Biomedical subjects
Publications and source records attributed to Ross F Waller.
One hundred diatom species have been selected for genome and transcriptome sequencing. The 100 Diatom Genomes Project aims to provide a scalable framework for understanding diatom biodiversity, ecology and evolution, and for investigating their use in biotechnology.
The mitochondrial gene for COXII is typically encoded in the organelle genome, however in some members of two unrelated groups, Apicomplexa and Chlorophyceae, cox2 is split into two genes, and both are encoded in the nucleus. Rare genomic changes (RGCs) have acquired popularity as phylogenetic markers, and accordingly this rearrangement of cox2 has been used to infer a possible source of the apicomplexan plastid, the apicoplast, a topic that continues to attract much debate. Accurate interpretation of RGCs, however, is critically dependent on appropriate sampling of the character state of interest amongst relevant taxa. Dinoflagellates form the sister taxon to Apicomplexa, and therefore the state of their cox2 is essential to the interpretation of this apparent RGC. Here we present the first complete cox2 data from dinoflagellates, that suggests despite the remarkable similarity of cox2 seen in Alveolates and Chlorophyceae, this gene reorganization arose independently in these two groups, not through lateral transfer as previously suggested.
Phosphatidylinositol mannosides (PIMs) are a major class of glycolipids in all mycobacteria. AcPIM2, a dimannosyl PIM, is both an end product and a precursor for polar PIMs, such as hexamannosyl PIM (AcPIM6) and the major cell wall lipoglycan, lipoarabinomannan (LAM). The mannosyltransferases that convert AcPIM2 to AcPIM6 or LAM are dependent on polyprenol-phosphate-mannose (PPM), but have not yet been characterized. Here, we identified a gene, termed pimE that is present in all mycobacteria, and is required for AcPIM6 biosynthesis. PimE was initially identified based on homology with eukaryotic PIG-M mannosyltransferases. PimE-deleted Mycobacterium smegmatis was defective in AcPIM6 synthesis, and accumulated the tetramannosyl PIM, AcPIM4. Loss of PimE had no affect on cell growth or viability, or the biosynthesis of other intracellular and cell wall glycans. However, changes in cell wall hydrophobicity and plasma membrane organization were detected, suggesting a role for AcPIM6 in the structural integrity of the cell wall and plasma membrane. These defects were corrected by ectopic expression of the pimE gene. Metabolic pulse-chase radiolabeling and cell-free PIM biosynthesis assays indicated that PimE catalyzes the alpha1,2-mannosyl transfer for the AcPIM5 synthesis. Mutation of an Asp residue in PimE that is conserved in and required for the activity of human PIG-M resulted in loss of PIM-biosynthetic activity, indicating that PimE is the catalytic component. Finally, PimE was localized to a distinct membrane fraction enriched in AcPIM4-6 biosynthesis. Taken together, PimE represents the first PPM-dependent mannosyl-transferase shown to be involved in PIM biosynthesis, where it mediates the fifth mannose transfer.
The number of cases of lateral or horizontal gene transfer in eukaryotic genomes is growing steadily, but in most cases, neither the donor nor the recipient is known, and the biological implications of the transfer are not clear. We describe a relatively well-defined case of transfer from a cyanobacterial source to an ancestor of dinoflagellates that diverged before Oxyrrhis but after Perkinsus. This case is also exceptional in that 2 adjacent genes, a paralogue of the shikimate biosynthetic enzyme AroB and an O-methyltransferase (OMT) were transferred together and formed a fusion protein that was subsequently targeted to the dinoflagellate plastid. Moreover, this fusion subsequently reverted to 2 individual genes in the genus Karlodinium, but both proteins maintained plastid localization with the OMT moiety acquiring its own plastid-targeting peptide. The presence of shikimate biosynthetic enzymes in the plastid is not unprecedented as this is a plastid-based pathway in many eukaryotes, but this species of OMT has not been associated with the plastid previously. It appears that the OMT activity was drawn into the plastid simply by virtue of its attachment to the AroB paralogue resulting from their cotransfer and once in the plastid performed some essential function so that it remained plastid targeted after it separated from AroB. Gene fusion events are considered rare and likely stable, and such an event has recently been used to argue for a root of the eukaryotic tree. Our data, however, show that exact reversals of fusion events do take place, and hence gene fusion data are difficult to interpret without knowledge of the phylogeny of the organisms--therefore their use as phylogenetic markers must be considered carefully.
