The impact of associated bacteria on morphology and physiology of the dinoflagellate Alexandrium tamarense

Despite their potential impact on phytoplankton dynamics and biogeochemical cycles, biological associations between algae and bacteria are still poorly understood. The aim of the present work was to characterize the influence of bacteria on the growth and function of the dinoflagellate Alexandrium tamarense. Axenic microalgal cultures were inoculated with a microbial community and the resulting cultures were monitored over a 15-month period, in order to allow for the establishment of specific algal–bacterial associations. Algal cells maintained in these new mixed cultures first experienced a period of growth inhibition. After several months, algal growth and cell volume increased, and indicators of photosynthetic function also improved. Our results suggest that community assembly processes facilitated the development of mutualistic relationships between A. tamarense cells and bacteria. These interactions had beneficial effects on the alga that may be only partly explained by mixotrophy of A. tamarense cells. The potential role of organic exudates in the establishment of these algal–bacterial associations is discussed. The present results do not support a role for algal–bacterial interactions in dinoflagellate toxin synthesis. However, variations observed in the toxin profile of A. tamarense cells during culture experiments give new clues for the understanding of biosynthetic pathways of saxitoxin, a potent phycotoxin.     

The Extent of Algal and Bacterial Endosymbioses in Protozoa1,2

Long neglected has been the extensive and more or less intimate association of protozoa with a wide variety of other cells, either prokaryotic or eukaryotic in nature. Yet study of such relationships can provide important information concerning certain basic aspects of cellular evolution in general. A survey is offered here of the whole range of such symbiotic associations (i.e. with species of protozoa serving as hosts) with the purposes of drawing attention to the exciting possibilities of such research and of reviewing significant findings made to date. Because of the vastness of the overall field, examples and discussion are primarily limited to consideration of the following major studies: methanogenic bacteria in certain ciliates, bacterial endosymbionts of the large freshwater amoeba Pelomyxa palustris (itself an amazing organism from an evolutionary/phylogenetic point of view), the rod-shaped bacteria found in Amoeba proteus, the “Greek-letter” prokaryotes of Paramecium species, the xenosomes (sensu stricto) of the marine scuticociliate Parauronema acutum, and the diverse algal endosymbionts of similarly diverse protozoan taxa–ciliates, flagellates, radiolarians, acantharians, and foraminifera.     

Aging of Utricularia traps and variability of microorganisms associated with that microhabitat

Various authors have described algae in aquatic Utricularia traps as commensals, as stress factors or as prey. This study examined the diversity and abundance of organisms (prey, algae, protozoa and bacteria) in the traps of aquatic Utricularia reflexa in relation to prey occurrence and trap age. The number of organisms increased with the trap age. In both young and old traps, phytoplankton dominated of all organisms found. In young traps, Scenedesmus spp. and Characiopsis sp. were the most abundant algae, while Scenedesmus spp. and the palmelloidal form of Euglena spp. dominated in the old traps. Most of the algal species found stayed alive in the trap environment. The number of living algae and ciliates inside the traps increased with the increasing trap age, too. As the number of Paramecium bursaria inside traps consistently increased with the trap age and number of bacteria, which serve as a food for them, ciliates can be regarded as commensals, but not as prey for the plant. The predominant organisms in the traps were those that can be considered either commensals or intruders, exceeding captured macroscopic prey.     

Symposium on “Symbiosis in Protozoa”: Introductory Remarks1,2

ABSTRACT. Attention, perhaps overdue, is drawn to the extent and significance of endosymbionts (xenosomes sensu lato) in the cytoplasm and nuclei of many protozoa from diverse taxonomic groups. Even more importantly, recent advances in the study of such intimate associations are reviewed and discussed and their impact on broader problems of cell biology and evolution are stressed. Workers inside and especially outside the fields of protozoology and parasitology have often neglected such data, failing to appreciate their relevance to significant problems in their own fields of investigation. The major topics covered by speakers in the Symposium (to which this paper serves only as an introduction) include the following, in order of their presentation: terminology for the symbiont-host relationship and a brief overview of the field; the evolutionary problem of the origin of contemporary associations, including cell organelles such as mitochondria and plastids; the adaptive value of endosymbionts to their protozoan hosts; mechanisms of establishment, maintenance, and integration of such foreign bodies/invaders in their unicellular eukaryotic host cells; and the extent of algal and bacterial endosymbioses in diverse protozoan groups. In all papers, the principal relatively well studied complexes used as examples are the following: various kinds of algae in the larger foraminifera and in ciliates, radiolarians, and acantharians; the several types of bacteria in the cytoplasm of Amoeba and of Pelomyxa; the endonuclear bacterial symbionts of Paramecium; the cytoplasmic prokaryotes in Paramecium and in Parauronema; and the methanogenic bacteria of certain ciliates.     

