Chromosome-scale assemblies of Acanthamoeba castellanii genomes provide insights into Legionella pneumophila infection-related chromatin reorganization.

The unicellular amoeba Acanthamoeba castellanii is ubiquitous in aquatic environments, where it preys on bacteria. The organism also hosts bacterial endosymbionts, some of which are parasitic, including human pathogens such as Chlamydia and Legionella spp. Here we report complete, high-quality genome sequences for two extensively studied A. castellanii strains, Neff and C3. Combining long- and short-read data with Hi-C, we generated near chromosome-level assemblies for both strains with 90% of the genome contained in 29 scaffolds for the Neff strain and 31 for the C3 strain. Comparative genomics revealed strain-specific functional enrichment, most notably genes related to signal transduction in the C3 strain and to viral replication in Neff. Furthermore, we characterized the spatial organization of the A. castellanii genome and showed that it is reorganized during infection by Legionella pneumophila Infection-dependent chromatin loops were found to be enriched in genes for signal transduction and phosphorylation processes. In genomic regions where chromatin organization changed during Legionella infection, we found functional enrichment for genes associated with metabolism, organelle assembly, and cytoskeleton organization. Given Legionella infection is known to alter its host's cell cycle, to exploit the host's organelles, and to modulate the host's metabolism in its favor, these changes in chromatin organization may partly be related to mechanisms of host control during Legionella infection.

Influenza A virus NS1 effector domain is required for PA-X-mediated host shutoff in infected cells.

Many viruses inhibit general host gene expression to limit innate immune responses and gain preferential access to the cellular translational apparatus for their protein synthesis. This process is known as host shutoff. Influenza A viruses (IAVs) encode two host shutoff proteins: nonstructural protein 1 (NS1) and polymerase acidic X (PA-X). NS1 inhibits host nuclear pre-messenger RNA maturation and export, and PA-X is an endoribonuclease that preferentially cleaves host spliced nuclear and cytoplasmic messenger RNAs. Emerging evidence suggests that in circulating human IAVs NS1 and PA-X co-evolve to ensure optimal magnitude of general host shutoff without compromising viral replication that relies on host cell metabolism. However, the functional interplay between PA-X and NS1 remains unexplored. In this study, we sought to determine whether NS1 function has a direct effect on PA-X activity by analyzing host shutoff in A549 cells infected with wild-type or mutant IAVs with NS1 effector domain deletion. This was done using conventional quantitative reverse transcription polymerase chain reaction techniques and direct RNA sequencing using nanopore technology. Our previous research on the molecular mechanisms of PA-X function identified two prominent features of IAV-infected cells: nuclear accumulation of cytoplasmic poly(A) binding protein (PABPC1) and increase in nuclear poly(A) RNA abundance relative to the cytoplasm. Here we demonstrate that NS1 effector domain function augments PA-X host shutoff and is necessary for nuclear PABPC1 accumulation. By contrast, nuclear poly(A) RNA accumulation is not dependent on either NS1 or PA-X-mediated host shutoff and is accompanied by nuclear retention of viral transcripts. Our study demonstrates for the first time that NS1 and PA-X may functionally interact in mediating host shutoff.IMPORTANCERespiratory viruses including the influenza A virus continue to cause annual epidemics with high morbidity and mortality due to the limited effectiveness of vaccines and antiviral drugs. Among the strategies evolved by viruses to evade immune responses is host shutoff-a general blockade of host messenger RNA and protein synthesis. Disabling influenza A virus host shutoff is being explored in live attenuated vaccine development as an attractive strategy for increasing their effectiveness by boosting antiviral responses. Influenza A virus encodes two proteins that function in host shutoff: the nonstructural protein 1 (NS1) and the polymerase acidic X (PA-X). We and others have characterized some of the NS1 and PA-X mechanisms of action and the additive effects that these viral proteins may have in ensuring the blockade of host gene expression. In this work, we examined whether NS1 and PA-X functionally interact and discovered that NS1 is required for PA-X to function effectively. This work significantly advances our understanding of influenza A virus host shutoff and identifies new potential targets for therapeutic interventions against influenza and further informs the development of improved live attenuated vaccines.

Heat stress response in the closest algal relatives of land plants reveals conserved stress signaling circuits.

