A cryptic algal group unveiled: a plastid biosynthesis pathway in the oyster parasite Perkinsus marinus

Plastids are widespread in plant and algal lineages. They are also exploited by some nonphotosynthetic protists, including malarial parasites, to support their diverse modes of life. However, cryptic plastids may exist in other nonphotosynthetic protists, which could be important in studies on the diversity and evolution of plastids. The parasite Perkinsus marinus, which causes mass mortality in oyster farms, is a nonphotosynthetic protist that is phylogenetically related to plastid-bearing dinoflagellates and apicomplexans. In this study, we searched for P. marinus methylerythritol phosphate (MEP) pathway genes, responsible for de novo isoprenoid synthesis in plastids, and determined the full-length gene sequences for 6 of 7 of these genes. Phylogenetic analyses revealed that each P. marinus gene clusters with orthologs from plastid-bearing eukaryotes, which have MEP pathway genes with essentially the same mosaic pattern of evolutionary origin. A new analytical method called sliding-window iteration of TargetP was developed to examine the distribution of targeting preferences. This analysis revealed that the sequenced genes encode bipartite targeting peptides that are characteristic of proteins targeted to secondary plastids originating from endosymbiosis of eukaryotic algae. These results support our idea that Perkinsus is a cryptic algal group containing nonphotosynthetic secondary plastids. In fact, immunofluorescent microscopy indicated that 1 of the MEP pathway enzymes, 1-deoxy-D-xylulose 5-phosphate reductoisomerase, was localized to small compartments near mitochondrion, which are possibly plastids. This tiny organelle seems to contain very low quantities of DNA or may even lack DNA entirely. The MEP pathway genes are a useful tool for investigating plastid evolution in both of the photosynthetic and nonphotosynthetic eukaryotes and led us to propose the hypothesis that ancestral "chromalveolates" harbored plastids before a secondary endosymbiotic event.     

5 The origin of the dinoflagellate plastid

The Dasycladales is an ancient order of tropical benthic marine green algae, unique in their radially arranged unicellular thalli and well-preserved fossil record due to extensive calcification of the thallus. The inference of an accurate phylogeny for the Dasycladales is important in order to better understand stratigraphy, character evolution, and classification. Previous analyses ( rbc L and 18S rDNA) suggested that the Family Acetabulariaceae is monophyletic, but that the Family Dasycladaceae is a basal paraphyletic assemblage. However, the two data sets disagreed regarding genus- and species-level relationships within the Dasycladales. For example, the placement of the genera, Halicoryne , Bornetella and Cymopolia were incongruent. Given the conflicting results of these previous analyses, the current project examined a third highly conserved nuclear-encoded gene, 26S rDNA. Aligned 26S rDNA sequences were analyzed with parsimony and model-based methods and compared to previous results based on18S and rbc L sequences. Family-level relationships based on 26S rDNA were congruent with previous studies: the Acetabulariaceae is monophyletic while the Dasycladaceae is paraphyletic. In addition, acetabulariacean genera are not monophyletic, suggesting that the presence of a corona inferior or calcification of gametes may not be appropriate to define genera. Within the Dasycladaceae, the basal position of Cymopolia is supported by 26S rDNA, a result congruent with rbcL and stratigraphy but not with 18S data. These results will be discussed in the context of morphological character evolution, fossil stratigraphy and family, tribal and generic relationships among these living algal fossils. Supported in part by NSF grant DEB-0128977 to FWZ.     

Molecular evidence for plastid robbery (Kleptoplastidy) in Dinophysis,a dinoflagellate causing diarrhetic shellfish poisoning

The dinoflagellate genus Dinophysis contains species known to cause diarrhetic shellfish poisoning. Although most photosynthetic dinoflagellates have plastids with peridinin, photosynthetic Dinophysis species have cryptophyte-like plastids containing phycobilin rather than peridinin. We sequenced nuclear- and plastid-encoded SSU rDNA from three photosynthetic species of Dinophysis for phylogenetic analyses. In the tree of nuclear SSU rDNA, Dinophysis was a monophyletic group nested with peridinin-containing dinoflagellates. However, in the tree of plastid SSU rDNA, the Dinophysis plastid lineage was within the radiation of cryptophytes and was closely related to Geminigera cryophila. These analyses indicate that an ancestor of Dinophysis, which may have originally possessed peridinin-type plastid and lost it subsequently, adopted a new plastid from a cryptophyte. Unlike dinoflagellates with fully integrated plastids, the Dinophysis plastid SSU rDNA sequences were identical among the three species examined, while there were species-specific base substitutions in their nuclear SSU rDNA sequences. Queries of the DNA database showed that the plastid SSU rDNA sequence of Dinophysis is almost identical to that of an environmental DNA clone of a <10 pm sized plankter, possibly a cryptophyte and a likely source of the Dinophysis plastid. The present findings suggest that these Dinophysis species engulfed and temporarily retained plastids from a cryptophyte.     

