Currently the shikimate pathway is reported as a metabolic feature of prokaryotes, ascomycete fungi, apicomplexans, and plants. The plant shikimate pathway enzymes have similarities to prokaryote homologues and are largely active in chloroplasts, suggesting ancestry from the plastid progenitor genome. Toxoplasma gondii, which also possesses an alga-derived plastid organelle, encodes a shikimate pathway with similarities to ascomycete genes, including a five-enzyme pentafunctional arom. These data suggests that the shikimate pathway and the pentafunctional arom either had an ancient origin in the eukaryotes or was conveyed by eukaryote-to-eukaryote horizontal gene transfer (HGT). We expand sampling and analyses of the shikimate pathway genes to include the oomycetes, ciliates, diatoms, basidiomycetes, zygomycetes, and the green and red algae. Sequencing of cDNA from Tetrahymena thermophila confirmed the presence of a pentafused arom, as in fungi and T. gondii. Phylogenies and taxon distribution suggest that the arom gene fusion event may be an ancient eukaryotic innovation. Conversely, the Plantae lineage (represented here by both Viridaeplantae and the red algae) acquired different prokaryotic genes for all seven steps of the shikimate pathway. Two of the phylogenies suggest a derivation of the Plantae genes from the cyanobacterial plastid progenitor genome, but if the full Plantae pathway was originally of cyanobacterial origin, then the five other shikimate pathway genes were obtained from a minimum of two other eubacterial genomes. Thus, the phylogenies demonstrate both separate HGTs and shared derived HGTs within the Plantae clade either by primary HGT transfer or secondarily via the plastid progenitor genome. The shared derived characters support the holophyly of the Plantae lineage and a single ancestral primary plastid endosymbiosis. Our analyses also pinpoints a minimum of 50 gene/domain loss events, demonstrating that loss and replacement events have been an important process in eukaryote genome evolution.

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Apicomplexan parasites, Eimeria tenella, Plasmodium spp. and Toxoplasma gondii, possess a homologous plastid-like organelle termed the apicoplast, derived from the endosymbiotic enslavement of a photosynthetic alga. However, currently no eimerian nuclear encoded apicoplast targeted proteins have been identified, unlike in Plasmodium spp. and T. gondii. In this study, we demonstrate that nuclear encoded enoyl reductase of E. tenella (EtENR) has a predicted N-terminal bipartite transit sequence, typical of apicoplast-targeted proteins. Using a combination of immunocytochemistry and EM we demonstrate that this fatty acid biosynthesis protein is located in the apicoplast of E. tenella. Using the EtENR as a tool to mark apicoplast development during the Eimeria lifecycle, we demonstrate that nuclear and apicoplast division appear to be independent events, both organelles dividing prior to daughter cell formation, with each daughter cell possessing one to four apicoplasts. We believe this is the first report of multiple apicoplasts present in the infectious stage of an apicomplexan parasite. Furthermore, the microgametes lacked an identifiable apicoplast consistent with maternal inheritance via the macrogamete. It was found that the size of the organelle and the abundance of EtENR varied with developmental stage of the E. tenella lifecycle. The high levels of EtENR protein observed during asexual development and macrogametogony is potentially associated with the increased synthesis of fatty acids required for the rapid formation of numerous merozoites and for the extracellular development and survival of the oocyst. Taken together the data demonstrate that the E. tenella apicoplast participates in type II fatty acid biosynthesis with increased expression of ENR during parasite growth. Apicoplast division results in the simultaneous formation of multiple fragments. The division mechanism is unknown, but is independent of nuclear division and occurs prior to daughter formation.

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