Diurnal changes in the xanthophyll cycle pigments of freshwater algae correlate with the environmental hydrogen peroxide concentration rather than non-photochemical quenching

Background and Aims In photosynthetic organisms exposure to high light induces the production of reactive oxygen species (ROS), such as hydrogen peroxide (H₂O₂), which in part is prevented by non-photochemical quenching (NPQ). As one of the most stable and longest-lived ROS, H₂O₂ is involved in key signalling pathways in development and stress responses, although in excess it can induce damage. A ubiquitous response to high light is the induction of the xanthophyll cycle, but its role in algae is unclear as it is not always associated with NPQ induction. The aim of this study was to reveal how diurnal changes in the level of H₂O₂ are regulated in a freshwater algal community.Methods A natural freshwater community of algae in a temporary rainwater pool was studied, comprising photosynthetic Euglena species, benthic Navicula diatoms, Chlamydomonas and Chlorella species. Diurnal measurements were made of photosynthetic performance, concentrations of photosynthetic pigments and H₂O₂. The frequently studied model organisms Chlamydomonas and Chlorella species were isolated to study photosynthesis-related H₂O₂ responses to high light.Key Results NPQ was shown to prevent H₂O₂ release in Chlamydomonas and Chlorella species under high light; in addition, dissolved organic carbon excited by UV-B radiation was probably responsible for a part of the H₂O₂ produced in the water column. Concentrations of H₂O₂ peaked at 2 µm at midday and algae rapidly scavenged H₂O₂ rather than releasing it. A vertical H₂O₂ gradient was observed that was lowest next to diatom-rich benthic algal mats. The diurnal changes in photosynthetic pigments included the violaxanthin and diadinoxanthin cycles; the former was induced prior to the latter, but neither was strictly correlated with NPQ.Conclusions The diurnal cycling of H₂O₂ was apparently modulated by the organisms in this freshwater algal community. Although the community showed flexibility in its levels of NPQ, the diurnal changes in xanthophylls correlated with H₂O₂ concentrations. Alternative NPQ mechanisms in algae involving proteins of the light-harvesting complex type and antioxidant protection of the thylakoid membrane by de-epoxidized carotenoids are discussed.     

Comparative and evolutionary aspects of the photosynthetic electron transfer system of purple bacteria

The cyclic electron transfer system in purple bacteria is composed of the photosynthetic reaction center, the cytochromebc ₁ complex, cytochromec ₂, and ubiquinone. These components share many characteristics with those of photosynthesis and respiration in other organisms. Studies of the cyclic electron transfer system have provided useful insights about the evolution and general mechanisms of membranous energy-conserving systems. The photosynthetic system in purple bacteria may represent a prototype of highly efficient, energy-conserving electron transfer systems in the organisms. Recipient of the Botanical Society Award of Young Scientists, 1992     

