Research Interests:
Research in the lab is generally related to the molecular evolution and cell biology of eukaryotes, in particular the protists (i.e., eukaryotes that are not animals, fungi, or plants). Protists are mostly single celled organisms, but are many are extremely complex and sophisticated despite their small size. Protists also represent the greatest part of eukaryotic diversity, although most protists groups are very poorly studied, especially at the molecular level. We use molecular biology, microscopy, and increasingly use genome wide analyses such as EST sequencing and genome sequence surveys to study a number of questions in different lineages of protists. Much of our research focuses on cellular organelles, in particular mitochondria and plastids. These organelles originated by endosymbiosis, or the uptake and retention of a bacterium (the endosymbiont) by a eukaryote (the host). In the case of mitochondria this involved an alpha-proteobacterium and took place around the origin of eukaryotes. Plastids originated more recently from a cyanobacterium. Plastids have also spread between eukaryotic lineages by a process called secondary endosymbiosis. In this case, a plastid-bearing alga is itself taken up by another eukaryote, and its photosynthetic apparatus is retained by this new, secondary, host. Another major focus of the research is the transition of free living organisms to parasitism and how this affects their organelles and metabolism. Below are short descriptions of several projects currently underway.

Current work:

1. Chlorarachnion EST project. Chlorarachniophytes are a group of amoeboflagellates and flagellates that have acquired a plastid by secondary endosymbiosis with a green alga. They are primarily distinguished by having also retained the nucleus and a relict genome of this alga, called the nucleomorph. Although this is a very difficult experimental system to work with in some ways, the presence of the nucleomorph and the fact that chlorarachniophytes photosynthesise and prey on other organisms at the same time offer a chance to study some questions that cannot be studied in other organisms. In particular, we are interested in the processes of lateral gene transfer and protein targeting. We are sequencing expressed sequence tags (ESTs) from the nuclear genome of the chlorarachniophyte recently named Bigalowiella natans (CCMP 621), focussing on genes encoded in the host nuclear genome. To date, slightly less than 4,000 ESTs have been sequenced. The process of lateral transfer has so far been examined in 80 genes for plastid targeted proteins, where we have found a significant fraction of these genes were derived from lateral gene transfer. We are now in the process of examining the targeting of these proteins to the plastid, and are characterising candidate proteins that appear to be targeted to the cytoplasm of the green algal endosymbiont. As little data were previously known from this group, an EST survey also yields a number of unanticipated but important discoveries. In this case, some of the more interesting findings include the discovery of a phylogenetic affinity between chlorarachniophytes , cercomonads and foraminifera and the discovery of a unique form of actin expression where all actins are fused to ubiquitin.

2. Protist EST Project – ESTs from algae with secondary plastids. In addition to Chlorarachnion, the lab is beginning EST projects on a number of other algae with secondary plastids though the Genome Canada Protist EST Project (PEP). The intent is to sequence relatively a small number of ESTs (about 5,000 each) from a variety of algae with secondary plastids, including haptophytes, heterokonts, cryptomonads, and chlorarachniophytes, as well as some of their non-photosynthetic relatives. Sequencing is underway for the haptophyte Isochrysis galbana, the dinoflagellate Heterocapsa triquetra, and library construction is underway for several other species. The goal of this work is similar to that for the Bigalowiella EST project, but with a much broader aim of understanding the history of all plastid types in eukaryotes.

3. Origin and evolution of the apicomplexa. This is a major and long-term focus of the lab. Apicomplexa are a group of intracellular parasites that include several major disease causing agents, such as Toxoplasma, Cryptosporidium, and the malaria parasite, Plasmodium. In the mid-1990s, apicomplexa were surprisingly shown to contain a plastid, although they are obviously not photosynthetic. The plastid function is now fairly well characterised in Plasmodium and Toxoplasma, where genes for nuclear-encoded plastid targeted proteins involved in the biosynthesis of heme, fatty acids and isoprenoids have been found. We are interested in the origin of this plastid and its metabolic enzymes, and also the distribution of the plastid in extant apicomplexa. To first end, we are studying the phylogeny of each enzyme involved in the major biochemical pathways presently recognised in the plastids of Toxoplasma and Plasmodium. To examine the distribution of the plastid, we are presently looking for evidence of a plastid or plastid genome in the earliest branching apicomplexa, the gregarines. We and other labs have also generated evidence that the apicomplexan plastid is of red algal origin and likely traces back to the same endosymbiotic event that gave rise to plastids in several algal groups, namely cryptomonads, heterokonts, and haptophytes. We are pursuing this question further by developing multigene data sets to test the relationship of these organisms to alveolates. In addition, we are also working to resolve the early branches of the apicomplexan tree to reconstruct the evolution of certain other characters involved in parasitism.

