Lacking catalase, a protistan parasite draws on its photosynthetic ancestry to complete an antioxidant repertoire with ascorbate peroxidase

Background: Antioxidative enzymes contribute to a parasite's ability to counteract the host's intracellular killing mechanisms. The facultative intracellular oyster parasite, Perkinsus marinus, a sister taxon to dinoflagellates and apicomplexans, is responsible for mortalities of oysters along the Atlantic coast of North America. Parasite trophozoites enter molluscan hemocytes by subverting the phagocytic response while inhibiting the typical respiratory burst. Because P. marinus lacks catalase, the mechanism(s) by which the parasite evade the toxic effects of hydrogen peroxide had remained unclear. We previously found that P. marinus displays an ascorbate-dependent peroxidase (APX) activity typical of photosynthetic eukaryotes. Like other alveolates, the evolutionary history of P. marinus includes multiple endosymbiotic events. The discovery of APX in P. marinus raised the questions: From which ancestral lineage is this APX derived, and what role does it play in the parasite's life history? Results: Purification of P. marinus cytosolic APX activity identified a 32 kDa protein. Amplification of parasite cDNA with oligonucleotides corresponding to peptides of the purified protein revealed two putative APX-encoding genes, designated PmAPX1 and PmAPX2. The predicted proteins are 93% identical, and PmAPX2 carries a 30 amino acid N-terminal extension relative to PmAPX1. The P. marinus APX proteins are similar to predicted APX proteins of dinoflagellates, and they more closely resemble chloroplastic than cytosolic APX enzymes of plants. Immunofluorescence for PmAPX1 and PmAPX2 shows that PmAPX1 is cytoplasmic, while PmAPX2 is localized to the periphery of the central vacuole. Three-dimensional modeling of the predicted proteins shows pronounced differences in surface charge of PmAPX1 and PmAPX2 in the vicinity of the aperture that provides access to the heme and active site. Conclusions: PmAPX1 and PmAPX2 phylogenetic analysis suggests that they are derived from a plant ancestor. Plant ancestry is further supported by the presence of ascorbate synthesis genes in the P. marinus genome that are similar to those in plants. The localizations and 3D structures of the two APX isoforms suggest that APX fulfills multiple functions in P. marinus within two compartments. The possible role of APX in free-living and parasitic stages of the life history of P. marinus is discussed.Fil: Schott, Eric. University Of Maryland. Biotechnology Institute. Center Of Marine Biotechnology; Estados UnidosFil: Di Lella, Santiago. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Bachvaroff, Tsvetan R.. University Of Maryland. Biotechnology Institute. Center Of Marine Biotechnology; Estados UnidosFil: Amzel, León Mario. University Johns Hopkins; Estados UnidosFil: Vasta, Gerardo. University Of Maryland. Biotechnology Institute. Center Of Marine Biotechnology; Estados Unido

From Stop to Start: Tandem Gene Arrangement, Copy Number and Trans-Splicing Sites in the Dinoflagellate Amphidinium carterae

Dinoflagellate genomes present unique challenges including large size, modified DNA bases, lack of nucleosomes, and condensed chromosomes. EST sequencing has shown that many genes are found as many slightly different variants implying that many copies are present in the genome. As a preliminary survey of the genome our goal was to obtain genomic sequences for 47 genes from the dinoflagellate Amphidinium carterae. A PCR approach was used to avoid problems with large insert libraries. One primer set was oriented inward to amplify the genomic complement of the cDNA and a second primer set would amplify outward between tandem repeats of the same gene. Each gene was also tested for a spliced leader using cDNA as template. Almost all (14/15) of the highly expressed genes (i.e. those with high representation in the cDNA pool) were shown to be in tandem arrays with short intergenic spacers, and most were trans-spliced. Only two moderately expressed genes were found in tandem arrays. A polyadenylation signal was found in genomic copies containing the sequence AAAAG/C at the exact polyadenylation site and was conserved between species. Four genes were found to have a high intron density (>5 introns) while most either lacked introns, or had only one to three. Actin was selected for deeper sequencing of both genomic and cDNA copies. Two clusters of actin copies were found, separated from each other by many non-coding features such as intron size and sequence. One intron-rich gene was selected for genomic walking using inverse PCR, and was not shown to be in a tandem repeat. The first glimpse of dinoflagellate genome indicates two general categories of genes in dinoflagellates, a highly expressed tandem repeat class and an intron rich less expressed class. This combination of features appears to be unique among eukaryotes