Gene organization and expression of the divalent cation transporter Nramp in the protistan parasite Perkinsus marinus

Trophozoites of the protistan parasite Perkinsus marinus reside and proliferate inside phagosomelike structures of hemocytes from the host, the eastern oyster Crassostrea virginica. In a murine model, it has been proposed that the outcome of intracellular parasite-host interactions is determined, at least in part, by the activity of the host's divalent cation transporter natural resistance-associated macrophage protein 1 (Nramp1). Although nucleotide sequences from members of the Nramp family in protozoan parasites have recently become available in public databases, little is known about their molecular, structural, and functional aspects that may relate to the parasite's survival of intracellular killing by the host. The complementary DNA (cDNA) sequence of the Nramp from P. marinus (PmNramp) was obtained by polymerase chain reaction amplification with degenerated primers, followed by rapid amplification of cDNA ends. The 2,082-bp cDNA sequence encoded a predicted protein of 558 amino acids. PmNramp is a single-copy gene composed of 7 exons and 6 short introns (44-61 bp) with the canonical splicing signal (GT/AG). A phylogenetic analysis indicates that P. marinus and apicomplexan Nramp genes derive from a common "archetype" Nramp ancestor. However, the apicomplexan Nramps are highly divergent from the P. marinus sequence and the rest of the archetype Nramp group. Preliminary studies suggest that expression of PmNramp in in vitro-cultured P. marinus trophozoites is modulated by metals and by exogenous oxidative stress.     

Dolphin genome provides evidence for adaptive evolution of nervous system genes and a molecular rate slowdown

Cetaceans (dolphins and whales) have undergone a radical transformation from the original mammalian bodyplan. In addition, some cetaceans have evolved large brains and complex cognitive capacities. We compared approximately 10 000 protein-coding genes culled from the bottlenose dolphin genome with nine other genomes to reveal molecular correlates of the remarkable phenotypic features of these aquatic mammals. Evolutionary analyses demonstrated that the overall synonymous substitution rate in dolphins has slowed compared with other studied mammals, and is within the range of primates and elephants. We also discovered 228 genes potentially under positive selection (dN/dS > 1) in the dolphin lineage. Twenty-seven of these genes are associated with the nervous system, including those related to human intellectual disabilities, synaptic plasticity and sleep. In addition, genes expressed in the mitochondrion have a significantly higher mean dN/dS ratio in the dolphin lineage than others examined, indicating evolution in energy metabolism. We encountered selection in other genes potentially related to cetacean adaptations such as glucose and lipid metabolism, dermal and lung development, and the cardiovascular system. This study underlines the parallel molecular trajectory of cetaceans with other mammalian groups possessing large brains.     

Migration of the plastid genome to the nucleus in a peridinin dinoflagellate

Dinoflagellate algae are important primary producers and of significant ecological and economic impact because of their ability to form "red tides". They are also models for evolutionary research because of an unparalleled ability to capture photosynthetic organelles (plastids) through endosymbiosis. The nature and extent of the plastid genome in the dominant perdinin-containing dinoflagellates remain, however, two of the most intriguing issues in plastid evolution. The plastid genome in these taxa is reduced to single-gene minicircles encoding an incomplete (until now 15) set of plastid proteins. The location of the remaining photosynthetic genes is unknown. We generated a data set of 6,480 unique expressed sequence tags (ESTs) from the toxic dinoflagellate Alexandrium tamarense (for details, see the Experimental Procedures in the Supplemental Data) to find the missing plastid genes and to understand the impact of endosymbiosis on genome evolution. Here we identify 48 of the non-minicircle-encoded photosynthetic genes in the nuclear genome of A. tamarense, accounting for the majority of the photosystem. Fifteen genes that are always found on the plastid genome of other algae and plants have been transferred to the nucleus in A. tamarense. The plastid-targeted genes have red and green algal origins. These results highlight the unique position of dinoflagellates as the champions of plastid gene transfer to the nucleus among photosynthetic eukaryotes.     

