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Changes in photosynthetic rate and pigment content of blue-green algae in Lake Mendota.

Blue-green algal blooms were present in Lake Mendota (Dane County, Wis.) from June to November 1976. Concentrations of total algal biomass and of particular algal species were monitored and compared with the pigment contents (chlorophyll a and phycocyanin) and photosynthetic rate of the algal populations. The specific photosynthetic rate (micrograms of C fixed per microgram of chlorophyll a per hour) was a good measure of the physiological state of the algae because this quantity increased just before each population increase and decreased before algal densities diminished. Since the quantity of light in the epilimnion which was available for photosynthesis by algal cells decreased in summer when the high algal densities attenuated incoming radiation, we investigated the possibility that the organisms would utilize lower light intensities more efficiently by increasing their pigment contents. Although some evidence of enhanced utilization of low light levels was found in the period from July to October, this result was not due to increasing chlorophyll and phycocyanin contents. There was a decrease in the phycocyanin content of the algae during this period, perhaps related to the availability of inorganic nitrogen.

Chlorophyll↗

Growth of Legionella pneumophila in association with blue-green algae (cyanobacteria).

Legionella pneumophila (Legionnaires disease bacterium) of serogroup 1 was isolated from an algal-bacterial mat community growing at 45 degrees C in a man-made thermal effluent. This isolate was grown in mineral salts medium at 45 degrees C in association with the blue-green alga (cyanobacterium) Fischerella sp. over a pH range of 6.9 to 7.6. L. pneumophila was apparently using algal extracellular products as its carbon and energy sources. These observations indicate that the temperature, pH, and nutritional requirements of L. pneumophila are not as stringent as those previously observed when cultured on complex media. This association between L. pneumophila and certain blue-green algae suggests an explanation for the apparent widespread distribution of the bacterium in nature.

Bacteria↗

Hemagglutination method for detection of freshwater cyanobacteria (blue-green algae) toxins.

Strains of the freshwater cyanobacteria (blue-green algae) Anabaena flosaquae and Microcystis aeruginosa produced toxins that caused intermittent but repeated cases of livestock, waterfowl, and other animal deaths. They also caused illness, especially gastrointestinal, in humans. The most common group of toxins produced by these two species were peptide toxins termed microcystin, M. Aeruginosa type c, and anatoxin-c. A method was found to detect the toxins which utilizes their ability to cause agglutination of isolated blood cells from mice, rats, and humans. The method could detect the toxin in samples from natural algal blooms, laboratory cultures, and toxin extracts. The method consists of: (i) washing lyophilized cyanobacteria cells with physiological saline (0.9% NaCl), (ii) centrifuging the suspension and then mixing portions of the cell-free supernatant with equal volumes of saline-washed erythrocytes in V-shaped microtiter plates, (iii) allowing the mixture to stand for 3 to 4 h, and (iv) scoring the presence of the toxin as indicated by blood cell agglutination. Nontoxic strains, as determined by intraperitoneal mouse bioassay of cyanobacteria or green algae, did not produce an agglutination response.

Bacterial Toxins↗

Diffusion through a Double-Sided Plate: Development of a Method to Study Alga-Bacterium Interactions.

Bacteria and algae isolated from a wastewater oxidation pond were inoculated onto opposing surfaces of double-layer agar plates (Lutri plates) to determine the usefulness of such plates for studying microbial interactions. The altered growth characteristics of various algae depending on the species of bacteria on the adjacent medium surface indicated that there was diffusion of extracellular products through the agar, suggesting that this simple assay can be used for screening potential interactions of actively growing organisms.

Journal Article↗

Molecular characterization of epiphytic bacterial communities on charophycean green algae

Epiphytic bacterial communities within the sheath material of three filamentous green algae, Desmidium grevillii, Hyalotheca dissiliens, and Spondylosium pulchrum (class Charophyceae, order Zygnematales), collected from a Sphagnum bog were characterized by PCR amplification, cloning, and sequencing of 16S ribosomal DNA. A total of 20 partial sequences and nine different sequence types were obtained, and one sequence type was recovered from the bacterial communities on all three algae. By phylogenetic analysis, the cloned sequences were placed into several major lineages of the Bacteria domain: the Flexibacter/Cytophaga/Bacteroides phylum and the alpha, beta, and gamma subdivisions of the phylum Proteobacteria. Analysis at the subphylum level revealed that the majority of our sequences were not closely affiliated with those of known, cultured taxa, although the estimated evolutionary distances between our sequences and their nearest neighbors were always less than 0.1 (i.e., greater than 90% similar). This result suggests that the majority of sequences obtained in this study represent as yet phenotypically undescribed bacterial species and that the range of bacterial-algal interactions that occur in nature has not yet been fully described.

