Extracellular DNA in aquatic ecosystems may in part be due to phycovirus activity

In a eutrophic lake, a crash of the algal population was followed by a significant increase in the number of virus-like particles (from ca. 1 10⁶ ml^–1 to ca. 26 10⁶ ml^–1), and soon thereafter by an increase of the amount of extracellular DNA (from ca. 20 µg l^–1 to ca. 40 µg l^–1). The same pattern of correlation between decrease of algae and increase of viruses and extracellular DNA could be demonstrated by an in vitro experiment with a Chlorella-virus-system. Lysis of algae by viruses increased both the number of viruses and the amount of DNA in the culture medium. Extracellular DNA mainly consisted of material with a molecular weight below 500 bp.The Chlorella-virus-system is discussed. It could be used as a model-system for studying the dynamics of interaction of viruses and algae in aquatic ecosystems.     

Characterization of the terminal inverted repeats and their neighboring tandem repeats in the Chlorella CVK1 virus genome

A unique group of large icosahedral viruses that infect a unicellular green alga (Chlorella sp. NC64A) were isolated from freshwater sources in Japan. These viruses contain a linear double-stranded DNA (dsDNA) genome with hairpin ends. A physical map was constructed for the genomic DNA of CVK1 (Chlorella virus isolated in Kyoto, no. 1) by pulsed-field gel electrophoresis of restriction fragments. The nucleotide sequences around both termini of the CVK1 DNA revealed the presence of inverted terminal repeats (ITR) of approximately 1.0 kb. Adjacent to the ITR, unique sequence elements of 10 to 20 by were directly repeated 20 to 30 times in tandem array. Several copies of these repeat elements were deleted in virus mutants that were occasionally generated from Chlorella cells that were in a putative CVK1 carrier state. These repeats might represent a hot spot of rearrangement in the CVK1 genome.     

A Functional Putative Phytochelatin Synthase from the Primitive Red Alga Cyanidioschyzon merolae

Phytochelatin synthase (PCS) catalyzes the synthesis of phytochelatins (PCs), which play a detoxification role in higher plants. Heterologous expression of CmPCS, a product of a PCS-like gene from the genomic DNA of the red alga Cyanidioschyzon merolae, rescued Cd²⁺-sensitive yeast from Cd²⁺ toxicity. The fact that these transformed cells synthesized PCs demonstrates that CmPCS is functional.     

Relationship between carbohydrate movement and the symbiosis in lichens with green algae

Summary When isolated in pure culture, four genera of lichen algae were able to produce the polyol which is known to move from the alga to the fungus in lichens with these algae. This conclusion corrects earlier suggestions that the mobile polyol is only formed by the alga in the lichen thallus. Stichococcus produced sorbitol and it is therefore suggested that, in lichens with this alga, sorbitol moves between the symbionts. Hyalococcus and Stichococcus had a similar pattern of incorporation of H¹⁴CO ₃ ^- in the light, suggesting a close relationship between these algae which are only separated now on morphological grounds.The pattern of incorporation of H¹⁴CO ₃ ^- in the light into Cladonia cristatella and its alga (Trebouxia erici) in culture indicates that in the cultured algae more ¹⁴C was incorporated into ethanol insoluble substances and lipids and less into ribitol than in the lichen. The pattern in a joint culture of the alga and the fungus of C. cristatella was approximately intermediate between that of the lichen and the alga. However, only a small amount of ¹⁴C fixed by the alga reached the fungus in the joint culture, and it is therefore suggested that the presence of the fungus without morphological differentiation into a lichen thallus is not sufficient to promote the alga to release carbohydrate.     

