Isolated in the Seoul,Taegu, Sendai and Bangkok Areas

Very efficient hydrogen producing photosynthetic bacteria, strains SL1, SL3, SL16 and TG28 newly isolated in Korea, and strain KM113 newly isolated in the Sendai area, were found to be Rhodopseudomonas spp. To examine the stability of cell suspensions of the cultures for hydrogen production, which is closely associated with light absorption, we conducted larger scale cultures under periodic illumination (12-hr intervals) without stirring at 30°C using strains SL1 and Rhodopseudomonas sphaeroides B5, the latter was isolated in the Bangkok area. Both strains gave homogeneous cell suspensions throughout the incubation period and larger amounts of hydrogen were produced in a shorter period of time by both cultures than obtained with Rhodopseudomonas sp. TN3, an isolate from the Sendai area which was reported previously. With the cells of the new isolates and strains TN3 and B5 grown on glutamate-malate medium at 30°C, we measured hydrogen production at 20, 30 and 40°C in the same medium. Among them, strains SL1, SL16 and KM113 showed the highest hydrogen production activity at 30°C. The maximum hydrogen production rates with these strains were over 130 µ1/hr/mg dry cells, but at 40°C, the highest activity (138 µl/hr/mg dry cells) was obtained with strain B5. Since strain B5 also showed good activities at 20 and 30°C, we suggest that this strain might be suitable for hydrogen production in outdoor cultures.     

A New Heterolobosean Amoeboflagellate,Tetramitus dokdoensis n. sp., Isolated from a Freshwater Pond on Dokdo Island in the East Sea,Korea

The genus Tetramitus is a representative amoeboflagellate group within the Heterolobosea, and currently contains over a dozen species. Here, a new heterolobosean amoeboflagellate was isolated from a freshwater pond on Dokdo Island, Korea. The amoebae have eruptive pseudopodia, no uroidal filament, and a nucleus with a central nucleolus. The length and width of the amoebae are 15.5–28.0 μm and 5.4–12.6 μm, respectively. The flagellates are conical, with 4 flagella of equal length (~10 μm). There is a discrete rostrum in the subapical region of the flagellate form. The cyst has thin endo‐ and ectocyst layers and no cyst pores. The amoeba shows slow movement at 37 °C, but does not move at 42 °C under a light microscope. Phylogenies of the 18S rRNA gene and the ITS1‐5.8S rRNA gene‐ITS2 sequence show that the strain belongs to a subclade of Tetramitus that includes Tetramitus rostratus, Tetramitus waccamawensis and Tetramitus entericus, amongst others. Nonetheless, the strain is distinct from other species in both molecular phylogenetic trees. Thus the strain isolated from the Dokdo Island is proposed as a novel species, Tetramitus dokdoensis n. sp.     

Characterization of Sphingomonas sp. Ant 17, an Aromatic Hydrocarbon-Degrading Bacterium Isolated from Antarctic Soil

Sphingomonas sp. strain Ant 17 was isolated from fuel-contaminated soil collected at Scott Base, Ross Island, Antarctica. We anticipated that Ant 17 would be a good model organism for studying cold climate bioremediation, and therefore determined its biodegradation capabilities and tolerance of potentially growth-limiting environmental conditions. Sphingomonas sp. Ant 17 degrades the aromatic fraction of several different crude oils, jet fuel, and diesel fuel at low temperatures and without nutrient amendment. It utilizes or transforms a broad range of pure aromatic substrates, including hydrocarbons, heterocycles, and aromatic acids and alcohols. Ant 17 grows at temperatures of 1 degree C to 35 degrees C and mineralizes radiolabeled phenanthrene over a range of more than 24 degrees C. This psychrotolerant isolate appears to utilize hydrocarbons more efficiently at low temperatures than would be predicted by mesophilic enzyme kinetics. The optimum pH for growth was 6.4 at 22 degrees C, with extended lag phases observed in more alkaline media. However, there was less effect of pH on lag phase at lower temperatures. Ant 17 displayed greater tolerance to UV irradiation and freeze-thaw cycles than the hydrocarbon-degrading isolate Sphingomonas sp. WPO-1, which may reflect adaptation to its Antarctic soil environment. However, it was more sensitive than expected to desiccation and to low concentrations of NaCl and CaCl(2). Ant 17 was phenotypically stable and lacked detectable plasmids, suggesting a chromosomal location for genes encoding aromatic degradation enzymes. Its broad aromatic substrate range and tolerance of low and fluctuating temperature and low nutrients make Sphingomonas sp. Ant 17 a valuable microbe for examining fuel spill bioremediation in cold soils.     