All land plants (embryophytes) share a common ancestor that likely evolved from a filamentous freshwater alga. Elucidating the transition from algae to embryophytes - and the eventual conquering of Earth's surface - is one of the most fundamental questions in plant evolutionary biology. Here, we investigated one of the organismal properties that might have enabled this transition: resistance to drastic temperature shifts. We explored the effect of heat stress in Mougeotia and Spirogyra, two representatives of Zygnematophyceae - the closest known algal sister lineage to land plants. Heat stress induced pronounced phenotypic alterations in their plastids, and high-performance liquid chromatography-tandem mass spectroscopy-based profiling of 565 transitions for the analysis of main central metabolites revealed significant shifts in 43 compounds. We also analyzed the global differential gene expression responses triggered by heat, generating 92.8 Gbp of sequence data and assembling a combined set of 8905 well-expressed genes. Each organism had its own distinct gene expression profile; less than one-half of their shared genes showed concordant gene expression trends. We nevertheless detected common signature responses to heat such as elevated transcript levels for molecular chaperones, thylakoid components, and - corroborating our metabolomic data - amino acid metabolism. We also uncovered the heat-stress responsiveness of genes for phosphorelay-based signal transduction that links environmental cues, calcium signatures and plastid biology. Our data allow us to infer the molecular heat stress response that the earliest land plants might have used when facing the rapidly shifting temperature conditions of the terrestrial habitat.

Re-examination of two diatom reference genomes using long-read sequencing.

BACKGROUND: The marine diatoms Thalassiosira pseudonana and Phaeodactylum tricornutum are valuable model organisms for exploring the evolution, diversity and ecology of this important algal group. Their reference genomes, published in 2004 and 2008, respectively, were the product of traditional Sanger sequencing. In the case of T. pseudonana, optical restriction site mapping was employed to further clarify and contextualize chromosome-level scaffolds. While both genomes are considered highly accurate and reasonably contiguous, they still contain many unresolved regions and unordered/unlinked scaffolds. RESULTS: We have used Oxford Nanopore Technologies long-read sequencing to update and validate the quality and contiguity of the T. pseudonana and P. tricornutum genomes. Fine-scale assessment of our long-read derived genome assemblies allowed us to resolve previously uncertain genomic regions, further characterize complex structural variation, and re-evaluate the repetitive DNA content of both genomes. We also identified 1862 previously undescribed genes in T. pseudonana. In P. tricornutum, we used transposable element detection software to identify 33 novel copia-type LTR-RT insertions, indicating ongoing activity and rapid expansion of this superfamily as the organism continues to be maintained in culture. Finally, Bionano optical mapping of P. tricornutum chromosomes was combined with long-read sequence data to explore the potential of long-read sequencing and optical mapping for resolving haplotypes. CONCLUSION: Despite its potential to yield highly contiguous scaffolds, long-read sequencing is not a panacea. Even for relatively small nuclear genomes such as those investigated herein, repetitive DNA sequences cause problems for current genome assembly algorithms. Determining whether a long-read derived genomic assembly is 'better' than one produced using traditional sequence data is not straightforward. Our revised reference genomes for P. tricornutum and T. pseudonana nevertheless provide additional insight into the structure and evolution of both genomes, thereby providing a more robust foundation for future diatom research.

RNA-Seq analysis reveals potential regulators of programmed cell death and leaf remodelling in lace plant (Aponogeton madagascariensis).