In fungi and animals the translocase in the outer mitochondrial membrane (TOM complex) consists of multiple components including the receptor subunit Tom70. Genome sequence analyses suggest no Tom70 receptor subunit exists in plants or protozoans, raising questions about its ancestry, function and the importance of its activity. Here we characterise the relationships within the Tom70 family of proteins. We find that in both fungi and animals, a conserved domain structure exists within the Tom70 family, with a transmembrane segment followed by 11 tetratricopeptide repeat motifs organised in three distinct domains. The C-terminal domain of Tom70 is highly conserved, and crucial for the import of hydrophobic substrate proteins, including those with and those without N-terminal presequences. Tom70 likely arose after fungi and animals diverged from other eukaryote lineages including plants, and subsequent gene duplication gave rise to a paralogue specific to the Saccharomyces group of yeasts. In animals and in fungi, Tom70 plays a fundamental role in the import of precursor proteins, by assisting relatively hydrophobic regions of substrate proteins into the translocation channel in the outer mitochondrial membrane. Proteins that function equivalently to Tom70 may have arisen independently in plants and protists.
The origin and subsequent spread of plastids by endosymbiosis had a major environmental impact and altered the course of a great proportion of eukaryotic biodiversity. The ancestor of dinoflagellates contained a secondary plastid that was acquired in an ancient endosymbiotic event, where a eukaryotic cell engulfed a red alga. This is known as secondary endosymbiosis and has happened several times in eukaryotic evolution. Certain dinoflagellates, however, are unique in having replaced this secondary plastid in an additional (tertiary) round of endosymbiosis. Most plastid proteins are encoded in the nucleus of the host and are targeted to the organelle. When secondary or tertiary endosymbiosis takes place, it is thought that these genes move from nucleus to nucleus, so the plastid retains the same proteome. We have conducted large-scale expressed sequence tag (EST) surveys from Karlodinium micrum, a dinoflagellate with a tertiary haptophyte-derived plastid, and two haptophytes, Isochrysis galbana and Pavlova lutheri. We have identified all plastid-targeted proteins, analysed the phylogenetic origin of each protein, and compared their plastid-targeting transit peptides. Many plastid-targeted genes in the Karlodinium nucleus are indeed of haptophyte origin, but some genes were also retained from the original plastid (showing the two plastids likely co-existed in the same cell), in other cases multiple isoforms of different origins exist. We analysed plastid-targeting sequences and found the transit peptides in K.micrum are different from those found in either dinoflagellates or haptophytes, pointing to a plastid with an evolutionarily chimeric proteome, and a massive remodelling of protein trafficking during plastid replacement.
The evolutionary history and relationship between plastids of dinoflagellate algae and apicomplexan parasites have been controversial both because the organelles are unusual and because their genomes contain few comparable genes. However, most plastid proteins are encoded in the host nucleus and targeted to the organelle, and several of these genes have proved to have interesting and informative evolutionary histories. We have used expressed sequence tag (EST) sequencing to generate gene sequence data from the nuclear genome of the dinoflagellate Heterocapsa triquetra and inferred phylogenies for the complete set of identified plastid-targeted proteins. Overall, dinoflagellate plastid proteins are most consistently related to homologues from the red algal plastid lineage (not green) and, in many of the most robust cases, they branch with other chromalveolate algae. In resolved phylogenies where apicomplexan data are available, dinoflagellates and apicomplexans are related. We also identified two cases of apparent lateral, or horizontal, gene transfer, one from the green plastid lineage and one from a bacterial lineage unrelated to plastids or cyanobacteria.
The ciliate Tetrahymena thermophila is a model organism for molecular and cellular biology. Like other ciliates, this species has separate germline and soma functions that are embodied by distinct nuclei within a single cell. The germline-like micronucleus (MIC) has its genome held in reserve for sexual reproduction. The soma-like macronucleus (MAC), which possesses a genome processed from that of the MIC, is the center of gene expression and does not directly contribute DNA to sexual progeny. We report here the shotgun sequencing, assembly, and analysis of the MAC genome of T. thermophila, which is approximately 104 Mb in length and composed of approximately 225 chromosomes. Overall, the gene set is robust, with more than 27,000 predicted protein-coding genes, 15,000 of which have strong matches to genes in other organisms. The functional diversity encoded by these genes is substantial and reflects the complexity of processes required for a free-living, predatory, single-celled organism. This is highlighted by the abundance of lineage-specific duplications of genes with predicted roles in sensing and responding to environmental conditions (e.g., kinases), using diverse resources (e.g., proteases and transporters), and generating structural complexity (e.g., kinesins and dyneins). In contrast to the other lineages of alveolates (apicomplexans and dinoflagellates), no compelling evidence could be found for plastid-derived genes in the genome. UGA, the only T. thermophila stop codon, is used in some genes to encode selenocysteine, thus making this organism the first known with the potential to translate all 64 codons in nuclear genes into amino acids. We present genomic evidence supporting the hypothesis that the excision of DNA from the MIC to generate the MAC specifically targets foreign DNA as a form of genome self-defense. The combination of the genome sequence, the functional diversity encoded therein, and the presence of some pathways missing from other model organisms makes T. thermophila an ideal model for functional genomic studies to address biological, biomedical, and biotechnological questions of fundamental importance.