Journal Article↗

Homogeneity of Danish environmental and clinical isolates of Shewanella algae.

Danish isolates of Shewanella algae constituted by whole-cell protein profiling a very homogeneous group, and no clear distinction was seen between strains from the marine environment and strains of clinical origin. Although variation between all strains was observed by ribotyping and random amplified polymorphic DNA analysis, no clonal relationship between infective strains was found. From several patients, clonally identical strains of S. algae were reisolated up to 8 months after the primary isolation, indicating that the same strain may be able to maintain the infection.

Bacterial Proteins↗

Development and application of a monoclonal-antibody technique for counting Aureococcus anophagefferens, an alga causing recurrent brown tides in the Mid-Atlantic United States.

A method was developed for the rapid detection and enumeration of Aureococcus anophagefferens, the cause of harmful algal blooms called "brown tides" in estuaries of the Mid-Atlantic United States. The method employs a monoclonal antibody (MAb) and a colorimetric, enzyme-linked immunosorbent assay format. The MAb obtained exhibits high reactivity with A. anophagefferens and very low cross-reactivities with a phylogenetically diverse array of other protists and bacteria. Standard curves are constructed for each 96-well microtiter plate by using known amounts of a preserved culture of A. anophagefferens. This approach allows estimation of the abundance of the alga in natural samples. The MAb method was compared to an existing method that employs polyclonal antibodies and epifluorescence microscopy and to direct microscopic counts of A. anophagefferens in samples with high abundances of the alga. The MAb method provided increased quantitative accuracy and greatly reduced sample processing time. A spatial survey of several Long Island estuaries in May 2000 using this new approach documented a range of abundances of A. anophagefferens in these bays spanning nearly 3 orders of magnitude.

Animals↗

Rapid quantification of the toxic alga Prymnesium parvum in natural samples by use of a specific monoclonal antibody and solid-phase cytometry.

The increasing incidence of harmful algal blooms around the world and their associated health and economic effects require the development of methods to rapidly and accurately detect and enumerate the target species. Here we describe use of a solid-phase cytometer to detect and enumerate the toxic alga Prymnesium parvum in natural samples, using a specific monoclonal antibody and indirect immunofluorescence. The immunoglobulin G antibody 16E4 exhibited narrow specificity in that it recognized several P. parvum strains and a Prymnesium nemamethecum strain but it did not cross-react with P. parvum strains from Scandinavia or any other algal strains, including species of the closely related genus Chrysochromulina. Prymnesium sp. cells labeled with 16E4 were readily detected by the solid-phase cytometer because of the large fluorescence signal and the signal/noise ratio. Immunofluorescence detection and enumeration of cultured P. parvum cells preserved with different fixatives showed that the highest cell counts were obtained when cells were fixed with either glutaraldehyde or formaldehyde plus the cell protectant Pluronic F-68, whereas the use of formaldehyde alone resulted in significantly lower counts. Immunofluorescence labeling and analysis with the solid-phase cytometer of fixed natural samples from a bloom of P. parvum occurring in Lake Colorado in Texas gave cell counts that were close to those obtained by the traditional method of counting using light microscopy. These results show that a solid-phase cytometer can be used to rapidly enumerate natural P. parvum cells and that it could be used to detect other toxic algae, with an appropriate antibody or DNA probe.

Antibodies, Monoclonal↗

Predicting production in light-limited continuous cultures of algae.

Equations relating productivity, growth rate, cell concentration, and light absorption lead to the prediction that, when incident light is below saturating intensity, maximal productivity will occur at half the maximal growth rate. The freshwater alga Chlorella pyrenoidosa TX71105 and the marine alga Dunaliella tertiolecta were grown in a small continuous culture apparatus with turbidostatic control. With both cultures, the cell concentration showed a linear decrease with dilution rate. Productivity was maximal at about one-half the maximal dilution rate. Average mass per cell increased near the maximal dilution rate, causing some asymmetry in the productivity versus dilution rate curve. The chlorophyll content per unit mass decreased in this region, but the chlorophyll content per cell remained constant. Best production rate in a light-limited algal culture was obtained when the growth rate at very low cell concentration was determined in the apparatus and the dilution rate was set at one-half that value.

Chemical Phenomena↗

Choline and inositol distribution in algae and fungi.