BACKGROUND: The lace plant (Aponogeton madagascariensis) is an aquatic monocot that develops leaves with uniquely formed perforations through the use of a developmentally regulated process called programmed cell death (PCD). The process of perforation formation in lace plant leaves is subdivided into several developmental stages: pre-perforation, window, perforation formation, perforation expansion and mature. The first three emerging "imperforate leaves" do not form perforations, while all subsequent leaves form perforations via developmentally regulated PCD. PCD is active in cells called "PCD cells" that do not retain the antioxidant anthocyanin in spaces called areoles framed by the leaf veins of window stage leaves. Cells near the veins called "NPCD cells" retain a red pigmentation from anthocyanin and do not undergo PCD. While the cellular changes that occur during PCD are well studied, the gene expression patterns underlying these changes and driving PCD during leaf morphogenesis are mostly unknown. We sought to characterize differentially expressed genes (DEGs) that mediate lace plant leaf remodelling and PCD. This was achieved performing gene expression analysis using transcriptomics and comparing DEGs among different stages of leaf development, and between NPCD and PCD cells isolated by laser capture microdissection. RESULTS: Transcriptomes were sequenced from imperforate, pre-perforation, window, and mature leaf stages, as well as PCD and NPCD cells isolated from window stage leaves. Differential expression analysis of the data revealed distinct gene expression profiles: pre-perforation and window stage leaves were characterized by higher expression of genes involved in anthocyanin biosynthesis, plant proteases, expansins, and autophagy-related genes. Mature and imperforate leaves upregulated genes associated with chlorophyll development, photosynthesis, and negative regulators of PCD. PCD cells were found to have a higher expression of genes involved with ethylene biosynthesis, brassinosteroid biosynthesis, and hydrolase activity whereas NPCD cells possessed higher expression of auxin transport, auxin signalling, aspartyl proteases, cysteine protease, Bag5, and anthocyanin biosynthesis enzymes. CONCLUSIONS: RNA sequencing was used to generate a de novo transcriptome for A. madagascariensis leaves and revealed numerous DEGs potentially involved in PCD and leaf remodelling. The data generated from this investigation will be useful for future experiments on lace plant leaf development and PCD in planta.

Embryophyte stress signaling evolved in the algal progenitors of land plants.

Streptophytes are unique among photosynthetic eukaryotes in having conquered land. As the ancestors of land plants, streptophyte algae are hypothesized to have possessed exaptations to the environmental stressors encountered during the transition to terrestrial life. Many of these stressors, including high irradiance and drought, are linked to plastid biology. We have investigated global gene expression patterns across all six major streptophyte algal lineages, analyzing a total of around 46,000 genes assembled from a little more than 1.64 billion sequence reads from six organisms under three growth conditions. Our results show that streptophyte algae respond to cold and high light stress via expression of hallmark genes used by land plants (embryophytes) during stress-response signaling and downstream responses. Among the strongest differentially regulated genes were those associated with plastid biology. We observed that among streptophyte algae, those most closely related to land plants, especially Zygnema, invest the largest fraction of their transcriptional budget in plastid-targeted proteins and possess an array of land plant-type plastid-nucleus communication genes. Streptophyte algae more closely related to land plants also appear most similar to land plants in their capacity to respond to plastid stressors. Support for this notion comes from the detection of a canonical abscisic acid receptor of the PYRABACTIN RESISTANCE (PYR/PYL/RCAR) family in Zygnema, the first found outside the land plant lineage. We conclude that a fine-tuned response toward terrestrial plastid stressors was among the exaptations that allowed streptophytes to colonize the terrestrial habitat on a global scale.

Extreme genome diversity in the hyper-prevalent parasitic eukaryote Blastocystis.

Blastocystis is the most prevalent eukaryotic microbe colonizing the human gut, infecting approximately 1 billion individuals worldwide. Although Blastocystis has been linked to intestinal disorders, its pathogenicity remains controversial because most carriers are asymptomatic. Here, the genome sequence of Blastocystis subtype (ST) 1 is presented and compared to previously published sequences for ST4 and ST7. Despite a conserved core of genes, there is unexpected diversity between these STs in terms of their genome sizes, guanine-cytosine (GC) content, intron numbers, and gene content. ST1 has 6,544 protein-coding genes, which is several hundred more than reported for ST4 and ST7. The percentage of proteins unique to each ST ranges from 6.2% to 20.5%, greatly exceeding the differences observed within parasite genera. Orthologous proteins also display extreme divergence in amino acid sequence identity between STs (i.e., 59%-61% median identity), on par with observations of the most distantly related species pairs of parasite genera. The STs also display substantial variation in gene family distributions and sizes, especially for protein kinase and protease gene families, which could reflect differences in virulence. It remains to be seen to what extent these inter-ST differences persist at the intra-ST level. A full 26% of genes in ST1 have stop codons that are created on the mRNA level by a novel polyadenylation mechanism found only in Blastocystis. Reconstructions of pathways and organellar systems revealed that ST1 has a relatively complete membrane-trafficking system and a near-complete meiotic toolkit, possibly indicating a sexual cycle. Unlike some intestinal protistan parasites, Blastocystis ST1 has near-complete de novo pyrimidine, purine, and thiamine biosynthesis pathways and is unique amongst studied stramenopiles in being able to metabolize α-glucans rather than ß-glucans. It lacks all genes encoding heme-containing cytochrome P450 proteins. Predictions of the mitochondrion-related organelle (MRO) proteome reveal an expanded repertoire of functions, including lipid, cofactor, and vitamin biosynthesis, as well as proteins that may be involved in regulating mitochondrial morphology and MRO/endoplasmic reticulum (ER) interactions. In sharp contrast, genes for peroxisome-associated functions are absent, suggesting Blastocystis STs lack this organelle. Overall, this study provides an important window into the biology of Blastocystis, showcasing significant differences between STs that can guide future experimental investigations into differences in their virulence and clarifying the roles of these organisms in gut health and disease.