Ciliates as engineers of phototrophic biofilms

1. Phototrophic biofilms consist of a matrix of phototrophs, non‐photosynthetic bacteria and extracellular polymeric substances (EPS) which is spatially structured. Despite widespread exploitation of algae and bacteria within phototrophic biofilms, for example by protozoans, the ‘engineering’ effects of these ciliates on the spatial heterogeneity of phototrophic biofilms are poorly studied. 2. We studied the potential engineering effects of two ciliates, Urostyla sp. and Paramecium bursaria, on the spatial heterogeneity of synthetic multispecies biofilms. Biomass of phototrophic organisms, EPS and bacteria was analysed three dimensionally using confocal laser scanning microscopy. Spatial heterogeneity and cover of the phototrophs, bacteria and EPS were determined at several depths within the biofilm. 3. Ciliate species did not interfere with the overall development of phototrophic microorganisms, because the thickness of the biofilm was equal whether the ciliates were present or not, even though their abundance did affect spatial heterogeneity of biofilm components. When Urostyla was present, it reduced aggregation in EPS and bacteria and increased EPS biovolume. This implies a local facilitating effect of ciliates on photosynthetic activity. Biofilms to which Paramecium was added did not differ from controls in terms of phototrophs, EPS cover and biovolume. Nevertheless, ciliates affected the spatial heterogeneity of these components as phototrophs and EPS became more evenly distributed. 4. This study shows that ecosystem engineering by organisms does not only occur at large spatial scales, as in grasslands and estuaries, but also plays a role at the microscopic scale of biofilms. This effect on spatial heterogeneity was not driven by substantial exploitation of biofilm components, but via the subtle engineering effects of ciliates.     

Intraspecific trait variation alters the outcome of competition in freshwater ciliates

Trait variation among heterospecific and conspecific organisms may substantially affect community and food web dynamics. While the relevance of competition and feeding traits have been widely studied for different consumer species, studies on intraspecific differences are more scarce, partly owing to difficulties in distinguishing different clones of the same species. Here, we investigate how intraspecific trait variation affects the competition between the freshwater ciliates Euplotes octocarinatus and Coleps hirtus in a nitrogen‐limited chemostat system. The ciliates competed for the microalgae Cryptomonas sp. (Cry) and Navicula pelliculosa (Nav), and the bacteria present in the cultures over a period of 33 days. We used monoclonal Euplotes and three different Coleps clones (Col 1, Col 2, and Col 3) in the experiment that could be distinguished by a newly developed rDNA‐based molecular assay based on the internal transcribed spacer (ITS) regions. While Euplotes feeds on Cry and on bacteria, the Coleps clones cannot survive on bacteria alone but feed on both Cry and Nav with clone‐specific rates. Experimental treatments comprised two‐species mixtures of Euplotes and one or all of the three different Coleps clones, respectively. We found intraspecific variation in the traits “selectivity” and “maximum ingestion rate” for the different algae to significantly affect the competitive outcome between the two ciliate species. As Nav quickly escaped top‐down control and likely reached a state of low food quality, ciliate competition was strongly determined by the preference of different Coleps clones for Cry as opposed to feeding on Nav. In addition, the ability of Euplotes to use bacteria as an alternative food source strengthened its persistence once Cry was depleted. Hence, trait variation at both trophic levels codetermined the population dynamics and the outcome of species competition.     

Sizing up the genomic footprint of endosymbiosis

A flurry of recent publications have challenged consensus views on the tempo and mode of plastid (chloroplast) evolution in eukaryotes and, more generally, the impact of endosymbiosis in the evolution of the nuclear genome. Endosymbiont‐to‐nucleus gene transfer is an essential component of the transition from endosymbiont to organelle, but the sheer diversity of algal‐derived genes in photosynthetic organisms such as diatoms, as well as the existence of genes of putative plastid ancestry in the nuclear genomes of plastid‐lacking eukaryotes such as ciliates and choanoflagellates, defy simple explanation. Collectively, these papers underscore the power of comparative genomics and, at the same time, reveal how little we know with certainty about the earliest stages of the evolution of photosynthetic eukaryotes. Editor's suggested further reading in BioEssays Early steps in plastid evolution: current ideas and controversies Abstract Dinoflagellate mitochondrial genomes: stretching the rules of molecular biology Abstract     

Lytic Organisms and Photooxidative Effects: Influence on Blue-Green Algae (Cyanobacteria) in Lake Mendota, Wisconsin

The effects of exposure to high light intensities on blue-green algal (cyanobacterial) populations were examined in Lake Mendota, Wis. The algal populations were shown to be susceptible to inhibition of photosynthetic activity and pigment bleaching as a result of exposure. These effects generally influence only a small percentage of the lake population and thus are probably not important in causing major declines in chlorophyll a. Lytic organisms were shown to increase in numbers in the lake in response to the seasonal development of blue-green algae, reaching values of greater than 1,000 plaque-forming units per ml in midsummer. Both bacteria and protozoa were observed in plaque zones, but it could not be determined whether these lytic organisms had a major role in algal biomass declines.     