4. Plastid function in Helicosporidium. Helicosporidia are little studied intracellular parasites of invertebrates with a complex infectious cyst. The origin of helicosporidia has not been clear historically, but recently they were shown by phylogenetic analyses to be highly derived green algae related to the vertebrate parasite Prototheca. There is no morphological evidence for a plastid in helicosporidian parasites, but plastids have been found in Prototheca and a number of other algal parasites, and there is now evidence for a plastid rRNA in Helicosporidium. To characterise the plastid and its functions in Helicosporidium, we have begun to sequence ESTs (again) in collaboration with the lab of Drion Boucias at the University of Florida. Important functional proteins in the plastid are likely encoded in the nuclear genome for the most part, and targeted to the plastid post-translationally. The aims of this work are to characterise as many genes for plastid targeted proteins as possible so as to determine the functional metabolic pathways that are housed in the plastid, and also to use sequences from plastid-encoded genes to identify the organelle by in situ hybridisation. This will offer a unique chance to compare plastids in highly adapted intracellular parasites that evolved from algae, but with very different ancestors. In the case of apicomplexa, the ancestor contained a red algal secondary plastid, while the Helicosporidium plastid is a primary green plastid.

5. Function of mitochondria in microsporidian spores. Microsporidia are a large and diverse group of intracellular parasites with a very complex and highly adapted mechanism of infection other cells. Microsporidia were thought for some time to be primitive eukaryotes that lacked a number of otherwise common features, in particular the mitochondrion. Now, however, molecular phylogenies have demonstrated that microsporidia are in fact highly derived fungi, and not especially ancient. In addition, several genes derived from the mitochondrial endosymbiont have now been found in microsporidian nuclear genomes. The first of these were HSP70 and pyruvate dehydrogenase, but the recent completion of the Encephalitozoon genome has provided several more. We are interested in how this organelle has adapted in microsporidia, and what it present functions are. We are using Nosema locustae as our model system, and have completed a genome sequence survey on this organism. We are now in the process of comparing this genome with that of Encephalitozoon, and characterising the gene sequence, expression pattern, and localisation of each mitochondrial protein.

6. Origin of non-canonical genetic codes and how they affect translation machinery. The genetic code is one of the most highly conserved characteristics of biological systems, but it does change very rarely. In cases where the code has changed, it differs only slightly from the “Universal Genetic Code”. It is clear that the last ancestor of all extant life used this standard code, and that all these variants arose by making small changes to this ancestral code. This non-canonical codes are very rare in eubacteria, unknown in archaebacteria, and relatively abundant in mitochondrial genomes. In eukaryotic nuclear genomes five lineages have been discovered to use non-canonical codes, and in one (ciliates) the code has changed several times independently. We recently discovered one of these five changes in the oxymond Streblomstix strix. In the standard code TAA and TAG are stop signals, but in Streblomastix uses TAA and TAG to encode the amino acid glutamine. Oxymonads are arguably the worst-studied group of protists. They are relatively diverse but are generally uncultivatable and only live in association with animals, Predominantly as symbionts or parasites in the gut of wood eating insects such as termites and roaches. Accordingly, very few molecular data are known from oxymonads, so the first goal of this project is to characterise the distribution of this code in oxymonads. Secondly, this non-canonical code is also found in some ciliates, hexamitid diplomonads, and certain green algae, and we are examining two translational proteins in these groups that are likely affected by the altered code, to determine if systematic changes take place in regions of these proteins that are important in maintaining the fidelity of translation.

Fast, N. M., Kissinger, J. C., Roos, D. S., & Keeling, P. J. 2001. Nuclear-encoded, plastid-targeted genes suggest a single common origin for apicomplexan and dinoflagellate plastids. Mol. Biol. Evol., 18, 418-426.

Fast, N. M. & Keeling, P. J. 2001. Alpha and beta subunits of pyruvate dehydrogenase E1 from the microsporidian Nosema locustae: Mitochondrion-derived carbon metabolism in microsporidia. Mol. Biochem. Parasitol. 117, 201-209.

Keeling, P. J. & Fast, N. M. 2002. Microsporidia: biology and evolution of highly reduced intracellular parasites. Ann. Rev. Microbiol., 56, 93-116.

Archibald, J. M. & Keeling, P. J. 2002. Recycled plastids: a green movement in eukaryotic evolution. Trends Genet., 18. 577-584.

Saldarriaga, J. F., McEwan, M. L., Fast, N. M., Taylor, F. J. R., & Keeling, P. J. 2003. Multiple protein phylogenies show that Oxyrrhis marina and Perkinsus marinus are early branches of the dinoflagellate lineage. Int. J. System. Evol. Microbiol., 53, 355-365.

Keeling, P. J. & Leander, B. S. 2003. Characterisation of a non-canonical genetic code in the oxymonad Streblomastix strix.. J. Mol. Biol., 326, 1337-1349.

Archibald, J. M., Teh, E. M. & Keeling, P. J. 2003. Novel ubiquitin fusion proteins: ribosomal protein P1 and actin. J. Mol. Biol., 328, 771-778.

Leander, B. S. & Keeling, P. J. 2003. Morphostasis in alveolate evolution. Trends Ecol. Evol., in press.

Archibald, J. M., Rogers, M. B., Toop, M., Ishida, K.-I., Keeling, P. J. 2003. Lateral gene transfer and the evolution of plastid-targeted proteins in the secondary-plastid-containing alga, Bigelowiella natans Proc. Natl. Acad. Sci. USA, in press.