Nuclear genome sequence of the plastid-lacking cryptomonad Goniomonas avonlea provides insights into the evolution of secondary plastids.

BACKGROUND: The evolution of photosynthesis has been a major driver in eukaryotic diversification. Eukaryotes have acquired plastids (chloroplasts) either directly via the engulfment and integration of a photosynthetic cyanobacterium (primary endosymbiosis) or indirectly by engulfing a photosynthetic eukaryote (secondary or tertiary endosymbiosis). The timing and frequency of secondary endosymbiosis during eukaryotic evolution is currently unclear but may be resolved in part by studying cryptomonads, a group of single-celled eukaryotes comprised of both photosynthetic and non-photosynthetic species. While cryptomonads such as Guillardia theta harbor a red algal-derived plastid of secondary endosymbiotic origin, members of the sister group Goniomonadea lack plastids. Here, we present the genome of Goniomonas avonlea-the first for any goniomonad-to address whether Goniomonadea are ancestrally non-photosynthetic or whether they lost a plastid secondarily. RESULTS: We sequenced the nuclear and mitochondrial genomes of Goniomonas avonlea and carried out a comparative analysis of Go. avonlea, Gu. theta, and other cryptomonads. The Go. avonlea genome assembly is ~ 92 Mbp in size, with 33,470 predicted protein-coding genes. Interestingly, some metabolic pathways (e.g., fatty acid biosynthesis) predicted to occur in the plastid and periplastidal compartment of Gu. theta appear to operate in the cytoplasm of Go. avonlea, suggesting that metabolic redundancies were generated during the course of secondary plastid integration. Other cytosolic pathways found in Go. avonlea are not found in Gu. theta, suggesting secondary loss in Gu. theta and other plastid-bearing cryptomonads. Phylogenetic analyses revealed no evidence for algal endosymbiont-derived genes in the Go. avonlea genome. Phylogenomic analyses point to a specific relationship between Cryptista (to which cryptomonads belong) and Archaeplastida. CONCLUSION: We found no convincing genomic or phylogenomic evidence that Go. avonlea evolved from a secondary red algal plastid-bearing ancestor, consistent with goniomonads being ancestrally non-photosynthetic eukaryotes. The Go. avonlea genome sheds light on the physiology of heterotrophic cryptomonads and serves as an important reference point for studying the metabolic "rewiring" that took place during secondary plastid integration in the ancestor of modern-day Cryptophyceae.

Algal genomes reveal evolutionary mosaicism and the fate of nucleomorphs.

Cryptophyte and chlorarachniophyte algae are transitional forms in the widespread secondary endosymbiotic acquisition of photosynthesis by engulfment of eukaryotic algae. Unlike most secondary plastid-bearing algae, miniaturized versions of the endosymbiont nuclei (nucleomorphs) persist in cryptophytes and chlorarachniophytes. To determine why, and to address other fundamental questions about eukaryote-eukaryote endosymbiosis, we sequenced the nuclear genomes of the cryptophyte Guillardia theta and the chlorarachniophyte Bigelowiella natans. Both genomes have >21,000 protein genes and are intron rich, and B. natans exhibits unprecedented alternative splicing for a single-celled organism. Phylogenomic analyses and subcellular targeting predictions reveal extensive genetic and biochemical mosaicism, with both host- and endosymbiont-derived genes servicing the mitochondrion, the host cell cytosol, the plastid and the remnant endosymbiont cytosol of both algae. Mitochondrion-to-nucleus gene transfer still occurs in both organisms but plastid-to-nucleus and nucleomorph-to-nucleus transfers do not, which explains why a small residue of essential genes remains locked in each nucleomorph.

