Unicellular Species Research Articles

Temperate and lytic cyanophages from the Gulf of Mexico

The unicellular cyanobacterial species Synechococcus and Prochlorococcus are known to be vital components of marine ecosystems, especially in the vast oligotrophic areas. Lytic cyanophages infecting unicellular phytoplankton are prevalent and have been demonstrated to act as important constraints on community composition contributing to the seasonal succession in genotypes. Lysogeny in Synechococcus has been documented experimentally in natural environments by prophage induction. At this time it is completely unknown how prevalent lysogeny is among Synechococcus populations. This study was performed to document important features such as size, morphology and the incidence of the T4-like capsid portal protein gene (g20) in a group of lytic Synechococcus cyanophages (35 isolates) isolated from the Gulf of Mexico. A group of Synechococcus isolates (24 strains) were isolated concurrently to investigate the virulence and cross-infectivity of the lytic cyanophages and to determine the frequency of lysogeny by detection of inducible prophage. The host range of the cyanophages toward these Synechococcus strains ranged from 1 of 25 (host of isolation only) to 17 of 25 (68%). Of the 35 cyanophage isolates the large majority were myoviruses (94%) and only two (6%) were of the podovirus type. The expected polymerase chain reaction product for g20 was detected in 20 of the phages (63%). The presence of a detectable g20 was associated with low-infectivity cyanophages at the 90% confidence interval. The Synechococcus strains varied in their resistance to lytic infection from 11% to resistance to all of the phage isolates utilized in testing. The prevalence of inducible prophage-like particles was determined in the Synechococcus strains using mitomycin C and enumerating viruses by epifluorescence microscopy. A statistically significant increase in viruses was detected in 11 of the strains (46%) in response to mitomycin C. There was no observed relationship between the occurrence of prophage induction in the Synechococcus isolates and their resistance to lytic infection. One putative lysogen was induced by continuous high light and contained a prophage-like particle with a single-stranded DNA (ssDNA) genome. Such a prophage-like particle is unlike any prophage described to date, implying that the process of lysogeny is unique in certain marine Synechococcus strains.

Regulating the Metazoan Cell Cycle

Although studies on unicellular yeast species have yielded considerable information on regulation of the cell cycle, multicellular organisms depend on additional regulatory mechanisms to determine the size of individual organs and the overall size of the organism. Björklund et al. combined RNA interference with flow cytometry to identify genes involved in regulating cell size, cytokinesis, cell death (or apoptosis or both), and progression through the cell cycle in Drosophila S2 cells. In addition to genes classically associated with cell cycle progression, unsupervised hierarchical clustering of genes associated with particular phenotypes implicated various other genes in regulation of the cell cycle, including genes associated with COP9 signalosome activity and with the Wnt, p38βMAPK, FRAP/TOR, and JAK/STAT signaling pathways. The authors conclude that their data will enable a systems biology analysis of cell cycle regulation in metazoans. M. Björklund, M. Taipale, M. Varjosalo, J. Saharinen, J. Lahdenperä, J. Taipale, Identification of pathways regulating cell size and cell-cycle progression by RNAi. Nature 439 , 1009-1013 (2006). [PubMed]

Identification of pathways regulating cell size and cell-cycle progression by RNAi

Many high-throughput loss-of-function analyses of the eukaryotic cell cycle have relied on the unicellular yeast species Saccharomyces cerevisiae and Schizosaccharomyces pombe. In multicellular organisms, however, additional control mechanisms regulate the cell cycle to specify the size of the organism and its constituent organs. To identify such genes, here we analysed the effect of the loss of function of 70% of Drosophila genes (including 90% of genes conserved in human) on cell-cycle progression of S2 cells using flow cytometry. To address redundancy, we also targeted genes involved in protein phosphorylation simultaneously with their homologues. We identify genes that control cell size, cytokinesis, cell death and/or apoptosis, and the G1 and G2/M phases of the cell cycle. Classification of the genes into pathways by unsupervised hierarchical clustering on the basis of these phenotypes shows that, in addition to classical regulatory mechanisms such as Myc/Max, Cyclin/Cdk and E2F, cell-cycle progression in S2 cells is controlled by vesicular and nuclear transport proteins, COP9 signalosome activity and four extracellular-signal-regulated pathways (Wnt, p38betaMAPK, FRAP/TOR and JAK/STAT). In addition, by simultaneously analysing several phenotypes, we identify a translational regulator, eIF-3p66, that specifically affects the Cyclin/Cdk pathway activity.

