65 research outputs found
A broad distribution of the alternative oxidase in microsporidian parasites
Microsporidia are a group of obligate intracellular parasitic eukaryotes that were considered to be amitochondriate until the recent discovery of highly reduced mitochondrial organelles called mitosomes. Analysis of the complete genome of Encephalitozoon cuniculi revealed a highly reduced set of proteins in the organelle, mostly related to the assembly of ironsulphur clusters. Oxidative phosphorylation and the Krebs cycle proteins were absent, in keeping with the notion that the microsporidia and their mitosomes are anaerobic, as is the case for other mitosome bearing eukaryotes, such as Giardia. Here we provide evidence opening the possibility that mitosomes in a number of microsporidian lineages are not completely anaerobic. Specifically, we have identified and characterized a gene encoding the alternative oxidase (AOX), a typically mitochondrial terminal oxidase in eukaryotes, in the genomes of several distantly related microsporidian species, even though this gene is absent from the complete genome of E. cuniculi. In order to confirm that these genes encode functional proteins, AOX genes from both A. locustae and T. hominis were over-expressed in E. coli and AOX activity measured spectrophotometrically using ubiquinol-1 (UQ-1) as substrate. Both A. locustae and T. hominis AOX proteins reduced UQ-1 in a cyanide and antimycin-resistant manner that was sensitive to ascofuranone, a potent inhibitor of the trypanosomal AOX. The physiological role of AOX microsporidia may be to reoxidise reducing equivalents produced by glycolysis, in a manner comparable to that observed in trypanosome
Distinct Gene Number-Genome Size Relationships for Eukaryotes and Non-Eukaryotes: Gene Content Estimation for Dinoflagellate Genomes
The ability to predict gene content is highly desirable for characterization of not-yet sequenced genomes like those of dinoflagellates. Using data from completely sequenced and annotated genomes from phylogenetically diverse lineages, we investigated the relationship between gene content and genome size using regression analyses. Distinct relationships between log10-transformed protein-coding gene number (Yβ²) versus log10-transformed genome size (Xβ², genome size in kbp) were found for eukaryotes and non-eukaryotes. Eukaryotes best fit a logarithmic model, Yβ²β=βln(-46.200+22.678Xβ², whereas non-eukaryotes a linear model, Yβ²β=β0.045+0.977Xβ², both with high significance (p<0.001, R2>0.91). Total gene number shows similar trends in both groups to their respective protein coding regressions. The distinct correlations reflect lower and decreasing gene-coding percentages as genome size increases in eukaryotes (82%β1%) compared to higher and relatively stable percentages in prokaryotes and viruses (97%β47%). The eukaryotic regression models project that the smallest dinoflagellate genome (3Γ106 kbp) contains 38,188 protein-coding (40,086 total) genes and the largest (245Γ106 kbp) 87,688 protein-coding (92,013 total) genes, corresponding to 1.8% and 0.05% gene-coding percentages. These estimates do not likely represent extraordinarily high functional diversity of the encoded proteome but rather highly redundant genomes as evidenced by high gene copy numbers documented for various dinoflagellate species
Genomics and transcriptomics yields a system-level view of the biology of the pathogen Naegleria fowleri
Background
The opportunistic pathogen Naegleria fowleri establishes infection in the human brain, killing almost invariably within 2 weeks. The amoeba performs piece-meal ingestion, or trogocytosis, of brain material causing direct tissue damage and massive inflammation. The cellular basis distinguishing N. fowleri from other Naegleria species, which are all non-pathogenic, is not known. Yet, with the geographic range of N. fowleri advancing, potentially due to climate change, understanding how this pathogen invades and kills is both important and timely.
Results
Here, we report an -omics approach to understanding N. fowleri biology and infection at the system level. We sequenced two new strains of N. fowleri and performed a transcriptomic analysis of low- versus high-pathogenicity N. fowleri cultured in a mouse infection model. Comparative analysis provides an in-depth assessment of encoded protein complement between strains, finding high conservation. Molecular evolutionary analyses of multiple diverse cellular systems demonstrate that the N. fowleri genome encodes a similarly complete cellular repertoire to that found in free-living N. gruberi. From transcriptomics, neither stress responses nor traits conferred from lateral gene transfer are suggested as critical for pathogenicity. By contrast, cellular systems such as proteases, lysosomal machinery, and motility, together with metabolic reprogramming and novel N. fowleri proteins, are all implicated in facilitating pathogenicity within the host. Upregulation in mouse-passaged N. fowleri of genes associated with glutamate metabolism and ammonia transport suggests adaptation to available carbon sources in the central nervous system.