Heme pathway evolution in kinetoplastid protists.

BACKGROUND: Kinetoplastea is a diverse protist lineage composed of several of the most successful parasites on Earth, organisms whose metabolisms have coevolved with those of the organisms they infect. Parasitic kinetoplastids have emerged from free-living, non-pathogenic ancestors on multiple occasions during the evolutionary history of the group. Interestingly, in both parasitic and free-living kinetoplastids, the heme pathway-a core metabolic pathway in a wide range of organisms-is incomplete or entirely absent. Indeed, Kinetoplastea investigated thus far seem to bypass the need for heme biosynthesis by acquiring heme or intermediate metabolites directly from their environment. RESULTS: Here we report the existence of a near-complete heme biosynthetic pathway in Perkinsela spp., kinetoplastids that live as obligate endosymbionts inside amoebozoans belonging to the genus Paramoeba/Neoparamoeba. We also use phylogenetic analysis to infer the evolution of the heme pathway in Kinetoplastea. CONCLUSION: We show that Perkinsela spp. is a deep-branching kinetoplastid lineage, and that lateral gene transfer has played a role in the evolution of heme biosynthesis in Perkinsela spp. and other Kinetoplastea. We also discuss the significance of the presence of seven of eight heme pathway genes in the Perkinsela genome as it relates to its endosymbiotic relationship with Paramoeba.

Nucleomorph and plastid genome sequences of the chlorarachniophyte Lotharella oceanica: convergent reductive evolution and frequent recombination in nucleomorph-bearing algae.

BACKGROUND: Nucleomorphs are residual nuclei derived from eukaryotic endosymbionts in chlorarachniophyte and cryptophyte algae. The endosymbionts that gave rise to nucleomorphs and plastids in these two algal groups were green and red algae, respectively. Despite their independent origin, the chlorarachniophyte and cryptophyte nucleomorph genomes share similar genomic features such as extreme size reduction and a three-chromosome architecture. This suggests that similar reductive evolutionary forces have acted to shape the nucleomorph genomes in the two groups. Thus far, however, only a single chlorarachniophyte nucleomorph and plastid genome has been sequenced, making broad evolutionary inferences within the chlorarachniophytes and between chlorarachniophytes and cryptophytes difficult. We have sequenced the nucleomorph and plastid genomes of the chlorarachniophyte Lotharella oceanica in order to gain insight into nucleomorph and plastid genome diversity and evolution. RESULTS: The L. oceanica nucleomorph genome was found to consist of three linear chromosomes totaling ~610 kilobase pairs (kbp), much larger than the 373 kbp nucleomorph genome of the model chlorarachniophyte Bigelowiella natans. The L. oceanica plastid genome is 71 kbp in size, similar to that of B. natans. Unexpectedly long (~35 kbp) sub-telomeric repeat regions were identified in the L. oceanica nucleomorph genome; internal multi-copy regions were also detected. Gene content analyses revealed that nucleomorph house-keeping genes and spliceosomal intron positions are well conserved between the L. oceanica and B. natans nucleomorph genomes. More broadly, gene retention patterns were found to be similar between nucleomorph genomes in chlorarachniophytes and cryptophytes. Chlorarachniophyte plastid genomes showed near identical protein coding gene complements as well as a high level of synteny. CONCLUSIONS: We have provided insight into the process of nucleomorph genome evolution by elucidating the fine-scale dynamics of sub-telomeric repeat regions. Homologous recombination at the chromosome ends appears to be frequent, serving to expand and contract nucleomorph genome size. The main factor influencing nucleomorph genome size variation between different chlorarachniophyte species appears to be expansion-contraction of these telomere-associated repeats rather than changes in the number of unique protein coding genes. The dynamic nature of chlorarachniophyte nucleomorph genomes lies in stark contrast to their plastid genomes, which appear to be highly stable in terms of gene content and synteny.

Draft genomes of Blastocystis subtypes from human samples of Colombia.

