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The interactions between marine prokaryotic and eukaryotic microorganisms are crucial to many biological and biogeochemical processes in the oceans. Often the interactions are mutualistic, as in the symbiosis between phytoplankton, e.g., the dinoflagellate Pfiesteria piscicida and Silicibacter sp. TM1040, a member of the Roseobacter taxonomic lineage. It is hypothesized that an important component of this symbiosis is bacterial production of tropodithietic acid (TDA), a biologically active tropolone compound whose synthesis requires the expression of tdaABCDEF (tdaA-F), as well as six additional genes (cysI, malY, paaIJK, and tdaH). The factors controlling tda gene expression are not known, although growth in laboratory standing liquid cultures drastically increases TDA levels. In this report, we measured the transcription of tda genes to gain a greater understanding of the factors controlling their expression. While the expression of tdaAB was constitutive, tdaCDE and tdaF mRNA increased significantly (3.7- and 17.4-fold, respectively) when cells were grown in standing liquid broth compared to their levels with shaking liquid culturing. No transcription of tdaC was detected when a tdaCp::lacZ transcriptional fusion was placed in 11 of the 12 Tda(-) mutant backgrounds, with cysI being the sole exception. The expression of tdaC could be restored to 9 of the remaining 11 Tda(-) mutants-tdaA and tdaH failed to respond-by placing wild-type (Tda(+)) strains in close proximity or by supplying exogenous TDA to the mutant, suggesting that TDA induces tda gene expression. These results indicate that TDA acts as an autoinducer of its own synthesis and suggest that roseobacters may use TDA as a quorum signal.

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Pfiesteria spp. are mixotrophic armored dinoflagellates populating the Atlantic coastal waters of the United States. They have been a focus of intense research due to their reported association with several fish mortality events. We have now used a clonal culture of Pfiesteria piscicida and several new environmental isolates to describe growth characteristics, feeding, and factors contributing to the encystment and germination of the organism in both laboratory and environmental samples. We also discuss applied methods of detection of the different morphological forms of Pfiesteria in environmental samples. In summary, Pfiesteria, when grown with its algal prey, Rhodomonas sp., presents a typical growth curve with lag, exponential, and stationary phases, followed by encystment. The doubling time in exponential phase is about 12 h. The profiles of proliferation under a standard light cycle and in the dark were similar, although the peak cell densities were markedly lower when cells were grown in the dark. The addition of urea, chicken manure, and soil extracts did not enhance Pfiesteria proliferation, but crude unfiltered spent aquarium water did. Under conditions of food deprivation or cold (4 degrees C), Pfiesteria readily formed harvestable cysts that were further analyzed by PCR and scanning electron microscopy. The germination of Pfiesteria cysts in environmental sediment was enhanced by the presence of live fish: dinospores could be detected 13 to 15 days earlier and reached 5- to 10-times-higher peak cell densities with live fish than with artificial seawater or f/2 medium alone. The addition of ammonia, urea, nitrate, phosphate, or surprisingly, spent fish aquarium water had no effect.

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Silicibacter sp. TM1040, originally isolated from a culture of the dinoflagellate Pfiesteria piscicida, senses and responds to the dinoflagellate secondary metabolite dimethylsulfoniopropionate (DMSP) by flagella-mediated chemotaxis behaviour. In this report we show that swimming motility is important for initiating the interaction between the bacterium and dinoflagellate. Following transposon mutagenesis, three mutants defective in wild-type swimming motility (Mot-) were identified. The defects in motility were found to be in homologues of cckA and ctrA, encoding a two-component regulatory circuit, and in a novel gene, flaA, likely to function in flagellar export or biogenesis. Mutation of flaA or cckA results in the loss of flagella and non-motile cells (Fla-), while CtrA- cells possess flagella, but have reduced motility due to increased cell length. All three Mot- mutants were defective in attaching to the dinoflagellate, particularly to regions that colocalized with intracellular organelles. The growth rate of the dinoflagellates was reduced in the presence of the Fla- mutants compared with Fla+ cells. These results indicate that bacterial motility is important for the Silicibacter sp. TM1040-P. piscicida interaction.