ANALYSIS OF THE EXPRESSION OF GENES FOR P-TYPE ATPASE IN THE HALOTOLERANT ALGA DUNALIELLA SALINA (CHLOROPHYCEAE)1

A partial complementary DNA (cDNA) (DSA8) for a P-type ATPase was obtained from the halotolerant alga Dunaliella salina (Dunal) Teod. (Chlorophyceae). The cDNA exhibited greater than 90% homology to the cDNA for a H⁺-ATPase in D. bioculata Butcher. The expression of the gene that corresponded to DSA8 was decreased strongly by increases in NaCl concentration. The expression of a gene that corresponded to another ATPase (DSA1; possibly for a Ca²⁺-ATPase) from D. salina did not show the same decrease as did the DSA8. However, increased osmotic pressure due to glycerol resulted in the same decrease in the DSA8 gene. Under salt or osmotic stress, the activity of a H⁺-ATPase from microsomes of this alga also decreased. We suggest that expression of the gene for the plasma membrane H⁺-ATPase of D. salina is regulated by osmotic pressure rather than by the concentration of NaCl.     

THE NUCLEAR CELL CYCLE IN CHLAMYDOMONAS (CHLOROPHYCEAE)1

The timing of replication and division of the Chlamydomonas Ehrenberg nucleus in the vegetative cell cycle and at gametogenesis was examined, using fluorescence microspectrophotometry with two fluorochromes, mithramycin and 4′,6-diamidino-2-phenylindole (DAPI). Under appropriate conditions, these bind specifically to DNA, and the fluorescence of the DNA fluorochrome complex is a quantitative measure of the DNA content. The alga is a haplont, which produces 2ⁿ daughter cells at the time of vegetative reproduction; cytokinesis and daughter cell release lag behind karyokinesis. No nucleus was found to contain more than the 2c quantity of DNA. Hence daughter cell production proceeds by doubling of the nuclear DNA followed by karyokinesis, in a repetitive sequence. As reported previously for C. reinhardtii Dangeard, the gametes of C. moewusii Gerloff contain the 1c amount of nuclear DNA. Several conflicting interpretations of the cell cycle sequence proposed in the literature were resolved.     

A linear DNA molecule of 5.9 kilobase-pairs is highly homologous to the chloroplast DNA in the green alga Chlamydomonas moewusii

Summary The unicellular green alga Chlamydomonas moewusii contains small DNA species of unknown cellular location. We report that the most abundant of these DNAs, here designated low-molecular-weight DNA (LMW DNA), is a linear molecule of 5.9 kilobase pairs (kbp). Southern blot hybridization and restriction enzyme analysis revealed that the LMW DNA sequence also exists as an integrated sequence in a discrete region of the chloroplast genome. We have confirmed earlier reports that small DNA species related to the LMW DNA are absent from Chlamydomonas eugametos, an alga which is interfertile with C. moewusii. In the C. eugametos chloroplast genome we found only remnants of the LMW DNA sequence.     

Rapid extraction of high-quality genomic DNA from Porphyra yezoensis (Bangiales, Rhodophyta)

We developed a simple, rapid and stable method for extraction of high molecular weight DNA from the marine red alga Porphyra yezoensis Ueda using both guanidium treatment and QIAGEN? kit (Funakoshi, Tokyo, Japan). The method does not require expensive equipment and complex steps. The DNA yield averaged 1.5 μg 100 mg^?1 of Porphyra tissue and the A260/A280 and A230/A260 ratios of the DNA were approximately 1.8 and 0.4, respectively. It was of sufficient quality to be used for not only polymerase chain reactions but also other DNA manipulation techniques such as restriction digestion and construction of genomic libraries.     

Screening of algal strains for metal removal capabilities

Eight algal species were tested for their ability to remove five toxic metalsduring 30-min exposures to single-metal (1 mg L^-1) solutions at pH7. Efficacy of metal bioremoval varied according to algal species and metal. Al⁺³ was best removed by the thermophilic blue-green alga(cyanobacterium) Mastigocladus laminosus, Hg⁺² and Zn⁺² by the thermophilic and acidophilic red alga Cyanidiumcaldarium, and Cd⁺² by C. caldarium and the green alga Scenedesmus quadricauda. All of these alga/metal combinations resultedin >90% metal removal. However, none of the eight algal speciesremoved more than 10% of Cr⁺⁶. Results indicate that some toxicmetals are more readily removed than others are by algae and that selectionof appropriate strains could potentially enhance bioremoval of specificmetals from wastewater at neutral pH.     