Species richness destabilizes ecosystem functioning in experimental aquatic microcosms

The relationship between species diversity and ecosystem stability has long interested ecologists, yet no consensus has been reached and the underlying mechanisms remain unclear. We used five unicellular algal species, cultured in all possible combinations, to assemble microcosms containing 1 to 5 algal species, on which a cold perturbation was imposed. Our aim was to find whether and how species richness begets ecosystem resistance and resilience. In the experiment, the species‐rich communities produced more biomass than the species‐poor ones, either in pre‐, under‐ or post‐perturbation conditions. The positive diversity–biomass relationship was weakened by the perturbation, and fully restored one week after the perturbation. The diverse communities showed greater absolute biomass reduction during the perturbation than did species‐poor systems. Resistance of community, measured by the relative change in biomass from pre‐ to under‐perturbation, decreased with species richness. All the species showed significant reduction in biomass when stressed, without any density compensation among species in diverse communities; and the ratio of biomass change in each species was independent of diversity. The overyielding effect, measured as relative yield total, remained constant from pre‐ to under‐perturbation; and the selection and complementarity effects played equal roles for the biodiversity effect on biomass production, and their relative importance was not altered by the perturbation. These results suggest that similar responses of different species to environmental perturbations may limit the insurance effect of biodiversity, and lead to an inverse diversity–resistance relationship.

Genome-wide analysis of restriction-modification system in unicellular and filamentous cyanobacteria

Cyanobacteria are an ancient group of gram-negative bacteria with strong genome size variation ranging from 1.6 to 9.1 Mb. Here, we first retrieved all the putative restriction-modification (RM) genes in the draft genome of Spirulina and then performed a range of comparative and bioinformatic analyses on RM genes from unicellular and filamentous cyanobacterial genomes. We have identified 6 gene clusters containing putative Type I RMs and 11 putative Type II RMs or the solitary methyltransferases (MTases). RT-PCR analysis reveals that 6 of 18 MTases are not expressed in Spirulina, whereas one hsdM gene, with a mutated cognate hsdS, was detected to be expressed. Our results indicate that the number of RM genes in filamentous cyanobacteria is significantly higher than in unicellular species, and this expansion of RM systems in filamentous cyanobacteria may be related to their wide range of ecological tolerance. Furthermore, a coevolutionary pattern is found between hsdM and hsdR, with a large number of site pairs positively or negatively correlated, indicating the functional importance of these pairing interactions between their tertiary structures. No evidence for positive selection is found for the majority of RMs, e.g., hsdM, hsdS, hsdR, and Type II restriction endonuclease gene families, while a group of MTases exhibit a remarkable signature of adaptive evolution. Sites and genes identified here to have been under positive selection would provide targets for further research on their structural and functional evaluations.

Characterization of different viruses infecting the marine harmful algal bloom species Phaeocystis globosa

Twelve lytic viruses (PgV) infecting the marine unicellular eukaryotic harmful algal bloom species Phaeocystis globosa were isolated from the southern North Sea in 2000–2001 and partially characterized. All PgV isolates shared common phenotypic features with other algal viruses belonging to the family Phycodnaviridae and could be categorized in four different groups. Two main groups (PgV Group I and II) were discriminated based on particle size (150 and 100 nm respectively), genome size (466 and 177 kb) and structural protein composition. The lytic cycle showed a latent period of 10 h for PgV Group I and latent periods of 12 h and 16 h for PgV Group IIA and IIB. Host specificity and temperature sensitivity finally defined a fourth group (PgV Group IIC). Our results imply that viral infection plays an important role not only in P. globosa dynamics but also in the diversity of both host and virus community.