Identification and Partial Characterization of a Dynamin-Like Protein,EhDLP1, from the Protist Parasite Entamoeba histolytica

The dynamin superfamily of proteins includes a large repertoire of evolutionarily conserved GTPases that interact with different subcellular organelle membranes in eukaryotes. Dynamins are thought to participate in a number of cellular processes involving membrane remodeling and scission. Dynamin-like proteins (DLPs) form a subfamily of this vast class and play important roles in cellular processes, such as mitochondrial fission, cytokinesis, and endocytosis. In the present study, a gene encoding a dynamin-like protein (EhDLP1) from the protist parasite Entamoeba histolytica was identified and the protein was partially characterized using a combination of in silico, biochemical, and imaging methods. The protein was capable of GTP binding and hydrolysis, lipid binding, and oligomerization. Immunofluorescence studies showed the protein to be associated with the nuclear membrane. A mutant of EhDLP1 lacking GTP binding and hydrolyzing activities did not associate with the nuclear membrane. The results suggest a nucleus-associated function for EhDLP1.Dynamins are a vast family of GTPases implicated in myriad processes, some of which lead to alteration of membrane structure (22). Classical dynamins, such as mammalian dynamins 1 to 3 (5) and the shibire protein from Drosophila melanogaster (29), are required mainly for scission of vesicles, acting as mechanoenzymes or molecular switches (12). In addition, several dynamin-like proteins (DLPs) have been identified in different organisms ranging from yeast to mammals. DLPs play a key role in the division of organelles such as chloroplasts, mitochondria, and peroxisomes (15, 22). For example, Candida albicans Vps1 has been shown to be associated with virulence-related phenotypes like filamentation and biofilm formation (2). DLPs have also been identified in protists. Downregulation or ablation of the gene products in protists by RNA interference or other methods has helped to decipher the multiple functions carried out by these proteins. These include mitochondrial division and endocytosis in Trypanosoma brucei (6, 20), cytokinesis in Dictyostelium discoideum (31), phagocytosis in Paramecium species (30), endocytic transport in Giardia lamblia (11), and biogenesis of secretory vesicles in Toxoplasma gondii (4). Apart from cellular membranes, some DLPs may also associate with nuclear membranes. Recently, a study on Tetrahymena thermophila reported the requirement of Drp6 for macronuclear development (23). The human DLP MxB has been shown previously to localize to the cytoplasmic face of the nuclear envelope and is involved in regulation of nuclear import (14). Dynamins and DLPs share a minimal domain architecture which includes an N-terminal GTPase domain, a middle domain, and a GTPase effector domain (GED). The GED is involved in enzyme oligomerization and the regulation of the GTPase activity. The GTPase domain contains a well-conserved GTP binding motif required for guanine-nucleotide binding and hydrolysis (22). DLPs lack a pleckstrin homology (PH) domain and a proline-rich domain (PRD), normally associated with protein-lipid and protein-protein interaction.The endocytic, secretory, and adhesion pathways of the parasite Entamoeba histolytica play crucial roles in nutrient uptake, host cell destruction, and the endocytosis of gut resident bacteria, erythrocytes, and cell debris (21). The trophozoites of E. histolytica are known to have robust endocytic capabilities, turning over approximately a third of their cellular volume every hour (1, 19). The presence of a classical receptor-mediated pathway has not yet been clearly demonstrated, though some of the molecules involved in this pathway, such as clathrin, have been identified in E. histolytica (28). Typical eukaryotic cytoplasmic organelles have not been observed in this organism. However, the functional equivalents of a Golgi network and an endoplasmic reticulum are reported to be present (3, 26). Entamoeba also contains a genomeless variant of mitochondria, termed mitosomes (17). The division or biogenesis of these organelles during cell division is not understood. Nuclear division in E. histolytica occurs without nuclear membrane dissolution and reassembly. Since dynamins and DLPs are known to be involved in endocytosis and organelle division, it is likely that these proteins may be performing similar functions in this organism. Although the E. histolytica genome encodes putative dynamins and DLPs, none of these have been characterized. In order to understand the roles of these molecules in amebic biology, we have initiated studies to characterize these proteins from E. histolytica. Here, we report the basic characterization of E. histolytica dynamin-like protein 1 (EhDLP1).     