ISOLATION AND PRELIMINARY CHARACTERIZATION OF THREE CHLAMYDOMONAS STRAINS INTERFERTILE WITH CHLAMYDOMONAS REINHARDTII (CHLOROPHYTA)1

Three new strains of the unicellular green alga Chlamydomonas reinhardtii Dangeard were isolated from soil. The isolates differed from one another and from standard laboratory strains of C. reinhardtii in a number of traits, including heavy metal resistance, protein composition, and mitochondrial DNA length. The new isolates also exhibited distinctive restriction fragment length polymorphisms in their nuclear, chloroplast, and mitochondrial genomes. The new isolates were interfertile with the standard laboratory strains and appeared to transfer chloroplast and mitochondrial genomes in a similar manner, that is, predominantly from the material (mt⁺) and paternal (mt^?) parents, respectively.     

Biochemical characterisation of LigN, an NAD+-dependent DNA ligase from the halophilic euryarchaeon Haloferax volcanii that displays maximal in vitro activity at high salt concentrations

Background  

DNA ligases are required for DNA strand joining in all forms of cellular life. NAD⁺-dependent DNA ligases are found primarily in eubacteria but also in some eukaryotic viruses, bacteriophage and archaea. Among the archaeal NAD⁺-dependent DNA ligases is the LigN enzyme of the halophilic euryarchaeon Haloferax volcanii, the gene for which was apparently acquired by Hfx.volcanii through lateral gene transfer (LGT) from a halophilic eubacterium. Genetic studies show that the LGT-acquired LigN enzyme shares an essential function with the native Hfx.volcanii ATP-dependent DNA ligase protein LigA.     

Ecology of phytoplankton communities dominated by Aureococcus anophagefferens: the role of viruses, nutrients, and microzooplankton grazing

We investigated the impact of viruses, nutrient loading, and microzooplankon grazing on phytoplankton communities in two New York estuaries that hosted blooms of the brown tide alga Aureococcus anophagefferens during 2000 and 2002. The absence of a bloom at one location during 2002 allowed for the fortuitous comparison of a bloom and non-bloom year at the same location as well as a comparison of two sites experiencing bloom and non-bloom conditions during the same year. During the study, blooms were found at locations with high levels of dissolved organic nitrogen and lower nitrate concentrations compared to a non-bloom location. Experimental additions of inorganic nitrogen and phosphorus yielded growth rates within the total phytoplankton community which significantly exceeded control treatments in 83% of experiments, while A. anophagefferens experienced significantly increased growth during only 20% of experimental inorganic nutrient additions. Consistent with prior research, these results suggest brown tides are not caused by eutrophication, but instead are more likely to occur when sources of labile DOM are readily available. Microzooplankton grazing rates on the total phytoplankton community during a bloom were lower than grazing rates at a non-bloom site, and grazing rates on A. anophagefferens were lower than grazing rates on the total community on some dates, suggesting that reduced grazing mortality may also promote brown tides. Mean densities of viruses during blooms (3 × 10⁸ ml⁻¹) were elevated compared to most estuarine environments and were twice the levels found at a non-bloom site. Experimental enrichment of the natural viral densities yielded a significant increase in A. anophagefferens growth rates relative to control treatments when background levels of viruses were low (<1.7 × 10⁸ ml⁻¹), suggesting that viruses may promote bloom occurrence by regenerating DOM or altering the composition of microbial communities.     