Inositol and choline were present in varying amounts among the species of Rhodophyta, Phaeophyta, Chlorophyta, and Euglenophyta examined. However, in the two members of the order Fucales (division Phaeophyta) examined, no detectable amounts of choline were found. In contrast, the species of Cyanophyta examined contained no detectable amounts of either choline or inositol. All species of the fungal classes Phycomyceteae, Ascomyceteae, and Basidiomyceteae collected contained both inositol and choline in varying amounts. The red, brown, and blue-green algae usually contained much less inositol and choline than do plant and animals sources, but the fungi and the algae Chlorella and Euglena contained amounts comparable to those present in plant sources.

Aspergillus↗

Effects of nitrogen limitation on the growth and composition of unicellular algae in continuous culture.

Since fat accumulation takes place in many algae as a response to exhaustion of the nitrogen supply, it has been suggested that this may provide a means of enhancing the potential food value of algae. To test this possibility, chemostatic continuous cultures of Chlorella sorokiniana and Oocystis polymorpha were subjected to successive reductions in influent nitrogen. As cellular nitrogen content decreased from about 10 to 4%, oxygen evolution, carbon dioxide uptake, chlorophyll content, and tissue production were drastically reduced, but total lipid content was essentially unchanged. Caloric values and C, H, and N analyses suggested a moderate increase in carbohydrate content, but gas chromatographic analyses revealed no significant qualitative or quantitative changes in the fatty acid fraction. In batch-cultured cells, nitrogen could be reduced to 3% of dry weight, causing a concomitant increase in total fatty acids and pronounced changes in the composition of the fatty acid fraction. These results suggest that cellular nitrogen must fall to approximately 3% of dry weight before appreciable increases in lipid synthesis can occur. Cell nitrogen is then apparently completely bound in essential cell constituents, and carbon subsequently fixed is converted into lipid products. The findings indicate that nitrogen limitation may be useful in increasing the food quality of batch-cultured cells, but the technique has little value for continuous culture systems per se.

Journal Article↗

Coevolution of a virus-alga system.

Plectonema boryanum, a filamentous blue-green alga, was cloned and then allowed to reach a steady state in a quasi-continuous culture in the presence of the algal virus, LPP-1. The culture was maintained for 3.5-month period during which time at least four distinct culture lysings were evident. After the fourth lysis the culture reached a steady-state level which was identical in its algal concentration to the preinfection level. Upon testing the characteristics of the evolved alga and virus variants, the following was determined: cell variants resistant to both the original virus and the derived virus had evolved, and there was no evidence of lysogeny present amony these cells. The evolved virus strains still grew on the parental algal strain, though with altered plaque morphology. Furthermore, they were antigenically similar to the parental virus, and showed no significant difference in adsorption rate or growth characteristics on parental cells. However, a low-grade chronic viral infection persisted in the culture. Rapid re-establishment of a dense, stable culture is apparantly the normal laboratory response of a procaryotic cell-virus system.

Adsorption↗

Enzymes of the heme biosynthetic pathway in the nonphotosynthetic alga Polytomella sp.

Heme biosynthesis involves a number of enzymatic steps which in eukaryotes take place in different cell compartments. Enzyme compartmentalization differs between photosynthetic and nonphotosynthetic eukaryotes. Here we investigated the structures and subcellular localizations of three enzymes involved in the heme pathway in Polytomella sp., a colorless alga evolutionarily related to the green alga Chlamydomonas reinhardtii. Functional complementation of Escherichia coli mutant strains was used to isolate cDNAs encoding three heme biosynthetic enzymes, glutamate-1-semialdehyde aminotransferase, protoporphyrinogen IX oxidase, and ferrochelatase. All three proteins show highest similarity to their counterparts in photosynthetic organisms, including C. reinhardtii. All three proteins have N-terminal extensions suggestive of intracellular targeting, and immunoblot studies indicate their enrichment in a dense cell fraction that is enriched in amyloplasts. These results suggest that even though the plastids of Polytomella sp. are not photosynthetically active, they are the major site of heme biosynthesis. The presence of a gene for glutamate-1-semialdehyde aminotransferase suggests that Polytomella sp. uses the five-carbon pathway for synthesis of the heme precursor 5-aminolevulinic acid.

Amino Acid Sequence↗

Multiple metabolic roles for the nonphotosynthetic plastid of the green alga Prototheca wickerhamii.