The selection of a model microalgal species as biomaterial for a novel aquatic phytotoxicity assay

A phytotoxicity assay based on the ToxY-PAM dual-channel yield analyser has been developed and successfully incorporated into field assessments for the detection of phytotoxicants in water. As a means of further exploring the scope of the assay application and of selecting a model biomaterial to complement the instrument design, nine algal species were exposed to four chemical substances deemed of priority for water quality monitoring purposes (chlorpyrifos, copper, diuron and nonylphenol ethoxylate). Inter-species differences in sensitivity to the four toxicants varied by a factor of 1.9–100. Measurements of photosystem-II quantum yield using these nine single-celled microalgae as biomaterial corroborated previous studies which have shown that the ToxY-PAM dual-channel yield analyser is a highly sensitive method for the detection of PS-II impacting herbicides. Besides Phaeodactylum tricornutum, the previously applied biomaterial, three other species consistently performed well ( Nitzschia closterium, Chlorella vulgaris and Dunaliella tertiolecta) and will be used in further test optimisation experiments. In addition to sensitivity, response time was evaluated and revealed a high degree of variation between species and toxicants. While most species displayed relatively weak and slow responses to copper, C. vulgaris demonstrated an IC 10 of 51 μg L −1, with maximum response measured within 25 minutes and inhibition being accompanied by a large decrease in fluorescence yield. The potential for this C. vulgaris-based bioassay to be used for the detection of copper is discussed. There was no evidence that the standard ToxY-PAM protocol, using these unicellular algae species, could be used for the detection of chlorpyrifos or nonylphenol ethoxylate at environmentally relevant levels.

An Inexpensive Apparatus for Growing Photosynthetic Microorganisms in Exotic Atmospheres

Given the need for a light source, cyanobacteria and other photosynthetic microorganisms can be difficult and expensive to grow in large quantities. Lighted growth chambers and incubators typically cost 50-100% more than standard microbiological incubators. Self-shading of cells in liquid cultures prevents the growth of dense suspensions. Growing liquid cultures on a shaker table or lighted shaker incubator achieves greater cell densities, but adds considerably to the cost. For experiments in which gases other than air are required, the cost for conventional incubators increases even more. We describe an apparatus for growing photosynthetic organisms in exotic atmospheres that can be built relatively inexpensively (approximately 100 dollars U.S.) using parts available from typical hardware or department stores (e.g., Wal-mart or K-mart). The apparatus uses microfiltered air (or other gases) to aerate, agitate, and mix liquid cultures, thus achieving very high cell densities (A750 > 3). Because gases are delivered to individual culture tubes, a variety of gas mixes can be used without the need for enclosed chambers. The apparatus works with liquid cultures of unicellular and filamentous species, and also works with agar slants.

Genomic Choice of Codons in 16 Microbial Species

We study the codon usage over whole set of ORFs of 16 unicellular microbial species: eight archaebacteria, seven eubacteria, and one eukarya. We first try to define, for each species, the neutral expected codon usage to better approach subsequently the influence of selection. Overlapping triplets counted from the complete DNA genomic sequence and mean amino acid composition of ORFs allow us to build satisfying expected codon usage for each species. Within species deviation from this neutral model is then studied through Correspondence Analysis and characterization with bias index, NC′ (effective number of codons reported to neutral model). Our results are compared to previously published ones for three species and let appear good agreement in spite of very different methods. We thus propose set of codons probably preferred by selection for nine other species. In the four last species, no clear preference can be evidenced. Finally, we characterize variation of codon usage over functional categories. We propose that the high degree of bias of proteins involved in translation, ribosomal structure and biogenesis has a positive influence on overexpression of the corresponding genes under optimum growth conditions and is a negative regulator of the same genes when amino acids become limited resources.

The Iron Superoxide Dismutase from the Filamentous Cyanobacterium Nostoc PCC 7120: LOCALIZATION, OVEREXPRESSION, AND BIOCHEMICAL CHARACTERIZATION