Protein trafficking pathways to plastids are directed by N-terminal targeting peptides. In plants this consists of a relatively simple transit peptide, while in organisms with secondary plastids (which reside within the endomembrane system) a signal peptide is appended to the transit peptide. Despite amino acid compositional differences between organisms, often due to nucleotide biases, the features of plastid targeting sequences are generally consistent within species. Dinoflagellate algae deviate from this trend. We have conducted an expressed sequence tag (EST) survey of the peridinin-plastid containing dinoflagellate Heterocapsa triquetra to identify and characterize numerous targeting presequences of plastid proteins encoded in the nucleus. Consistent with targeting systems present in other secondary plastid-containing organisms, these all possess a canonical signal peptide at their N termini, however two major classes of transit peptides occur. Both classes possess a common N-terminal portion of the transit peptide, but one class of transit peptides contains a hydrophobic domain that has been reported to act as a stop-transfer membrane anchor, temporarily arresting protein insertion into the endoplasmic reticulum. A second class of transit peptide lacks this feature. These two classes are represented approximately equally, and for any given protein the class is conserved across all dinoflagellate taxa surveyed to date. This dichotomy suggests that two mechanisms, perhaps even trafficking routes, may direct proteins to dinoflagellate plastids. A four-residue phenylalanine-based motif is also a consistent feature of H. triquetra transit peptides, which is an ancient feature predating red algae and galucophytes that was lost in green plastids.
The plasma membrane of Mycobacterium sp. is the site of synthesis of several distinct classes of lipids that are either retained in the membrane or exported to the overlying cell envelope. Here, we provide evidence that enzymes involved in the biosynthesis of two major lipid classes, the phosphatidylinositol mannosides (PIMs) and aminophospholipids, are compartmentalized within the plasma membrane. Enzymes involved in the synthesis of early PIM intermediates were localized to a membrane subdomain termed PMf, that was clearly resolved from the cell wall by isopyknic density centrifugation and amplified in rapidly dividing Mycobacterium smegmatis. In contrast, the major pool of apolar PIMs and enzymes involved in polar PIM biosynthesis were localized to a denser fraction that contained both plasma membrane and cell wall markers (PM-CW). Based on the resistance of the PIMs to solvent extraction in live but not lysed cells, we propose that polar PIM biosynthesis occurs in the plasma membrane rather than the cell wall component of the PM-CW. Enzymes involved in phosphatidylethanolamine biosynthesis also displayed a highly polarized distribution between the PMf and PM-CW fractions. The PMf was greatly reduced in non-dividing cells, concomitant with a reduction in the synthesis and steady-state levels of PIMs and amino-phospholipids and the redistribution of PMf marker enzymes to non-PM-CW fractions. The formation of the PMf and recruitment of enzymes to this domain may thus play a role in regulating growth-specific changes in the biosynthesis of membrane and cell wall lipids.
Tom20 is the master receptor for protein import into mitochondria. Analysis of motifs present in Tom20 sequences from fungi and animals found several highly conserved regions, including features of the transmembrane segment, the ligand-binding domain and functionally important flexible segments at the N terminus and the C terminus of the protein. Hidden Markov model searches of genome sequence data revealed novel isoforms of Tom20 in vertebrate and invertebrate animals. A three-dimensional comparative model of the novel type I Tom20, based on the structurally characterized type II isoform, shows important differences in the amino acid residues lining the ligand-binding groove, where the type I protein from animals is more similar to the fungal form of Tom20. Given that the two receptor types from mouse interact with the same set of precursor protein substrates, comparative analysis of the substrate-binding site provides unique insight into the mechanism of substrate recognition. No Tom20-related protein was found in genome sequence data from plants or protozoans, suggesting the receptor Tom20 evolved after the split of animals and fungi from the main lineage of eukaryotes.
The apicoplast is a plastid organelle, homologous to chloroplasts of plants, that is found in apicomplexan parasites such as the causative agents of Malaria Plasmodium spp. It occurs throughout the Apicomplexa and is an ancient feature of this group acquired by the process of endosymbiosis. Like plant chloroplasts, apicoplasts are semi-autonomous with their own genome and expression machinery. In addition, apicoplasts import numerous proteins encoded by nuclear genes. These nuclear genes largely derive from the endosymbiont through a process of intracellular gene relocation. The exact role of a plastid in parasites is uncertain but early clues indicate synthesis of lipids, heme and isoprenoids as possibilities. The various metabolic processes of the apicoplast are potentially excellent targets for drug therapy.