Conclusions
In-depth analysis of Naegleria genomes and transcriptomes provides a model of cellular systems involved in opportunistic pathogenicity, uncovering new angles to understanding the biology of a rare but highly fatal pathogen.publishedVersio
Function and Regulation of Vibrio campbellii Proteorhodopsin: Acquired Phototrophy in a Classical Organoheterotroph
Proteorhodopsins (PRs) are retinal-binding photoproteins that mediate light-driven proton translocation across prokaryotic cell membranes. Despite their abundance, wide distribution and contribution to the bioenergy budget of the marine photic zone, an understanding of PR function and physiological significance in situ has been hampered as the vast majority of PRs studied to date are from unculturable bacteria or culturable species that lack the tools for genetic manipulation. In this study, we describe the presence and function of a horizontally acquired PR and retinal biosynthesis gene cluster in the culturable and genetically tractable bioluminescent marine bacterium Vibrio campbellii. Pigmentation analysis, absorption spectroscopy and photoinduction assays using a heterologous over-expression system established the V. campbellii PR as a functional green light absorbing proton pump. In situ analyses comparing PR expression and function in wild type (WT) V. campbellii with an isogenic ΞpR deletion mutant revealed a marked absence of PR membrane localization, pigmentation and light-induced proton pumping in the ΞpR mutant. Comparative photoinduction assays demonstrated the distinct upregulation of pR expression in the presence of light and PR-mediated photophosphorylation in WT cells that resulted in the enhancement of cellular survival during respiratory stress. In addition, we demonstrate that the master regulator of adaptive stress response and stationary phase, RpoS1, positively regulates pR expression and PR holoprotein pigmentation. Taken together, the results demonstrate facultative phototrophy in a classical marine organoheterotrophic Vibrio species and provide a salient example of how this organism has exploited lateral gene transfer to further its adaptation to the photic zone
Evolutionary Convergence on Highly-Conserved 3β² Intron Structures in Intron-Poor Eukaryotes and Insights into the Ancestral Eukaryotic Genome
The presence of spliceosomal introns in eukaryotes raises a range of questions about genomic evolution. Along with the fundamental mysteries of introns' initial proliferation and persistence, the evolutionary forces acting on intron sequences remain largely mysterious. Intron number varies across species from a few introns per genome to several introns per gene, and the elements of intron sequences directly implicated in splicing vary from degenerate to strict consensus motifs. We report a 50-species comparative genomic study of intron sequences across most eukaryotic groups. We find two broad and striking patterns. First, we find that some highly intron-poor lineages have undergone evolutionary convergence to strong 3β² consensus intron structures. This finding holds for both branch point sequence and distance between the branch point and the 3β² splice site. Interestingly, this difference appears to exist within the genomes of green alga of the genus Ostreococcus, which exhibit highly constrained intron sequences through most of the intron-poor genome, but not in one much more intron-dense genomic region. Second, we find evidence that ancestral genomes contained highly variable branch point sequences, similar to more complex modern intron-rich eukaryotic lineages. In addition, ancestral structures are likely to have included polyT tails similar to those in metazoans and plants, which we found in a variety of protist lineages. Intriguingly, intron structure evolution appears to be quite different across lineages experiencing different types of genome reduction: whereas lineages with very few introns tend towards highly regular intronic sequences, lineages with very short introns tend towards highly degenerate sequences. Together, these results attest to the complex nature of ancestral eukaryotic splicing, the qualitatively different evolutionary forces acting on intron structures across modern lineages, and the impressive evolutionary malleability of eukaryotic gene structures
The Marine Microbial Eukaryote Transcriptome Sequencing Project (MMETSP): illuminating the functional diversity of eukaryotic life in the oceans through transcriptome sequencing
International audienceCurrent sampling of genomic sequence data from eukaryotes is relatively poor, biased, and inadequate to address important questions about their biology, evolution, and ecology; this Community Page describes a resource of 700 transcriptomes from marine microbial eukaryotes to help understand their role in the world's oceans
The Marine Microbial Eukaryote Transcriptome Sequencing Project (MMETSP): illuminating the functional diversity of eukaryotic life in the oceans through transcriptome sequencing.
Microbial ecology is plagued by problems
of an abstract nature. Cell sizes are so
small and population sizes so large that
both are virtually incomprehensible. Niches
are so far from our everyday experience
as to make their very definition elusive.