The nitrogen-fixing filamentous cyanobacterium Nostoc PCC 7120 (formerly named Anabaena PCC 7120) possesses two genes for superoxide dismutase, a unique membrane-associated manganese superoxide dismutase (MnSOD) and a soluble iron superoxide dismutase (FeSOD). A phylogenetic analysis of FeSODs shows that cyanobacterial enzymes form a well separated cluster with filamentous species found in one subcluster and unicellular species in the other. Activity staining, inhibition patterns, and immunogold labeling show that FeSOD is localized in the cytosol of vegetative cells and heterocysts (nitrogenase containing specialized cells formed during nitrogen-limiting conditions). The recombinant Nostoc FeSOD is a homodimeric, acidic enzyme exhibiting the characteristic iron peak at 350 nm in its ferric state, an almost 100% occupancy of iron per subunit, a specific activity using the ferricytochrome assay of (2040 +/- 90) units mg(-1) at pH 7.8, and a dissociation constant Kd of the azide-FeSOD complex of 2.1 mM. Using stopped flow spectroscopy it was shown that the decay of superoxide in the presence of various FeSOD concentrations is first-order in enzyme concentration allowing the calculation of the catalytic rate constants, which increase with decreasing pH: 5.3 x 10(9) M(-1) s(-1) (pH 7) to 4.8 x 10(6) M(-1) s(-1) (pH 10). FeSOD and MnSOD complement each other to keep the superoxide level low in Nostoc PCC 7120, which is discussed with respect to the fact that Nostoc PCC 7120 exhibits oxygenic photosynthesis and oxygen-dependent respiration within a single prokaryotic cell and also has the ability to form differentiated cells under nitrogen-limiting conditions.

Inheritance of organelles in artificial hybrids of the isogamous multicellular chromist alga Ectocarpus siliculosus (Phaeophyceae)

Our knowledge about the sexual transmission of mitochondria and plastids (hereafter organelles) in isogamous eukaryotes comes mostly from studies of the yeast Saccharomyces cerevisiae and the green alga Chlamydomonas reinhardtii which are both unicellular species. To investigate organelle inheritance in a multicellular organism with morphological isogamy, we studied the filamentous brown alga Ectocarpus siliculosus, in which each gamete contributes one plastid and at least one mitochondrion to the zygote. We crossed strains whose organelle genotypes were distinguishable by PCR. Hybrids contained only maternal mitochondrial genotypes, indicating uniparental inheritance of this organelle. In contrast, hybrids were chimerical for the plastid genome. In 64 to 75% of cases examined, the two zygotic plastids were partitioned into the two halves of the sporophyte, which developed from the zygote. A smaller number of hybrids deviated from this pattern, suggesting more complex mechanisms such as irregular division, segregation, or exchange of genetic material between plastids. E. siliculosus is the first isogamous heterokont eukaryote in which inheritance of organelles has been determined with molecular markers. The deterministic pseudo-Mendelian mechanism of plastid inheritance in E. siliculosus is unusual among eukaryotes.

Phenotypic yeast growth analysis for chronic toxicity testing

In Saccharomyces cerevisiae the pH-dependent growth inhibition of the heavy metals Cu 2+, Cr6+, Zn2+, Co2+, and Cd2+ was examined in comparison to that of organic solvents and pure compounds DMSO, MNNG, 4-NQO, MTBE, ethanol, and 2-AA. The assay was based on both S. cerevisiae wild-type and genetically modified cells deleted in the transporters Pdr5, Snq2, and Yor1 that facilitate pleiotropic drug resistance to explore the potential for short-term chronic aquatic toxicity tests. The strain deleted in the proteins that mediate the efflux of structurally diverse hydrophobic compounds exhibited high sensitive growth inhibition at low (0.04mg/L 4-NQO) to moderate (5.5mg/L DMSO) organic compound exposure. At pH 6.4 the EC50's, for all tested heavy metals were significantly low, in contrast to acidic pH conditions, in which both strains were able to grow in the presence of high concentrations of the transition metals Cu2+, Zn2+, and Co2+, with the pdr5 yor1 snq2 mutant being more tolerant. Cd2+ exerted the highest toxicity, with an EC50 of 0.49mg/L. Obtained results were compared with data determined from growth-inhibition tests involving other unicellular species. The comparison provided evidence that yeast is a sensitive and practical model system for toxicological risk assessment.