Phototaxis in a dinoflagellate: Action spectra as evidence for a two-pigment system

Summary Action spectra were determined in the UV region of the spectrum for the first phase of the phototactic response (stop response) and for the phytochrome pigment associated with this response in the dinoflagellate Gyrodinium dorsum Kofoid. Differences between these action spectra indicate the participation of two pigments in phototaxis. Following R (620 nm) irradiation of the phytochrome, the stop response maxima occur at 470 and 280-nm; after FR irradiation they shift to 490 and 300–310 nm. These maxima suggest that the photoreceptor pigment for phototaxis is a carotenoprotein. The action spectrum shift following the different phytochrome conversions may represent a trans to cis isomer change by the carotenoid. The absorption maximum of P_R in the UV appears to be at 320 nm, which is consistent with the shift of the R absorption maximum to shorter wavelengths (620 nm) as compared to higher plants. The P_FR absorption maximum appears as a broad band between 360 and 390 nm. Comparison of P_R to P_FR conversions by different intensities of 620-nm and 320-nm light indicates that at lower intensities the logarithm of the threshold for the stop response is inversely proportional to the logarithm of the intensity of the sensitizing light. The ratio of response activation by R and UV light is about 4:1.Abbreviations FR far-red - R red - P_FR far-red-absorbing form of phytochrome - P_R red-absorbing form of phytochrome - UV ultraviolet     

A two-alpha-helix extra domain mediates the halophilic character of a plant-type ferredoxin from halophilic archaea

The [2Fe-2S] ferredoxin (HsFdx) of the halophilic archaeon Halobacterium salinarum exhibits a high degree of sequence conservation with plant-type ferredoxins except for an insertion of 30 amino acids near its N-terminus which is extremely rich in acidic amino acids. Unfolding studies reveal that HsFdx has an unfolding temperature of approximately 85 degrees C in 4.3 M NaCl, but of only 50 degrees C in low salinity, revealing its halophilic character. The three-dimensional structure of HsFdx was determined by NMR spectroscopy, resulting in a backbone rmsd of 0.6 A for the diamagnetic regions of the protein. Whereas the overall structure of HsFdx is very similar to that of the plant-type ferredoxins, two additional alpha-helices are found in the acidic extra domain. (15)N NMR relaxation studies indicate that HsFdx is rigid, and the flexibility of residues is similar throughout the molecule. Monitoring protein denaturation by NMR did not reveal differences between the core fold and the acidic domain, suggesting a cooperative unfolding of both parts of the molecule. A mutant of the HsFdx in which the acidic domain is replaced with a short loop of the nonhalophilic Anabaena ferredoxin shows a considerably changed expression pattern. The halophilic wild-type protein is readily expressed in large amounts in H. salinarum, but not in Escherichia coli, whereas the mutant ferredoxin could only be overexpressed in E. coli. The salt concentration was also found to play a critical role for the efficiency of cluster reconstitution: the cluster of HsFdx could be reconstituted only in a solution containing molar concentrations of NaCl, while the reconstitution of the cluster in the mutant protein proceeds efficiently in low salt. These findings suggest that the acidic domain mediates the halophilic character which is reflected in its thermostability, the exclusive expression in H. salinarum, and the ability to efficiently reconstitute the iron-sulfur cluster only at high salt concentrations.     

Perkinsus marinus, a protozoan parasite of the eastern oyster, has a requirement for dietary sterols

Perkinsus marinus, a protozoan parasite of the eastern oyster, Crassostrea virginica, causes high mortality in its host along the Atlantic and Gulf coasts of North America. P. marinus meronts cultured in vitro in medium containing complete lipid supplement (cod liver oil, cholesterol and alpha tocopherol acetate in detergent) are able to synthesize a wide variety of lipids, yet cultures cannot be maintained in lipid-free medium. To determine P. marinus lipid requirements meronts were inoculated into media containing different combinations of lipid components in detergent. Treatments included complete lipid supplement (positive control), detergent only (negative control), cholesterol in detergent, alpha tocopherol acetate in detergent and cholesterol+alpha tocopherol acetate in detergent. Meronts proliferated in the positive control medium and media containing cholesterol or cholesterol+alpha tocopherol acetate, but failed to proliferate in the negative control medium and the medium containing just alpha tocopherol acetate. Gas chromatography analysis of P. marinus meronts grown in medium with added (13)C sodium acetate (0.5 mg mL(-1)) revealed the presence of fatty acids containing (13)C, but the only sterol present was cholesterol containing no (13)C. These results suggest that P. marinus cannot synthesize sterols and must sequester them from its host.     