Multiple genes of apparent algal origin suggest ciliates may once have been photosynthetic

Plantae (as defined by Cavalier-Smith, 1981) plastids evolved via primary endosymbiosis whereby a heterotrophic protist enslaved a photosynthetic cyanobacterium. This "primary" plastid spread into other eukaryotes via secondary endosymbiosis. An important but contentious theory in algal evolution is the chromalveolate hypothesis that posits chromists (cryptophytes, haptophytes, and stramenopiles) and alveolates (ciliates, apicomplexans, and dinoflagellates) share a common ancestor that contained a red-algal-derived "secondary" plastid. Under this view, the existence of several later-diverging plastid-lacking chromalveolates such as ciliates and oomycetes would be explained by plastid loss in these lineages. To test the idea of a photosynthetic ancestry for ciliates, we used the 27,446 predicted proteins from the macronuclear genome of Tetrahymena thermophila to query prokaryotic and eukaryotic genomes. We identified 16 proteins of possible algal origin in the ciliates Tetrahymena and Paramecium tetraurelia. Fourteen of these are present in other chromalveolates. Here we compare and contrast the likely scenarios for algal-gene origin in ciliates either via multiple rounds of horizontal gene transfer (HGT) from algal prey or symbionts, or through endosymbiotic gene transfer (EGT) during a putative photosynthetic phase in their evolution.     

SIGNIFICANCE OF BACTERIAL-ANABAENA (CYANOPHYCEAE) ASSOCIATIONS WITH RESPECT TO N2 FIXATION IN FRESHWATER1, 2

The functional aspects of specific associations between bluegreen algae and bacteria were investigated using both naturally occurring and cultured species of Anabaena. In take waters where bacteria were associated with Anabaena heterocysls, the bacteria exhibited a chemotactic response to a variety of amino acids and glucose. Earlier autoradiographic evidence that bacteria associated with heterocysts incorporate identical substrates indicates that associated bacteria probably benefit by utilizing algal excretion products. In return, the bacteria stimulate algal N₂fixation. The most likely mechanism explaining such stimulation appeared to be bacterial oxygen removal in microzones (< 3 μm diam) bordering heterocysts during periods of high ambient oxygen concentrations. In the presence of bacteria, Anabaena rapidly overcame nitrogenase- inhibiting concentrations of oxygen. Axenic cullures had more extensive nitrogenase inhibition, and took longer to recover in response to oxygenation. Algal-bacterial mutualism aids Anabaena in maintaining concurrent optimal N2 fixation and high photosynthetic rates in highly oxygenated surface waters.     

Lysine biosynthesis and the evolution-of pennate marine diatoms

The classification of lysine biosynthetic pathways in various organisms have been used to investigate their descent in evolution. We have attempted these determinations in the diatoms Amphora coffeaeformis var:perpusilla (Grunow Cleve.) and Phaeodactylum tricornutum (Bohlin). Additionally, we have verified earlier results of Vogel in a green alga, Chlorella pyrenoidosa strain Tx 71105 (Texas Culture Collection). Our research indicates that the diaminopimelic acid route is involved in all three organisms. While these studies do not exclude the possible co-existence of the α-aminoadipic acid route, the results imply a closer evolutionary relationship of pennate diatoms to bacteria and “classical” photosynthetic plants rather than to heterotrophic or mixotrophic fungi and atypical algal strains such as the Euglenophyta.     

Endosymbiotic purple non-sulphur bacteria in an anaerobic ciliated protozoon

Abstract The marine ciliate Strombidium purpureum Kahl harbours endosymbiotic purple non-sulphur bacteria. The bacteria contain bacteriochlorophyll a and the carotenoid spirilloxanthin, and they have photosynthetic membranes and cell walls. The ciliates require light for survival and growth under anaerobic conditions; in the dark the cells prefer microaerobic conditions. The ciliates show a photosensory behaviour, and they accumulate in light at wave lenghts corresponding to the absorption spectrum of the symbionts. The findings are discussed in terms of theories on the endosymbiotic origin of mitochondria.     