Broad Phylogenomic Sampling and the Sister Lineage of Land Plants

The tremendous diversity of land plants all descended from a single charophyte green alga that colonized the land somewhere between 430 and 470 million years ago. Six orders of charophyte green algae, in addition to embryophytes, comprise the Streptophyta s.l. Previous studies have focused on reconstructing the phylogeny of organisms tied to this key colonization event, but wildly conflicting results have sparked a contentious debate over which lineage gave rise to land plants. The dominant view has been that ‘stoneworts,’ or Charales, are the sister lineage, but an alternative hypothesis supports the Zygnematales (often referred to as “pond scum”) as the sister lineage. In this paper, we provide a well-supported, 160-nuclear-gene phylogenomic analysis supporting the Zygnematales as the closest living relative to land plants. Our study makes two key contributions to the field: 1) the use of an unbiased method to collect a large set of orthologs from deeply diverging species and 2) the use of these data in determining the sister lineage to land plants. We anticipate this updated phylogeny not only will hugely impact lesson plans in introductory biology courses, but also will provide a solid phylogenetic tree for future green-lineage research, whether it be related to plants or green algae

The sorting of different actin clones between the two clusters of copies.

<p>The different cluster assignments for genomic culture DNA, single cell genomic, and two different cDNA amplicons (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002929#pone-0002929-g003" target="_blank">Figure 3</a> for arrangement) are shown, asterisks indicate significant differences at p<0.05. Below each category the total number of clones sequenced is given. The cluster A and B assignments refer to the location of the clones on <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002929#pone-0002929-g003" target="_blank">figure 3</a>.</p

A scaled schematic representation of the polyketide synthase (PKS) genomic and cDNA sequence.

<p>Exons, donor sites, and the start codon are shown, the genomic sequence does not extend to the stop codon, but does extend well upstream of the start codon. The location of the amplicon from a second toxin-producing strain of <i>Amphidinium carterae</i> CCMP121 is also shown. When atypical intron donors are present the actual dinucleotide donor is shown at the edge of the intron.</p

A schematic representation of the different actin amplicons.

<p>At the top is a hypothetical arrangement of tandem gene copies in the genome showing a long array of repeated gene copies, with short intergenic regions between them, below are different actin amplicons shown at the same scale. A. An mRNA schematic based on an assembly of 24 different ESTs showing stop and start codons, and the polyA tail. B. Two different cDNA amplicons, the shorter of which was amplified with two gene specific primers (33 were sequenced), and a longer amplicon using the <i>trans</i>-spliced leader primer with a gene specific reverse primer (34 were sequenced). C. The genomic amplicon bridging between two adjacent gene copies (46 were sequenced). All schematics are drawn to an equal scale except for the proposed arrangement at the top.</p

Genes selected for this survey.

a<p>Inferred evolutionary source of gene.</p>b<p>Number of ESTs in a previous survey <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002929#pone.0002929-Bachvaroff1" target="_blank">[10]</a>.</p>c<p>Tandem repeat genes are repeats of the same gene in a series demonstrated with PCR and sequencing using outwardly directed primers.</p>d<p>PCR and sequencing with a general spliced leader primer and a gene specific reverse primer. If this PCR was unsuccessful, but was amplified from the same cDNA using gene specific primers then the entry is marked “present”.</p>e<p>If genomic amplification using inwardly directed primers, followed by sequencing produced a genomic complement to the EST.</p>f<p>The number of introns found by comparing the expressed and amplified portions of the genomic sequences.</p>g<p>The intergenic amplicon was used as a proxy for a forward genomic amplicon, since the intergenic amplicon covered almost the entire coding region of these genes.</p

A distance tree using both genomic and expressed versions of the actin gene.

<p>The tree was based on an 800 base coding region common to all amplicons (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002929#pone-0002929-g003" target="_blank">Figure 3</a>). The two genomic clusters were cleanly divided based on 45 synonymous substitutions over 800 bases, with no intermediate sequences found in the genomic clones (exemplified by the stop codons in this figure). Other features such as intron sequence, and intergenic spacer sequence also sort the two genomic clusters. Multiple pseudogene versions were also found and are underlined. Expressed sequences are marked with colored boxes and were mostly drawn from a single cluster with several chimeric sequences having features of both genomic clusters (marked with a star).</p

A sequence logo showing the putative polyadenylation sequence for dinoflagellates.

<p>This logo is based on an alignment of the intergenic spacer genomic amplicons of 25 different intergenic spacer sequences from 14 genes. The polyadenylation site was inferred by comparing ESTs to the genomic sequence. The same motif is present in <i>Karlodinium veneficum</i> and <i>Lingulodinium polyedrum</i>.</p