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Proliferating cell nuclear antigen (PCNA), a co-factor of DNA polymerases delta and epsilon, is essential for DNA replication and repair. Understanding the structure and expression characteristics of this gene in dinoflagellates would enable us to gain insights into how the cell cycle in these enigmatic eukaryotes is regulated and whether this gene can be a growth marker of these ecologically important organisms. We analyzed pcna and its encoded protein from Pfiesteria piscicida (Ppi_PCNA). Using reverse transcription-polymerase chain reaction (RT-PCR) and RNA ligase mediated-rapid amplification of cDNA ends (RLM-RACE) methods, Ppi_pcna cDNA was isolated; it contained a coding region for 258 amino acid residues (aa) preceded by various 5'- and 3'-untranslated ends. The deduced protein length was similar to that of typical vertebrate and plant PCNA. PCR using genomic DNA as the template yielded multiple products whose sequences revealed multiple copies of pcna in tandem repeats separated by an unknown sequence. Using real-time PCR, we estimated 41+/-7 copies of this gene in each P. piscicida cell. Reverse transcription real-time PCR indicated a similar pcna mRNA level between the exponential and the stationary growth phases. Western blot analysis revealed a slightly higher PCNA level (<2-fold) in the exponential than in the stationary growth phases. We conclude that (1) P. piscicida possesses a typical eukaryote PCNA; (2) unlike in other eukaryotes, pcna in P. piscicida occurs in multiple copies arranged in tandem; and (3) regulation of P. piscicida PCNA probably lies in post-translational modification.

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Molecular methods offer an efficient alternative to microscopic identification of dinoflagellate cysts in natural sediments. Unfortunately, amplification of DNA also detects the presence of dead cells and is not a reliable indication of cyst viability. Because mRNA transcripts are more labile than DNA, the presence of specific transcripts may be used as a proxy for cyst viability. Here, we evaluate mRNA detection capabilities for identification of viable cysts of the dinoflagellate, Pfiesteria piscicida, in natural sediment samples. We targeted transcripts for cytochrome c oxidase subunit 1, cytochrome b (COB), and Tags 343 and 277, recently identified by serial analysis of gene expression. Expression was confirmed in laboratory cultures and compared with natural sediment samples. Three of the transcripts were detected in sediments by RT-PCR. The fourth transcript, for COB, was not detected in sediments, perhaps because of down-regulation of the gene in anoxic conditions. Our results suggest that methods targeting specific mRNA transcripts may be useful for detection of viable cysts in natural sediment samples. In addition, dinoflagellate cysts, which sustain extended periods of anoxia, may provide an important source of data for studies of anoxia tolerance by microbial eukaryotes.

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This study was undertaken to assess whether amoebae commonly found in mesohaline environments are in fact stages in the life cycles of Pfiesteria and Pfiesteria-like dinoflagellates. Primary isolations of amoebae and dinoflagellates were made from water and sediment samples from five tributaries of the Chesapeake Bay. Additional amoebae were also cloned from bioassay aquaria where fish mortality was attributed to Pfiesteria. Electron microscopy and small subunit (SSU) rRNA gene sequence analysis of these isolates clearly demonstrated that the commonly depicted amoeboid form of Pfiisteria is very likely a species of Korotnevella and is unrelated to Pfiesteria or Pfiesteria-like dinoflagellates. We have determined that the Pfiesteria and Pfiesteria-like dinoflagellates examined in this study undergo a typical homothallic life cycle without amoeboid stages. Furthermore, we have demonstrated that cloned amoebae sharing morphological characteristics described for stages in the life cycle of Pfiesteria do not transform into dinozoites. The strict clonal isolation and cultivation techniques used in this study substantially support the conclusion that the amoebae and some of the flagellates depicted in the life cycle of Pfiesteria are environmental contaminants of the Pfiesteria culture system and that the Ambush Predator Hypothesis needs to be rigorously reevaluated.

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The dinoflagellate Pfiesteria piscicida coexists with bacteria in aquatic environments and as such, may interact with them at the physiological level. This study was designed to investigate the influence of bacteria, present in a clonal culture of Pfiesteria piscicida, on the predator/prey relationship of this dinoflagellate with the alga Rhodomonas. A series of replenishment experiments with bacteria isolated from P. piscicida clonal culture and the bacteria-free P. piscicida derived from the same culture were carried out. In the presence of bacteria, the number of P. piscicida increased significantly when incubated with alga Rhodomonas. This enhanced growth was almost entirely due to the increased consumption rate of Rhodomonas by P. piscicida since in bacteria-free (axenic) cultures Rhodomonas were consumed at significantly reduced rates relative to cultures with bacteria. Subsequent replenishment experiments with individual bacterial isolates showed that a single isolate was responsible for the increased predation rate of P. piscicida. The presence or absence of this specific bacterium determined the outcome of the interaction between P. piscicida and Rhodomonas. Partial sequence analysis of the 16S rDNA of this isolate indicated that it was a novel marine alpha proteobacterium with sequence similarities to a Roseobacter sp. and a bacterium recently isolated from a toxic dinoflagellate Alexandrium sp.