BACKGROUND: Blastocystis is one of the most common eukaryotic microorganisms colonizing the intestines of both humans and animals, but the conditions under which it may be a pathogen are unclear. METHODS: To study the genomic characteristics of circulating subtypes (ST) in Colombia, we established nine xenic cultures from Blastocystis isolated from human fecal samples, we identified 10 different subtypes, since one sample had a mixed infection. Thus, the genomes of the subtypes ST1 (n = 3), ST2 (n = 1), ST3 (n = 2), ST6 (n = 1), ST7 (n = 1), and ST8 (n = 2) were sequenced using Illumina and Oxford Nanopore Technologies (ONT). RESULTS: Analyses of these draft nuclear genomes indicated remarkable diversity in terms of genome size and guanine-cytosine (GC) content among the compared STs. Illumina sequencing-only draft genomes contained 824 to 2077 scaffolds, with total genome size ranging from 12 to 13.2 Mb and N50 values ranging from 10,585 to 29,404 base pairs (bp). The genome of one ST1 isolate was sequenced using ONT. This assembly was more contiguous, with a size of 20 million base pairs (Mb) spread over 116 scaffolds, and an N50 of 248,997 bp. CONCLUSION: This work represents one of the few large-scale comparative genomic analyses of Blastocystis isolates, providing an additional glimpse into its genomic diversity.

TreeTuner: A pipeline for minimizing redundancy and complexity in large phylogenetic datasets.

Various bioinformatics protocols have been developed for trimming the number of operational taxonomic units (OTUs) in phylogenetic datasets, but they typically require significant manual intervention. Here we present TreeTuner, a semiautomated pipeline that allows both coarse and fine-scale tuning of large protein sequence phylogenetic datasets via the minimization of OTU redundancy. TreeTuner facilitates preliminary investigation of such datasets as well as more rigorous downstream analysis of specific subsets of OTUs. For complete details on the use and execution of this protocol, please refer to Maruyama et al. (2013) and Sibbald et al. (2019).

Genomic analysis finds no evidence of canonical eukaryotic DNA processing complexes in a free-living protist.

Cells replicate and segregate their DNA with precision. Previous studies showed that these regulated cell-cycle processes were present in the last eukaryotic common ancestor and that their core molecular parts are conserved across eukaryotes. However, some metamonad parasites have secondarily lost components of the DNA processing and segregation apparatuses. To clarify the evolutionary history of these systems in these unusual eukaryotes, we generated a genome assembly for the free-living metamonad Carpediemonas membranifera and carried out a comparative genomics analysis. Here, we show that parasitic and free-living metamonads harbor an incomplete set of proteins for processing and segregating DNA. Unexpectedly, Carpediemonas species are further streamlined, lacking the origin recognition complex, Cdc6 and most structural kinetochore subunits. Carpediemonas species are thus the first known eukaryotes that appear to lack this suite of conserved complexes, suggesting that they likely rely on yet-to-be-discovered or alternative mechanisms to carry out these fundamental processes.

Nucleomorph genome of Hemiselmis andersenii reveals complete intron loss and compaction as a driver of protein structure and function.

Nucleomorphs are the remnant nuclei of algal endosymbionts that took up residence inside a nonphotosynthetic eukaryotic host. The nucleomorphs of cryptophytes and chlorarachniophytes are derived from red and green algal endosymbionts, respectively, and represent a stunning example of convergent evolution: their genomes have independently been reduced and compacted to <1 megabase pairs (Mbp) in size (the smallest nuclear genomes known) and to a similar three-chromosome architecture. The molecular processes underlying genome reduction and compaction in eukaryotes are largely unknown, as is the impact of reduction/compaction on protein structure and function. Here, we present the complete 0.572-Mbp nucleomorph genome of the cryptophyte Hemiselmis andersenii and show that it is completely devoid of spliceosomal introns and genes for splicing RNAs-a case of complete intron loss in a nuclear genome. Comparison of H. andersenii proteins to those encoded in the slightly smaller (0.551-Mbp) nucleomorph genome of another cryptophyte, Guillardia theta, and to their homologs in the unicellular red alga Cyanidioschyzon merolae reveal that (i) cryptophyte nucleomorph genomes encode proteins that are significantly smaller than those in their free-living algal ancestors, and (ii) the smaller, more compact G. theta nucleomorph genome encodes significantly smaller proteins than that of H. andersenii. These results indicate that genome compaction can eliminate both coding and noncoding DNA and, consequently, drive the evolution of protein structure and function. Nucleomorph proteins have the potential to reveal the minimal functional units required for basic eukaryotic cellular processes.