Double-stranded RNA replicons associated with chloroplasts of a green alga,Bryopsis cinicola

Double-stranded RNAs (dsRNAs) associated with chloroplasts and mitochondria have been found in the coenocytic green alga Bryopsis cinicola. In this study we report molecular properties of the four chloroplast-associated dsRNAs (BDRC1 to BDRC4) The longest dsRNA molecule (BDRC1) was sequenced entirely (1959 bp) and a single large ORF of 1722 bp was found within it. Database searches revealed similarities between the deduced amino acid sequence of this ORF and RNA-dependent RNA polymerase (RdRp) sequences from several RNA viruses. The most similar sequence in the database was the RdRp of beet cryptic virus 3. Phylogenetic analysis revealed that the RdRp-like sequence of BDRC1 can be placed in the Partitiviridae clade. To detect autonomous replication of these dsRNAs, RdRp assays were carried out with actinomycin D, which is an inhibitor of DNA-dependent RNA synthesis. Incorporation of [-³²P]UTP was detected specifically in the chloroplast and mitochondrial dsRNAs, indicating that both the chloroplast dsRNAs (BDRCs) and the mitochondrial dsRNA (BDRM) of B. cinicola are RNA replicons. The green alga B. cinicola harbors different dsRNA replicons in its chloroplasts and mitochondria.     

IDENTIFICATION OF CYTOKININS IN SARGASSUM MUTICUM (PHAEOPHYTA) AND PORPHYRA PERFORATA (RHODOPHYTA)1

Combined gas chromatography-mass spectrometry (GCMS) was used to identify and quantify specific cytokinins from Porphyra perforate J. Ag. and Sargassum muticum (Yendo) Fensh. The level of isopentenyladenosine was estimated to be 0.6 μ·kg^?1 fresh weight in Porphyra and 0.9 μ·kg^?1 fresh weight in Sargassum. The level of cis-zeatin riboside was estimated to be 0.2 μ·kg^?1 fresh weight in Sargassum. This is the first definitive identification of a cytokinin from a red alga, and the second report from a brown alga.     

Development and evaluation of a new detection tool—visual DNA microarray for simultaneous and specific detection of human immunodeficiency virus type-1 and hepatitis C virus

Human immunodeficiency virus type-1 (HIV-1) and hepatitis C virus (HCV) are transfusion-transmitted human pathogens that have a major impact on blood safety and public health. Based on multiplex asymmetrical PCR and coupled with gold labelled silver stain (GLSS), we developed the visual DNA microarray for sensitive and specific detection of these two viruses. Capturing probes of 5′-end-amino-modified oligonucleotides were immobilized on glass surface to bind the complement biotinylated target DNA. The Au–streptavidin probe was introduced to the microarray for specific binding to biotin. Black images of microarray spots which result from the precipitation of silver onto Au–streptavidin probes, were visualized by naked eyes. In order to improve the efficiency of microarray hybridization, triplex asymmetrical PCR of HIV-1, HCV and Human enterovirus 71 (EV-71, used as positive control) were performed to prepare abundant biotinylated single-stranded target DNA. The sensitivity of visual DNA microarray (10³ copies/ml) was higher than conventional PCR (10⁴ copies/ml) and was identical to FQ-PCR (10³ copies/ml). Total 152 blood samples containing the two viruses were tested using the DNA microarray and fluorescence quantitative real-time PCR (FQ-PCR). The results were identical (P > 0.05). So this system has high sensitivity and may have potential in clinical applications.     

Isolation and Phylogenetic Analysis of Novel Viruses Infecting the Phytoplankton Phaeocystis globosa (Prymnesiophyceae)

Viruses infecting the harmful bloom-causing alga Phaeocystis globosa (Prymnesiophyceae) were readily isolated from Dutch coastal waters (southern North Sea) in 2000 and 2001. Our data show a large increase in the abundance of putative P. globosa viruses during blooms of P. globosa, suggesting that viruses are an important source of mortality for this alga. In order to examine genetic relatedness among viruses infecting P. globosa and other phytoplankton, DNA polymerase gene (pol) fragments were amplified and the inferred amino acid sequences were phylogenetically analyzed. The results demonstrated that viruses infecting P. globosa formed a closely related monophyletic group within the family Phycodnaviridae, with at least 96.9% similarity to each other. The sequences grouped most closely with others from viruses that infect the prymnesiophyte algae Chrysochromulina brevifilum and Chrysochromulina strobilus. Whether the P. globosa viruses belong to the genus Prymnesiovirus or form a separate group needs further study. Our data suggest that, like their phytoplankton hosts, the Chrysochromulina and Phaeocystis viruses share a common ancestor and that these prymnesioviruses and their algal host have coevolved.     