Bioavailability, Bioaccumulation and Tolerance of Chromium: Consequences in the Food Chain of Freshwater Ecosystems

Many abiotic and biotic factors can influence the partitioning equilibrium of heavy metals, thus influencing metal impact on aquatic environments. Unicellular algal species release soluble organic substances able to complex metals. In our laboratory a Cr-tolerant strain was selected and isolated from a wild type strain of Scenedesmus acutus. The exudates released by the two strains counteracted the growth inhibition caused by Cr(VI) and the exudates of the Cr-tolerant strain were more effective. On the contrary, the exudates did not reduce chromium toxicity to the cladoceran Daphnia magna. The reduction of chromium effect on algae seems the consequence of an algae-specific interaction among Cr(VI), exudates and algal cells. Chromium uptake resulted to be energy-dependent since bioaccumulation rate in subdued light condition was lower than at high light intensity. The effect of Cr(VI) on algae changed depending on metabolism of the cells and in particular it seemed to be related to the bioaccumulation rate. Tolerance in the selected strain could not be ascribed to a lower uptake of chromium. The difference in sensitivity to chromium between the two strains was exploited to evaluate if tolerance acquired by algae could have consequences for Daphnia. After treatment with Cr(VI), the two strains of S. acutus were used as food source for D. magna. The results indicate that chromium is accumulated by algae in a form not available for daphnids and that Cr tolerance acquired by the algae can be of some advantage to the consumer organism.

Algae affecting lettuce growth in hydroponic systems

SummaryHydroponic systems, especially those with recirculating nutrient solution, are an ideal environment for algal growth. It is not clear if this contaminant has an effect on the crop. Therefore, a greenhouse and a climate chamber experiment were carried out to monitor algal development and composition as well as their effect on crop performance. In addition, antifungal activity of algae and their aqueous extracts against root pathogens was tested. In open troughs planted with lettuce, algal density was 1.6 104 to 4 104 cells ml–1 in the greenhouse and 1.3 106 to 3.2 106 cells ml–1 in containers used in the climate chamber. Algal density and composition was influenced by initial density in the rainwater, temperature, and nutrient solution concentration during the experiments. The diversity of the algal community was poor. Exclusively unicellular species were observed with Chlamydomonas spp. and Scenedesmus spp. as main representatives. Lettuce fresh weight, shoot/root ratio, water and nitrogen uptake were reduced significantly in the presence of algae compared with treatments which excluded algae. Aqueous extracts prepared from algae collected from the greenhouse significantly inhibited growth of Fusarium oxysporum on slow nutrient agar but not growth of Pythium aphanidermatum. The presence of algae in containers with lettuce plants inoculated with P. aphanidermatum showed no effect on growth characteristics. It is assumed that the algae present do not release antifungal compounds. Overall, it appears that algae have more negative effects on crop yield in hydroponic systems. The adverse effect on the water supply system, which is usually cited as the main problem caused by algae, was not encountered in our experiments.

English

Cyanobacteria are one of the earth's most ancient life forms. Evidence of their existence on earth, derived from fossil records, encompasses a period of some 3.5 billion years in the late Precambrian era. Cyanobacteria are the dominant phytoplanton group in eutrophic freshwater bodies worldwide. They have caused animal poisoning in many parts of the world and may present risks to human health through drinking and recreational activity. Cyanobacteria produce two main groups of toxin namely neurotoxins and peptide hepatotoxins. They were first characterized from the unicellular species, Microcystis aeruginosa , which is the most common toxic cyanobacterium in eutrophic freshwater. The association of environmental parameters with cyanobacterial blooms and the toxicity of microcystin are discussed. Also, the synthesis of the microcystins, as well as the mode of action, control and analysis methods for quantitation of the toxin is reviewed. Key Words: Cyanobacteria, microcystins, mcyB gene, PCR-RFLP. African Journal of Biotechnology Vol.3(3) 2004: 159-168

Apocalypse soon?

Apocalypse soon?

Immunocytochemical evidence for acidic thylakoids in intact diatom cells

Using the weak base 3-(2,4-dinitroanilino)-3'-amino-N-methyl propylamine (DAMP) in in situ probing experiments, ultrastructural evidence for the acidic nature of the thylakoids in whole cells has been obtained in three unicellular pennate diatom species, with special emphasis on Navicula salinarum. Positive internal controls were provided by compartments known to be acidic (microbodies, lysosomal active vacuoles, and silica deposition vesicle). Negative internal controls were circumneutral compartments (nucleus and mitochondria). The predictable labelling of these compartments demonstrated the translocation of unprotonated DAMP over membranes and into acidic environments, where it became protonated. In cells that were treated with ionophores to disrupt pH gradients after incubation with DAMP, DAMP was no longer confined specifically to thylakoids and appeared to be more randomly distributed within the chloroplasts, indicating that the pH gradient of the thylakoids was affected. After densitometry, the transthylakoid ΔpH was estimated to vary between 1.0 and 2.0, coinciding with a moderate lumen acidity. The results obtained provide what we believe is the first clear evidence of the acidic nature of the thylakoid lumen in living cells at an ultrastructural level.