The genome of Thermosipho africanus TCF52B: lateral genetic connections to the Firmicutes and Archaea.

Lateral gene transfers (LGT) (also called horizontal gene transfers) have been a major force shaping the Thermosipho africanus TCF52B genome, whose sequence we describe here. Firmicutes emerge as the principal LGT partner. Twenty-six percent of phylogenetic trees suggest LGT with this group, while 13% of the open reading frames indicate LGT with Archaea.

Nucleomorph Small RNAs in Cryptophyte and Chlorarachniophyte Algae.

The regulation of gene expression and RNA maturation underlies fundamental processes such as cell homeostasis, development, and stress acclimation. The biogenesis and modification of RNA is tightly controlled by an array of regulatory RNAs and nucleic acid-binding proteins. While the role of small RNAs (sRNAs) in gene expression has been studied in-depth in select model organisms, little is known about sRNA biology across the eukaryotic tree of life. We used deep sequencing to explore the repertoires of sRNAs encoded by the miniaturized, endosymbiotically derived "nucleomorph" genomes of two single-celled algae, the cryptophyte Guillardia theta and the chlorarachniophyte Bigelowiella natans. A total of 32.3 and 35.3 million reads were generated from G. theta and B. natans, respectively. In G. theta, we identified nucleomorph U1, U2, and U4 spliceosomal small nuclear RNAs (snRNAs) as well as 11 C/D box small nucleolar RNAs (snoRNAs), five of which have potential plant and animal homologs. The snoRNAs are predicted to perform 2'-O methylation of rRNA (but not snRNA). In B. natans, we found the previously undetected 5S rRNA as well as six orphan sRNAs. Analysis of chlorarachniophyte snRNAs shed light on the removal of the miniature 18-21 nt introns found in B. natans nucleomorph genes. Neither of the nucleomorph genomes appears to encode RNA pseudouridylation machinery, and U5 snRNA cannot be found in the cryptophyte G. theta. Considering the central roles of U5 snRNA and RNA modifications in other organisms, cytoplasm-to-nucleomorph RNA shuttling in cryptophyte algae is a distinct possibility.

Complete sequence and analysis of the mitochondrial genome of Hemiselmis andersenii CCMP644 (Cryptophyceae).

BACKGROUND: Cryptophytes are an enigmatic group of unicellular eukaryotes with plastids derived by secondary (i.e., eukaryote-eukaryote) endosymbiosis. Cryptophytes are unusual in that they possess four genomes-a host cell-derived nuclear and mitochondrial genome and an endosymbiont-derived plastid and 'nucleomorph' genome. The evolutionary origins of the host and endosymbiont components of cryptophyte algae are at present poorly understood. Thus far, a single complete mitochondrial genome sequence has been determined for the cryptophyte Rhodomonas salina. Here, the second complete mitochondrial genome of the cryptophyte alga Hemiselmis andersenii CCMP644 is presented. RESULTS: The H. andersenii mtDNA is 60,553 bp in size and encodes 30 structural RNAs and 36 protein-coding genes, all located on the same strand. A prominent feature of the genome is the presence of a approximately 20 Kbp long intergenic region comprised of numerous tandem and dispersed repeat units of between 22-336 bp. Adjacent to these repeats are 27 copies of palindromic sequences predicted to form stable DNA stem-loop structures. One such stem-loop is located near a GC-rich and GC-poor region and may have a regulatory function in replication or transcription. The H. andersenii mtDNA shares a number of features in common with the genome of the cryptophyte Rhodomonas salina, including general architecture, gene content, and the presence of a large repeat region. However, the H. andersenii mtDNA is devoid of inverted repeats and introns, which are present in R. salina. Comparative analyses of the suite of tRNAs encoded in the two genomes reveal that the H. andersenii mtDNA has lost or converted its original trnK(uuu) gene and possesses a trnS-derived 'trnK(uuu)', which appears unable to produce a functional tRNA. Mitochondrial protein coding gene phylogenies strongly support a variety of previously established eukaryotic groups, but fail to resolve the relationships among higher-order eukaryotic lineages. CONCLUSION: Comparison of the H. andersenii and R. salina mitochondrial genomes reveals a number of cryptophyte-specific genomic features, most notably the presence of a large repeat-rich intergenic region. However, unlike R. salina, the H. andersenii mtDNA does not possess introns and lacks a Lys-tRNA, which is presumably imported from the cytosol.