Organisms that may be abundant and
critical to our survival are little understood,
seldom described and/or cultured,
and sometimes yet to be even seen. One
way to confront these problems is to use
data of an even more abstract nature:
molecular sequence data. Massive environmental
nucleic acid sequencing, such
as metagenomics or metatranscriptomics,
promises functional analysis of microbial
communities as a whole, without prior
knowledge of which organisms are in the
environment or exactly how they are
interacting. But sequence-based ecological
studies nearly always use a comparative
approach, and that requires relevant
reference sequences, which are an extremely
limited resource when it comes to
microbial eukaryotes.
In practice, this means sequence databases
need to be populated with enormous
quantities of data for which we have
some certainties about the source. Most
important is the taxonomic identity of
the organism from which a sequence is
derived and as much functional identification
of the encoded proteins as possible. In
an ideal world, such information would be
available as a large set of complete, well curated,
and annotated genomes for all the
major organisms from the environment
in question. Reality substantially diverges
from this ideal, but at least for bacterial
molecular ecology, there is a database
consisting of thousands of complete genomes
from a wide range of taxa,
supplemented by a phylogeny-driven approach
to diversifying genomics [2]. For
eukaryotes, the number of available genomes
is far, far fewer, and we have relied
much more heavily on random growth of
sequence databases, raising the
question as to whether this is fit for
purpose
Inter-Species Complementation of the Translocon Beta Subunit Requires Only Its Transmembrane Domain
In eukaryotes, proteins enter the secretory pathway through the translocon pore of the endoplasmic reticulum. This protein translocation channel is composed of three major subunits, called Sec61Ξ±, Ξ² and Ξ³ in mammals. Unlike the other subunits, the Ξ² subunit is dispensable for translocation and cell viability in all organisms studied. Intriguingly, the knockout of the Sec61Ξ² encoding genes results in different phenotypes in different species. Nevertheless, the Ξ² subunit shows a high level of sequence homology across species, suggesting the conservation of a biological function that remains ill-defined. To address its cellular roles, we characterized the homolog of Sec61Ξ² in the fission yeast Schizosaccharomyces pombe (Sbh1p). Here, we show that the knockout of sbh1+ results in severe cold sensitivity, increased sensitivity to cell-wall stress, and reduced protein secretion at 23Β°C. Sec61Ξ² homologs from Saccharomyces cerevisiae and human complement the knockout of sbh1+ in S. pombe. As in S. cerevisiae, the transmembrane domain (TMD) of S. pombe Sec61Ξ² is sufficient to complement the phenotypes resulting from the knockout of the entire encoding gene. Remarkably, the TMD of Sec61Ξ² from S. cerevisiae and human also complement the gene knockouts in both yeasts. Together, these observations indicate that the TMD of Sec61Ξ² exerts a cellular function that is conserved across species
Inter-Species Complementation of the Translocon Beta Subunit Requires Only Its Transmembrane Domain
In eukaryotes, proteins enter the secretory pathway through the translocon pore of the endoplasmic reticulum. This protein translocation channel is composed of three major subunits, called Sec61Ξ±, Ξ² and Ξ³ in mammals. Unlike the other subunits, the Ξ² subunit is dispensable for translocation and cell viability in all organisms studied. Intriguingly, the knockout of the Sec61Ξ² encoding genes results in different phenotypes in different species. Nevertheless, the Ξ² subunit shows a high level of sequence homology across species, suggesting the conservation of a biological function that remains ill-defined. To address its cellular roles, we characterized the homolog of Sec61Ξ² in the fission yeast Schizosaccharomyces pombe (Sbh1p). Here, we show that the knockout of sbh1+ results in severe cold sensitivity, increased sensitivity to cell-wall stress, and reduced protein secretion at 23Β°C. Sec61Ξ² homologs from Saccharomyces cerevisiae and human complement the knockout of sbh1+ in S. pombe. As in S. cerevisiae, the transmembrane domain (TMD) of S. pombe Sec61Ξ² is sufficient to complement the phenotypes resulting from the knockout of the entire encoding gene. Remarkably, the TMD of Sec61Ξ² from S. cerevisiae and human also complement the gene knockouts in both yeasts. Together, these observations indicate that the TMD of Sec61Ξ² exerts a cellular function that is conserved across species
Frequency of intron loss correlates with processed pseudogene abundance: a novel strategy to test the reverse transcriptase model of intron loss
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