Characterization of Methanosarcina mazei N2M9705 Isolated from an Aquaculture Fishpond

A new methanogenic isolate, designated as strain N2M9705 (=OCM 668), was isolated from an aquaculture fishpond near Wang-gong, Taiwan. This strain grew on trimethylamine and methanol, but it did not catabolize H₂-CO₂, acetate, or formate. The cells were stained Gram-negative, nonmotile, irregular coccus 0.6–0.8 μm in diameter. Gas vacuoles were observed and cell aggregated to form various sizes of granules. Cells grew optimally at 32°–37°C with 1% NaCl. The pH range of growth was 6.2–7.4, and higher pH inhibited the cell growth. The cells grew well in minimal medium, but growth was greatly stimulated by yeast extract and peptone. A comparison of 16S rDNA sequences of this organism phylogenetically related to Methanosarcina mazei. This is the first report of methyltrophic methanogenic isolated from an aquaculture fishpond. Received: 16 March 1999 / Accepted: 16 April 1999     

Identification and Characterization of Leuconostoc fallax Strains Isolated from an Industrial Sauerkraut Fermentation

Lactic acid bacterial strains were isolated from brines sampled after 7 days of an industrial sauerkraut fermentation, and six strains were selected on the basis of susceptibility to bacteriophages. Bacterial growth in cabbage juice was monitored, and the fermentation end products were identified, quantified, and compared to those of Leuconostoc mesenteroides. Identification by biochemical fingerprinting, endonuclease digestion of the 16S-23S intergenic transcribed spacer region, and sequencing of variable regions V1 and V2 of the 16S rRNA gene indicated that the six selected sauerkraut isolates were Leuconostoc fallax strains. Random amplification of polymorphic DNA fingerprints indicated that the strains were distinct from one another. The growth and fermentation patterns of the L. fallax isolates were highly similar to those of L. mesenteroides. The final pH of cabbage juice fermentation was 3.6, and the main fermentation end products were lactic acid, acetic acid, and mannitol for both species. However, none of the L. fallax strains exhibited the malolactic reaction, which is characteristic of most L. mesenteroides strains. These results indicated that in addition to L. mesenteroides, a variety of L. fallax strains may be present in the heterofermentative stage of sauerkraut fermentation. The microbial ecology of sauerkraut fermentation appears to be more complex than previously indicated, and the prevalence and roles of L. fallax require further investigation.     

Biochemical Characterization and Substrate Specificity of Autophagin-2 from the Parasite Trypanosoma cruzi

The genome of the parasite Trypanosoma cruzi encodes two copies of autophagy-related cysteine proteases, Atg4.1 and Atg4.2. T. cruzi autophagin-2 (TcAtg4.2) carries the majority of proteolytic activity and is responsible for processing Atg8 proteins near the carboxyl terminus, exposing a conserved glycine. This enables progression of autophagy and differentiation of the parasite, which is required for successful colonization of humans. The mechanism of substrate hydrolysis by Atg4 was found to be highly conserved among the species as critical mutations in the TcAtg4.2, including mutation of the conserved Gly-244 residue in the hinge region enabling flexibility of the regulatory loop, and deletion of the regulatory loop, completely abolished processing capacity of the mutants. Using the positional scanning-substrate combinatorial library (PS-SCL) we determined that TcAtg4.2 tolerates a broad spectrum of amino acids in the P4 and P3 positions, similar to the human orthologue autophagin-1 (HsAtg4B). In contrast, both human and trypanosome Atg4 orthologues exhibited exclusive preference for aromatic amino acid residues in the P2 position, and for Gly in the P1 position, which is absolutely conserved in the natural Atg8 substrates. Using an extended P2 substrate library, which also included the unnatural amino acid cyclohexylalanine (Cha) derivative of Phe, we generated highly selective tetrapeptide substrates acetyl-Lys-Lys-Cha-Gly-AFC (Ac-KKChaG-AFC) and acetyl-Lys-Thr-Cha-Gly-AFC (Ac-KTChaG-AFC). Althoughthese substrates were cleaved by cathepsins, making them unsuitable for analysis of complex cellular systems, they were recognized exclusively by TcAtg4.2, but not by HsAtg4B nor by the structurally related human proteases SENP1, SENP2, and UCH-L3.     

Production and Characterization of a New Fungal Metabolite,Cotylenol

A new metabolite, cotylenol, has been isolated from the culture filtrate of a fungal strain, 501–7 w. Production and characterization of cotylenol are described. The chemical relationship between cotylenol and cotylenin A has been elucidated.