An assessment of long-term post-restoration water quality trends in a shallow, subtropical, urban hypereutrophic lake

City Park Lake is a shallow urban hypereutrophic lake located in Baton Rouge, Louisiana, with a surface area of 0.23 km 2 and a mean depth of 1.2 m. By the late 1970s, the lake had become highly eutrophic and suffered from frequent and severe algal blooms and fish kills. A major restoration effort was undertaken in 1983 that consisted of dredging and the repair of sewage infrastructure. Immediate improvements in water quality were observed following restoration; algal blooms and fish kills were virtually eliminated for nearly a decade. However, large floating mats of filamentous algae periodically occurred during the early 1990s. Results of a water quality sampling program conducted in 2000 and 2001 indicated that phosphorus has once again reached pre-restoration levels, and nitrogen levels have decreased well below those observed during pre-restoration years. Whereas phosphorus-limited conditions predominated in the years preceding the 1983 restoration, results of the 2000–2001 sampling program indicate that the lake has become nitrogen-limited with respect to photosynthetic activity. This trend in nutrient levels has likely influenced the recent predominance of filamentous over unicellular species of algae observed during the last decade. Nearly 4 years of drought-like conditions beginning in 1998 have resulted in an overall increase in the hydraulic retention time of the lake. This condition has resulted in organic staining of the lake waters, or the development of a tea-like color due to the decomposition of organic compounds. This phenomenon has played a major role in inhibiting the sunlight available for filamentous algal growth since 1998 and the absence of filamentous algae during the 2000–2001 sampling program.

Aging in fungi: role of mitochondria in Podospora anserina

In experimental gerontology, there is a long tradition in the use of both unicellular and filamentous species of fungi. In the last three decades, biochemical, genetic and molecular approaches have proved very fruitful in elucidating different aspects of ageing. It was shown that various genes and molecular pathways are involved in life span control. The oxygenic energy metabolism plays a central role. During mitochondrial energy transduction, reactive oxygen species (ROS) are generated as by-products. These molecules are able to damage all cellular compounds leading to cellular dysfunctions. Within certain limits, however, cells are able to cope with ROS-related problems. First, ROS scavengers can be induced which are effective in lowering the molecular burden of ROS on cellular functions. Second, if damage occurs, specific repair mechanisms and the general turnover of affected molecules can maintain cellular functions. Finally, if damage of essential components is too severe, cells may induce specific pathways to compensate for the corresponding impairments. A coordinated interaction between different cellular compartments is involved in these processes. In this review I shall concentrate on the ageing in the filamentous ascomycete Podospora anserina. It is clear that both environmental as well as genetic traits are involved in the control of life span and that mitochondrial-nuclear interactions play a paramount role.

Screening of cyanophytes from mosquito breeding places (mostly rice fields) with emphasis on unicellular species - potential recipients of Bti genes

35 genera with more than 60 species of cyanophytes were determined in samples collected from rice fields primarily in the malaria areas of Indonesia, China, French Guiana, Japan and the south-east Turkey and in various Anopheles breeding places in Cameroon, Uganda, and Syria. The most abundant unicellular genera observed were Synechococcus, Merismopedia, and Synechocystis which often occurred in high concentrations (approximately 102 cells per ml). Arthrospira platensis collected in Cameroon was the only species forming a water bloom. We successfully isolated 5 unicellular strains which occurred the most frequently (Synechococcus sp., Microcystis sp., Merismopedia tenuissima, Chroococcus minor, and Synechocystis sp.). Isolated strains grew well in culture and were kept and available in the Culture Collection of Autotrophic Organisms (CCALA) at Trebon. Two unicellular strains, i.e. Chroococcus minor strain CEPAK 1993/1 and Synechococcus sp. strain CEPAK 1996/1 were prospective for Bti gene introcuction due to their easy ingestibility and digestibility and rapid growth in culture.