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The Roseobacter clade of marine bacteria is often found associated with dinoflagellates, one of the major producers of dimethylsulfoniopropionate (DMSP). In this study, we tested the hypothesis that Roseobacter species have developed a physiological relationship with DMSP-producing dinoflagellates mediated by the metabolism of DMSP. DMSP was measured in Pfiesteria and Pfiesteria-like (Cryptoperidiniopsis) dinoflagellates, and the identities and metabolic potentials of the associated Roseobacter species to degrade DMSP were determined. Both Pfiesteria piscicida and Pfiesteria shumwayae produce DMSP with an average intracellular concentration of 3.8 microM. Cultures of P. piscicida or Cryptoperidiniopsis sp. that included both the dinoflagellates and their associated bacteria rapidly catabolized 200 microM DMSP (within 30 h), and the rate of catabolism was much higher for P. piscicida cultures than for P. shumwayae cultures. The community of bacteria from P. piscicida and Cryptoperidiniopsis cultures degraded DMSP with the production of dimethylsulfide (DMS) and acrylate, followed by 3-methylmercaptopropionate (MMPA) and methanethiol (MeSH). Four DMSP-degrading bacteria were isolated from the P. piscicida cultures and found to be taxonomically related to Roseobacter species. All four isolates produced MMPA from DMSP. Two of the strains also produced MeSH and DMS, indicating that they are capable of utilizing both the lyase and demethylation pathways. The diverse metabolism of DMSP by the dinoflagellate-associated Roseobacter spp. offers evidence consistent with a hypothesis that these bacteria benefit from association with DMSP-producing dinoflagellates.

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The potential use of clays to control harmful algal blooms (HABs) has been explored in East Asia, Australia, the United States, and Sweden. In Japan and South Korea, minerals such as montmorillonite, kaolinite, and yellow loess, have already been used in the field effectively, to protect fish mariculture from Cochlodinium spp. and other blooms. Cell removal occurs through the flocculation of algal and mineral particles, leading to the formation of larger aggregates (i.e. marine snow), which rapidly settle and further entrain cells during their descent. In the U.S., several clays and clay-rich sediments have shown high removal abilities (e.g. > 80% cell removal efficiency) against Karenia brevis, Heterosigma akashiwo, Pfiesteria piscicida and Aureococcus anophagefferens. In some cases, the removal ability of certain clays was further enhanced with chemical flocculants, such as polyaluminum chloride (PAC), to increase their adhesiveness. However, cell removal was also affected by bloom concentration, salinity, and mixing. Cell mortality was observed after clay addition, and increased with increasing clay concentration, and prolonged exposure to clays in the settled layer. Mesocosm, field enclosure, and flume experiments were also conducted to address cell removal with increasing scale and flow, water-column impacts, and the possible benthic effects from clay addition. Results from these studies will be presented, especially those in regards to water quality, seawater chemistry, bottom erodibility and faunal impacts in the benthos. At this time, clay dispersal continues to be a promising method for controlling HABs and mitigating their impacts based on existing information and experimental data.

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Pfiesteria piscicida and Pfiesteria shumwayae are estuarine dinoflagellates thought to be responsible for massive fish deaths and associated human illnesses in the southeastern United States. These dinoflagellates are described as having a complex life cycle involving flagellated zoospores, cysts, and amoeboid stages. Although no Pfiesteria toxin has been identified, certain strains of these dinoflagellates are thought to produce a water-soluble toxin that can kill fish and cause human illness. Recent reports show no evidence for amoeboid stages and indicate that a much more simplified life cycle exists. In addition, researchers have shown that P. shumwayae only kills fish through direct contact that does not necessarily involve the production of one or more toxins. This review summarizes these and other recent findings with an emphasis on establishing basic facts regarding the toxicity and life history of Pfiesteria dinoflagellates.