EVOLUTIONARY RELATIONSHIPS BETWEEN FOUR SPECIES OF CLADOPHORA (CLADOPHORALES,CHLOROPHYTA) BASED ON DNA–DNA HYBRIDIZATION1

Analysis of the reassociation kinetics of the DNA from Cladophora pellucida (Huds.) Kütz. indicates that the genome of this benthic alga is comprised of approximately 75% repetitive sequences. Single-copy sequences reassociated with a rate constant of 1.8 × 10^?3 M^?1· s^?1, which corresponds to a haploid genome size of 4.7 × 10⁸ bp. Genotypic relationships between members of the form section Longiarticulatae were determined by the method of DNA–DNA hybridization. No significant divergence was observed between the single-copy sequences of C. pellucida isolates from the East Atlantic coast and Mediterranean Sea. Cladophora feredayi Harv. and C. att. ad pellucida from Australia and C. pellucidoidea van den Hoek from the West Atlantic coast were highly and about equally divergent from C. pellucida. The data support the hypothesis that the West Atlantic–West Pacific divergence reflects the middle Miocene closure of the Mediterranean–Indo-Pacific seaways, and the hypothesis that the Northwest Atlantic–Northeast Atlantic divergence reflects the middle Miocene thermal separation of these coasts.     

Caribbean corals exhibit species-specific differences in competitive abilities with an aggressive encrusting alga,Ramicrusta textilis

Caribbean coral cover has decreased substantially in recent decades, with much of the live coral being replaced by macroalgae. Encrusting red algae in the genus Ramicrusta have become abundant throughout the region and have demonstrated widespread harm to corals by overgrowing living tissue, causing colony mortality, and impairing coral recruitment. In this research, Ramicrusta textilis was identified by morpho-anatomy and DNA sequencing from nine sites around St. Thomas, US Virgin Islands, and 3D photogrammetry was used to measure the rate of algal growth on stony corals. 3D models of individual coral colonies (five species plus controls, N = 72) competing with R. textilis revealed differential competitive abilities among taxa, with Siderastrea siderea being the only species capable of inhibiting overgrowth by the alga (mean linear algal growth − 1.1 mm yr⁻¹). Important reef building coral species such as Orbicella annularis and Orbicella faveolata were poor competitors (mean linear algal growth + 15 mm yr⁻¹ and + 7.7 mm yr⁻¹, respectively), indicating that the emergence of the alga could have significant impacts on Caribbean coral reef species diversity, community composition, and structural complexity.

     

Characterization of rapidly labelled RNA associated with DNA in Chlorella

Summary After pulse-labelling with ³²P-orthophosphate and fractionation of the nucleic acids from synchronously cultured cells of the green alga Chlorella pyrenoidosa on methylated serum albumin and kieselgur (MAK) the DNA contained a species of ³²P-RNA. About 3% of the total ³²P radioactivity incorporated in the cells' RNA were confined to this DNA-associated component. Its base ratio differed significantly from that of soluble and ribosomal RNA but varied only slightly during the life-cycle of the cells. About 4% of the DNA-associated ³²P-RNA resisted ribonuclease digestion suggesting a stable binding of RNA to DNA in the form of a complex. Gel filtration and sucrose gradient centrifugation of the nucleic acids isolated in the DNA region during previous MAK column chromatography resulted in a separation of most of the ³²P-RNA from the DNA. The remaining ribonuclease-resistent but alkali labile ³²P radioactivity bound to the latter was in the order of 4%. No evidence has been obtained so far that it represents rapidly synthesized RNA associated with DNA in stable and functional complex.