The surface expression of 9-O-acetylated sialic acid (9-OAcSA) is elevated on hematopoietic cells and erythrocytes of visceral leishmaniasis (VL) patients. In this study, we show that VL patients contain elevated levels of IgM antibodies directed against 9-O-acetylated sialoglycoconjugates (9-OAcSG). These antibodies were affinity purified with bovine submaxillary protein as the affinity matrix containing the terminal epitope, 9-OAcSAalpha2-6GalNAc. They also bound to 9-OAcSGs on hematopoietic cells of patients with VL and to epitopes in the cytosol of Leishmania donovani promastigotes. A novel enzyme-linked immunosorbent assay was employed that showed 4-fold higher anti-OAcSG titers in VL patients (n=38), mean +/- S.E.M. being 0.83 +/- 0.09 vs. 0.21 +/- 0.04 detected in normal donors (n=20) and patients with cross-reactive diseases such as malaria (n=4) or tuberculosis (n=4). Assay specificity and sensitivity was 100% and 92%, respectively, whereas positive and negative predictive values were 100% and 90%, respectively. Significantly, anti-OAcSG titers declined 30 days after completion of anti-leishmanial treatment, indicating that monitoring of anti-9-OAcSGs may be a valuable alternative toward increasing the efficiency of diagnosis and follow-up of VL.
Trypanosomatid parasites are disease agents with an extraordinarily broad host range including humans, livestock and plants. Recent work has revealed that trypanosomatids harbour numerous genes sharing apparent common ancestry with plants and/or bacteria. Although there is no evidence of a plastid (chloroplast-like organelle) in trypanosomatids, the presence of such genes suggests lateral gene transfer from some photosynthetic organism(s) during trypanosomatid evolution. Remarkably, many products of these horizontally acquired genes now function in the glycosome, a highly modified peroxisome unique to trypanosomatids and their near relatives.
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Upregulation of the multidrug resistance protein 1 (LeMDR1) in the protozoan parasite, Leishmania enriettii, confers resistance to hydrophobic drugs such as vinblastine, but increases the sensitivity of these parasites to the mitochondrial drug, rhodamine 123. In order to investigate the mechanism of action of LeMDR1, the subcellular localization of green fluorescent protein (GFP)-tagged versions of LeMDR1 and the fate of the traceable-fluorescent LeMDR1 substrate calcein AM were examined in both Leishmania mexicana and L. enriettii LeMDR1 -/- and overexpressing cell lines. The LeMDR1-GFP chimera was localized by fluorescence microscopy to a number of secretory and endocytic compartments, including the Golgi apparatus, endoplasmic reticulum (ER) and a multivesicular tubule (MVT)-lysosome. Pulse-chase labelling experiments with calcein AM suggested that the Golgi and ER pools, but not the MVT-lysosome pool, of LeMDR1 were active in pumping calcein AM out of the cell. Cells labelled with calcein AM under conditions that slow vesicular transport (low temperature and stationary growth) inhibited export and resulted in the accumulation of fluorescent calcein in both the Golgi and the mitochondria. We propose that LeMDR1 substrates are pumped into secretory compartments and exported from the parasite by exocytosis. Accumulation of MDR substrates in the ER can result in alternative transport to the mitochondrion, explaining the reciprocal sensitivity of drug-resistant Leishmania to vinblastine and rhodamine 123.
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Mycobacteria are the causative agents of tuberculosis and several other significant diseases in humans. All species of mycobacteria synthesize abundant cell-wall mannolipids (phosphatidylinositol mannosides, lipoarabinomannan), a cytoplasmic methylmannose polysaccharide and O-mannosylated glycoproteins. To investigate whether these molecules are essential for mycobacterial growth, we have generated a Mycobacterium smegmatis mannose auxotroph by targeted deletion of the gene encoding phosphomannose isomerase (PMI). The PMI deletion mutant displayed a mild hyperseptation phenotype, but grew normally in media containing an exogenous source of mannose. When this mutant was suspended in media without mannose, ongoing synthesis of both the mannolipids and methylmannose polysaccharides was halted and the hyperseptation phenotype became more pronounced. These changes preceded a dramatic loss of viability after 10 h in mannose-free media. Mannose starvation did not lead to detectable changes in cell-wall ultrastructure or permeability to hydrophobic drugs, or to changes in the rate of biosynthesis of other plasma-membrane or wall-associated phospholipids. These results show that mannose metabolism is required for growth of M. smegmatis and that one or more mannose-containing molecules may play a role in regulating septation and cell division in these bacteria.