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A full-length cDNA (1,434 bp) of mitogen-activated protein kinase (MAPK), a key molecule of a signal transduction cascade, was isolated from the estuarine heterotrophic dinoflagellate Pfiesteria piscicida. This cDNA (Ppmapk1) encoded a protein (PpMAPK1) of 428 amino acid residues that shared about 30 to 40% amino acid similarity with MAPKs in other organisms. Phylogenetic analysis indicated that PpMAPK1 was tightly clustered with MAPK3 in protozoans. Using reverse transcription-PCR, expression of this gene was evaluated for P. piscicida cultures grown under different conditions. While salinity shock, heat shock, starvation, and a subsequent encounter with prey did not appear to affect expression of this gene, Ppmapk1 expression level was correlated with growth rate, suggesting involvement of this gene in the regulation of cell proliferation in the organism.

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We describe the two species of the toxic Pfiesteria complex to date (Pfiesteria piscicida and Pfiesteria shumwayae), their complex life cycles, and the characteristics required for inclusion within this complex. These species resemble P. piscicida Steidinger & Burkholder and also have a) strong attraction to fresh fish tissues and excreta, b) toxic activity stimulated by live fish, and c) production of toxin that can cause fish death and disease. Amoeboid stages were verified in 1992-1997 by our laboratory (various stages from toxic cultures) and that of K. Steidinger and co-workers (filose amoebae in nontoxic cultures), and in 2000 by H. Marshall and co-workers (various stages from toxic cultures), from clonal Pfiesteria spp. cultures, using species-specific polymerase chain reaction-based molecular probes with cross-confirmation by an independent specialist. Data were provided from tests of the hypothesis that Pfiesteriastrains differ in response to fresh fish mucus and excreta, algal prey, and inorganic nutrient (N, P) enrichment, depending on functional type or toxicity status. There are three functional types: TOX-A, in actively toxic, fish-killing mode; TOX-B, temporarily nontoxic, without access to live fish for days to weeks, but capable of toxic activity if fish are added; and NON-IND, noninducible with negligible toxicity in the presence of live fish. NON-IND Pfiesteria attained highest zoospore production on algal prey without or without inorganic nitrogen or inorganic phosphorus enrichment. TOX-B Pfiesteria was intermediate and TOX-A was lowest in zoospore production on algal prey with or without nutrients. TOX-A Pfiesteria spp. showed strong behavioral attraction to fresh fish mucus and excreta in short-term trials, with intermediate attraction of TOX-B zoospores and relatively low attraction of NON-IND cultures when normalized for cell density. The data for these clones indicated a potentially common predatory behavioral response, although differing in intensity distinct from a toxicity effect, in attack of fish prey. The data also demonstrated that functional types of Pfiesteria spp. show distinct differences in response to fish, algal prey, and inorganic nutrient enrichment. Collectively, the experiments indicate that NON-IND strains should not be used in research to gain insights about environmental controls on toxic strains of Pfiesteria spp.

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Dinoflagellates (Eukaryota; Alveolata; Dinophyceae) are single-cell eukaryotic microorganisms implicated in many toxic outbreaks in the marine and estuarine environment. Co-existing with dinoflagellate communities are bacterial assemblages that undergo changes in species composition, compete for nutrients and produce bioactive compounds, including toxins. As part of an investigation to understand the role of the bacteria in dinoflagellate physiology and toxigenesis, we have characterized the bacterial community associated with laboratory cultures of four 'Pfiesteria-like' dinoflagellates isolated from 1997 fish killing events in Chesapeake Bay. A polymerase chain reaction with oligonucleotide primers specific to prokaryotic 16S rDNA gene sequences was used to characterize the total bacterial population, including culturable and non-culturable species, as well as possible endosymbiotic bacteria. The results indicate a diverse group of over 30 bacteria species co-existing in the dinoflagellate cultures. The broad phylogenetic types of dinoflagellate-associated bacteria were generally similar, although not identical, to those bacterial types found in association with other harmful algal species. Dinoflagellates were made axenic, and the culturable bacteria were added back to determine the contribution of the bacteria to dinoflagellate growth. Confocal scanning laser fluorescence microscopy with 16S rDNA probes was used to demonstrate a physical association of a subset of the bacteria and the dinoflagellate cells. These data point to a key component in the bacterial community being species in the marine alpha-proteobacteria group, most closely associated with the alpha-3 or SAR83 cluster.

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