The independence of photosynthesis and aerobiosis from sterol biosynthesis in bacteria

Sterols were present in neither of two representative species of photosynthetic bacteria, Rhodopseudomonas spheroides and Chromatium vinosum. These organisms were grown under conditions commonly viewed as anaerobic. However, such conditions did not prevent Saccharomyces cerevisiae from biosynthesizing sterols, although they did induce accumulation of both 4,4-dimethyl and 4-desmethyl intermediates. Since the photosynthetic organisms did not biosynthesize sterols, bacterial photosynthesis must not be mated genetically or functionally to sterol biosynthesis. In contrast to what the literature records, Escherichia coli, grown under fully aerobic conditions, also failed to contain sterols which indicates that bacterial aerobiosis does not necessarily imply either the presence of sterol biosynthesis or a requirement for an exogenous source of sterols. Among the lipids of E. coli was a substance with the formula C₁₆H₃₂O₂ which moved in silica gel TLC at a rate similar to that of sterols and may have been a keto-alcohol of the same formula already isolated from coliforms. In the photosynthetic bacteria the major neutral lipid after saponification was phytol, in agreement with expectation based on the presence of bacteriochlorophyll-a.     

Use of deuterium labelling from deuterium oxide to demonstrate carotenoid transformations in photosynthetic bacteria

Carotenoid biosynthesis in many purple photosynthetic bacteria of the Rhodospirillaceae is inhibited by nicotine, and biosynthetic intermediates accumulate. If the inhibitor is removed and the bacteria are then incubated in buffered 99.6% deuterium oxide, deuterium is incorporated specifically into the C-2 position in both cyclic and acyclic carotenoids that are then formed from the previously accumulated hydrocarbon precursors. The deuterated molecular species can be detected and assayed by mass spectrometry. By use of this procedure, direct proof has been obtained for the conversion of lycopene into β-carotene and rhodopin in Rhodomicrobium vannielii, of neurosporene into spheroidene in Rhodopseudomonas sphaeroides and of spheroidene into hydroxyspheroidene in Rps. gelatinosa. The results confirm the operation of the biosynthetic pathways postulated for these organisms, and prove that formation of the acyclic 1-hydroxy-1,2-dihydro end-group characteristic of the carotenoids of photosynthetic bacteria occurs by addition of water to the C-1,2 double band.     

Effects of acidic conditions on the physiology of a green alga (Micractinium sp.) before and after a 5-year interaction with Tetrahymena thermophila in an experimental microcosm

The mechanisms through which algae evolve physiological characteristics related to endosymbiotic associations with heterotrophic organisms remain unclear. We previously showed that a green alga (Micractinium sp.) was able to evolve a host (ciliate)-benefiting phenotype that prolonged the longevity of Tetrahymena thermophila in the absence of bacteria during a 5-year culture period in an experimental microcosm. Comparative experiments between the ancestral alga (i.e. original) and evolved algal clones of the same lineage can be performed to analyse the mechanisms that underlie algal evolution during interactions with ciliates. Here, we investigated the effects of acidic conditions on algal physiology because the acidic conditions within the food vacuoles of Tetrahymena could potentially affect algal evolution during long-term interactions. It might be expected that algal clones isolated from T. thermophila hosts would have developed the ability to resist acidic conditions in order to establish within the host, when compared with the ancestral strain. Unexpectedly, comparative analyses demonstrated that acidic conditions with pH values ranging from 3.9 to 4.3 limited the growth of the isolated algal clones SC9-1 and SC10-2, whereas the ancestral strain could grow under these conditions. Acidic conditions were responsible for significantly lower chlorophyll a/chlorophyll b ratios in the isolated algal clones, and the isolated clone SC9-1 exhibited a high cellular content of chlorophyllide a during a short exposure to acidic conditions, suggesting that degradation of chlorophyll a occurred. Furthermore, acidic conditions increased the release of extracellular glycerol and sucrose in the SC9-1 clone. These results indicated that the ancestral strain showed phenotypic changes related to acidic conditions, which may reflect the ongoing development of an algal phenotype suited to symbiosis. Hypotheses are proposed to explain the limited growth of the isolated clones, and implications for the Micractinium–Tetrahymena association are discussed.     