The presence of plastids in diverse eukaryotic lineages that have lost the capacity for photosynthesis is well documented. The metabolic functions of such organelles, however, are poorly understood except in the case of the apicoplast in the Apicomplexa, a group of intracellular parasites including Plasmodium falciparum, and the plastid of the green alga Helicosporidium sp., a parasite for which the only host-free stage identified in nature so far is represented by cysts. As a first step in the reconstruction of plastid functions in a nonphotosynthetic, predominantly free-living organism, we searched for expressed sequence tags (ESTs) that correspond to nucleus-encoded plastid-targeted polypeptides in the green alga Prototheca wickerhamii. From 3,856 ESTs, we found that 71 unique sequences (235 ESTs) correspond to different nucleus-encoded putatively plastid-targeted polypeptides. The identified proteins predict that carbohydrate, amino acid, lipid, tetrapyrrole, and isoprenoid metabolism as well as de novo purine biosynthesis and oxidoreductive processes take place in the plastid of P. wickerhamii. Mg-protoporphyrin accumulation and, therefore, plastid-to-nucleus signaling might also occur in this nonphotosynthetic organism, as we identified a transcript which encodes subunit I of Mg-chelatase, the enzyme which catalyzes the first committed step in chlorophyll synthesis. Our data indicate a far more complex metabolism in P. wickerhamii's plastid compared with the metabolic pathways predicted to be located in the apicoplast of P. falciparum and the plastid of Helicosporidium sp.

Algal Proteins↗

Characteristics of a stable, filamentous mutant of a coccoid blue-green alga.

Filamentous mutants were induced in a coccoid blue-green alga, Agmenellum quadruplicatum strain BG1, after treatment with N-methyl-N'-nitro-N-nitrosoguanidine (NTG). The mutants fall into two general classes: filaments with cross walls and filaments without cross walls. All mutants of these general types derived from BG1 are stable and have growth rates the same as or very similar to the wild type under a variety of conditions. Detailed examination of one mutant, 53SB2, revealed no difference in deoxyribonucleic acid content nor in base ratios. Mutant 53SB2 did not revert to the normal cell size and shape when grown under different physical conditions nor upon the addition of potential reversing agents to the basal medium. It is our general experience that filamentous mutants such as those described here in BG1 are commonly induced in other coccoid blue-green algae after NTG treatment.

Cell Nucleus↗

Endogenous dark respiration of the blue-green alga, Plectonema boryanum.

Endogenous dark respiration in the blue-green alga Plectonema boryanum is markedly affected by preincubation in the light: it can be increased from a basal rate of 5 nmoles of O(2) to 55 nmoles of O(2) per mg of cell protein per min after exposure of the cells to light for 8 to 10 hr. Under conditions of enhanced dark respiration, cyanophage multiplication in the dark increases drastically and approaches the cyanophage yields obtained in photosynthesizing Plectonema cells. This implies that the biosynthetic capabilities of the algal cells, at least with respect to viral synthesis, can be similar in the dark to those in the light. The enhanced endogenous respiration rate was found to be dependent on photoassimilation of CO(2) and on protein synthesis. The implications of these findings with respect to obligate photoautotrophic metabolism in blue-green algae are discussed.

Carbon Dioxide↗

Ribosomal ribonucleic acid synthesis and maturation in the blue-green alga Anacystis nidulans.

Methods are described for preparation of pulse-labeled ribonucleic acid (RNA) from the blue-green alga Anacystis nidulans. Synthesis of labeled RNA was found to be in part dependent on concurrent photosynthesis and was inhibited by the antibiotic streptolydigin. Mature 23S ribosomal RNA (rRNA) appeared before mature 16S rRNA. Formation of either molecule was inhibited by chloramphenicol, and RNA species of lesser mobility accumulated. These species may be precursors of the mature forms. Maturation of 16S rRNA was also inhibited by streptolydigin. (The effect of this antibiotic on 23S rRNA maturation was not examined). In many respects, ribosomal RNA synthesis and maturation in this blue-green alga appear to follow the pattern already established for bacteria.

Anti-Bacterial Agents↗

Photooxidative death in blue-green algae.

When incubated in the light under 100% oxygen, wild-type blue-green algae (Anacystis nidulans, Synechococcus cedrorum) die out rapidly at temperatures of 4 to 15 C, and at 35 C (or at 26 C in the case of S. cedrorum) in the absence of CO(2). Photosynthesis is impaired in these cells long before they die. Blocking of photosystem II at high temperatures in the presence of CO(2) sensitizes the algae to photooxidative death. Photooxidative death and bleaching of photosynthetic pigments are separable phenomena. Photooxidative conditions were demonstrated in Israeli fish ponds using A. nidulans as the test organism during dense summer blooms, when dissolved CO(2) is low, and in winter, when water temperatures generally drop below 15 C. This finding suggests that photooxidative death may be responsible for the sudden decomposition of blue-green blooms in summer, and may be a factor in the absence of blue-green blooms in winter.

Cyanobacteria↗