The genome of Aeromonas salmonicida subsp. salmonicida A449: insights into the evolution of a fish pathogen.

BACKGROUND: Aeromonas salmonicida subsp. salmonicida is a Gram-negative bacterium that is the causative agent of furunculosis, a bacterial septicaemia of salmonid fish. While other species of Aeromonas are opportunistic pathogens or are found in commensal or symbiotic relationships with animal hosts, A. salmonicida subsp. salmonicida causes disease in healthy fish. The genome sequence of A. salmonicida was determined to provide a better understanding of the virulence factors used by this pathogen to infect fish. RESULTS: The nucleotide sequences of the A. salmonicida subsp. salmonicida A449 chromosome and two large plasmids are characterized. The chromosome is 4,702,402 bp and encodes 4388 genes, while the two large plasmids are 166,749 and 155,098 bp with 178 and 164 genes, respectively. Notable features are a large inversion in the chromosome and, in one of the large plasmids, the presence of a Tn21 composite transposon containing mercury resistance genes and an In2 integron encoding genes for resistance to streptomycin/spectinomycin, quaternary ammonia compounds, sulphonamides and chloramphenicol. A large number of genes encoding potential virulence factors were identified; however, many appear to be pseudogenes since they contain insertion sequences, frameshifts or in-frame stop codons. A total of 170 pseudogenes and 88 insertion sequences (of ten different types) are found in the A. salmonicida genome. Comparison with the A. hydrophila ATCC 7966T genome reveals multiple large inversions in the chromosome as well as an approximately 9% difference in gene content indicating instances of single gene or operon loss or gain.A limited number of the pseudogenes found in A. salmonicida A449 were investigated in other Aeromonas strains and species. While nearly all the pseudogenes tested are present in A. salmonicida subsp. salmonicida strains, only about 25% were found in other A. salmonicida subspecies and none were detected in other Aeromonas species. CONCLUSION: Relative to the A. hydrophila ATCC 7966T genome, the A. salmonicida subsp. salmonicida genome has acquired multiple mobile genetic elements, undergone substantial rearrangement and developed a significant number of pseudogenes. These changes appear to be a consequence of adaptation to a specific host, salmonid fish, and provide insights into the mechanisms used by the bacterium for infection and avoidance of host defence systems.

Anticancer effects of Annona glabra plant extracts in human leukemia cell lines.

Annona glabra (pond apple), a tropical tree growing wild in the Americas and Asia, is used in traditional medicine against several human ailments, including cancer. To validate the ethnopharmacological claims against cancer, the anticancer effects of alcoholic extracts prepared from pond apple leaves, pulp and seed, were investigated in human leukemia cell lines. The alcoholic extracts were not cytotoxic to normal human lymphocytes. However, extracts were highly cytotoxic to drug sensitive (CEM) and multidrug-resistant leukemia (CEM/VLB) cell lines. The seed extract was more potent than leaf and pulp extracts, and the cytotoxicity values were significantly lower than that for adriamycin. The seed extract caused a concentration-dependent increase in the percentage of the sub G0/G1, as well as G0/G1 cell population, contributing to the cytotoxicity. The sub G0/G1 population increased from 2.2 to 7.0% in CEM and from 1.0 to 10.7% in CEM/VLB cell lines, when the cells were treated with 0-10 Bg/ml seed extract. Treatment of CEM and CEM/VLB cells with seed extract induced apoptosis and necrosis in both sensitive and resistant leukemia cells in a concentration-dependent manner. The seed extract at 2 and 5 Bg/ml enhanced cellular daunorubicin accumulation, indicating the competitive P-glycoprotein binding ability and drug-resistance reversal effect. Treatment of CEM and CEM/VLB cells with seed extracts also up-regulated the expression of cyclin kinase inhibitor (WAF1/p21) contributing to the arrest of cells at the G0/G1 phase of the cell cycle. These results support the traditional use of A. glabra and the alcoholic seed extract is a potent source of anticancer compounds that could be utilized pharmaceutically.