Real-Time PCR for Detection and Quantification of the Protistan Parasite Perkinsus marinus in Environmental Waters

The protistan parasite Perkinsus marinus is a severe pathogen of the oyster Crassostrea virginica along the east coast of the United States. Very few data have been collected, however, on the abundance of the parasite in environmental waters, limiting our understanding of P. marinus transmission dynamics. Real-time PCR assays with SybrGreen I as a label for detection were developed in this study for quantification of P. marinus in environmental waters with P. marinus species-specific primers and of Perkinsus spp. with Perkinsus genus-specific primers. Detection of DNA concentrations as low as the equivalent of 3.3 × 10⁻² cell per 10-μl reaction mixture was obtained by targeting the multicopy internal transcribed spacer region of the genome. To obtain reliable target quantification from environmental water samples, removal of PCR inhibitors and efficient DNA recovery were two major concerns. A DNA extraction kit designed for tissues and another designed for stool samples were tested on environmental and artificial seawater (ASW) samples spiked with P. marinus cultured cells. The stool kit was significantly more efficient than the tissue kit at removing inhibitors from environmental water samples. With the stool kit, no significant difference in the quantified target concentrations was observed between the environmental and ASW samples. However, with the spiked ASW samples, the tissue kit demonstrated more efficient DNA recovery. Finally, by performing three elutions of DNA from the spin columns, which were combined prior to target quantification, variability of DNA recovery from different samples was minimized and more reliable real-time PCR quantification was accomplished.     

Real-time PCR for detection and quantification of the protistan parasite Perkinsus marinus in environmental waters

The protistan parasite Perkinsus marinus is a severe pathogen of the oyster Crassostrea virginica along the east coast of the United States. Very few data have been collected, however, on the abundance of the parasite in environmental waters, limiting our understanding of P. marinus transmission dynamics. Real-time PCR assays with SybrGreen I as a label for detection were developed in this study for quantification of P. marinus in environmental waters with P. marinus species-specific primers and of Perkinsus spp. with Perkinsus genus-specific primers. Detection of DNA concentrations as low as the equivalent of 3.3 x 10(-2) cell per 10-microl reaction mixture was obtained by targeting the multicopy internal transcribed spacer region of the genome. To obtain reliable target quantification from environmental water samples, removal of PCR inhibitors and efficient DNA recovery were two major concerns. A DNA extraction kit designed for tissues and another designed for stool samples were tested on environmental and artificial seawater (ASW) samples spiked with P. marinus cultured cells. The stool kit was significantly more efficient than the tissue kit at removing inhibitors from environmental water samples. With the stool kit, no significant difference in the quantified target concentrations was observed between the environmental and ASW samples. However, with the spiked ASW samples, the tissue kit demonstrated more efficient DNA recovery. Finally, by performing three elutions of DNA from the spin columns, which were combined prior to target quantification, variability of DNA recovery from different samples was minimized and more reliable real-time PCR quantification was accomplished.     

Mapping of protein-protein interaction sites in the plant-type [2Fe-2S] ferredoxin

Knowing the manner of protein-protein interactions is vital for understanding biological events. The plant-type [2Fe-2S] ferredoxin (Fd), a well-known small iron-sulfur protein with low redox potential, partitions electrons to a variety of Fd-dependent enzymes via specific protein-protein interactions. Here we have refined the crystal structure of a recombinant plant-type Fd I from the blue green alga Aphanothece sacrum (AsFd-I) at 1.46 Å resolution on the basis of the synchrotron radiation data. Incorporating the revised amino-acid sequence, our analysis corrects the 3D structure previously reported; we identified the short α-helix (67-71) near the active center, which is conserved in other plant-type [2Fe-2S] Fds. Although the 3D structures of the four molecules in the asymmetric unit are similar to each other, detailed comparison of the four structures revealed the segments whose conformations are variable. Structural comparison between the Fds from different sources showed that the distribution of the variable segments in AsFd-I is highly conserved in other Fds, suggesting the presence of intrinsically flexible regions in the plant-type [2Fe-2S] Fd. A few structures of the complexes with Fd-dependent enzymes clearly demonstrate that the protein-protein interactions are achieved through these variable regions in Fd. The results described here will provide a guide for interpreting the biochemical and mutational studies that aim at the manner of interactions with Fd-dependent enzymes.     

Proteomic analysis of the Cyanophora paradoxa muroplast provides clues on early events in plastid endosymbiosis

Glaucophytes represent the first lineage of photosynthetic eukaryotes of primary endosymbiotic origin that diverged after plastid establishment. The muroplast of Cyanophora paradoxa represents a primitive plastid that resembles its cyanobacterial ancestor in pigment composition and the presence of a peptidoglycan wall. To attain insights into the evolutionary history of cyanobiont integration and plastid development, it would thus be highly desirable to obtain knowledge on the composition of the glaucophyte plastid proteome. Here, we provide the first proteomic analysis of the muroplast of C. paradoxa. Mass spectrometric analysis of the muroplast proteome identified 510 proteins with high confidence. The protein repertoire of the muroplast revealed novel paths for reduced carbon flow and export to the cytosol through a sugar phosphate transporter of chlamydial origin. We propose that C. paradoxa possesses a primordial plastid mirroring the situation in the early protoalga.