Macroalgal decomposition: Laboratory studies with particular regard to microorganisms and meiofauna

The microbial degradation of North Sea macroalgae was studied in laboratory microcosms, containing autoclaved seawater and a mixture of equal parts of air-driedDelesseria sanguinea, Ulva lactuca, andLaminaria saccharina (red, green and brown algae, respectively). To determine the influence of different organisms on the decomposition rate (expressed in terms of algal dry weight loss relative to the material present at time zero) and their development during decomposition processes, yeast, flagellates, ciliates, nematodes and a harpacticoid copepod species were introduced to the microcosms. Results show that microbial degradation compared to the controls was enhanced in the presence of non-axenic nematodes (Monhystera sp.) and protozoans, including bacterivorous ciliates (Euplotes sp. and aUronema-like sp.) and flagellates. No enhancement occurred with yeast (Debaryomyces hansenii) or with the harpacticoid copepodTisbe holothuriae. The most rapid algal dry weight loss (78.7% after 14 d at 18°C) occurred with the addition of raw seawater sampled near benthic algal vegetation and containing only the natural microorganisms present. These consisted mainly of bacteria with different morphological properties, whereby their numbers alone (viable counts) could not be correlated with algal dry weight loss. Although no single dominant species could be determined, lemon yellow pigmented colonies were frequently found. During decomposition in all microcosms the formation of algal particles 40–400 μm was observed, which were rapidly colonized by the other organisms present.     

Algal photosynthetic aeration increases the capacity of bacteria to degrade organics in wastewater

Wastewater treatment is an energy-intensive process and a net emitter of greenhouse gas emissions. A large fraction of these emissions is due to intensive aeration of aerobic bacteria to facilitate break-down of organic compounds. Algae can generate dissolved oxygen at levels in excess of saturation, and therefore hold the potential to partially displace or complement mechanical aeration in wastewater treatment processes. The objective of this study was to develop an internally consistent experimental and modeling approach to test the hypothesis that algal photosynthetic aeration can speed the removal of organic constituents by bacteria. This framework was developed using a simplified wastewater treatment process consisting of a model bacteria (Escherichia coli), a model algae (Auxenochlorella protothecoides), and a single carbon source that was consumable by bacteria only. This system was then tested both with and without the presence of algae. A MATLAB model that considered mass transfer and biological kinetics was used to estimate the production and consumption of O₂ and CO₂ by algae and bacteria. The results indicated that the presence of algae led to 18–66% faster removal of COD by bacteria, and that roughly one-third of biochemical oxygen demand was offset by algal photosynthetic aeration.     

Ciliate grazing on <Emphasis Type="Italic">Nitrosomonas europaea</Emphasis> and <Emphasis Type="Italic">Nitrospira</Emphasis><Emphasis Type="Italic">moscoviensis</Emphasis>: Is selectivity a factor for the nitrogen cycle in natural aquatic systems?

Ciliated protists are important predators of bacteria in many aquatic habitats, including sediments. Since, many biochemical transformations within the nitrogen cycle are performed by bacteria, ciliates could have an indirect impact on the nitrogen cycle through selective grazing on nitrogen-transforming bacteria. As a case study, we examined ciliate grazing on nitrifying bacteria of the genera Nitrosomonas and Nitrospira. All experiments were designed as in vitro-experiments with cultures of different bacteria and ciliate species. The nitrifying bacteria used in our experiments were Nitrosomonas europaea [Winogradsky 1892] and Nitrospira moscoviensis [Ehrich 2001]. The ciliates comprised of four species that are known as efficient bacterivores and common members of the protist community in aquatic systems: Paramecium aurelia [Müller 1773], Euplotes octocarinatus [Carter 1972], Tetrahymena pyriformis [Ehrenberg 1830] and Cyclidium glaucoma [Müller 1786]. Our experimental approach, using a combination of DAPI and FISH staining, was successful in allowing the observation of ingestion of specific bacteria and their detection within ciliate food vacuoles. However, the ciliates in this study showed no significant selective grazing. No food preferences for a any bacterial taxon or any size class or morphotype were detected. Correlation with time between ciliate abundance and bacterial abundance or biovolume, using log transformed growth rates of ciliates and bacteria, showed no significant results. On the bacterial side, neither an active defence mechanism of the nitrifying bacteria against ciliate grazing, such as changes in morphology, nor competition for resources were observed. These results suggest that in our in vitro-experiments grazing by ciliates has no influence on abundance and growth of nitrifying bacteria and nitrification.