Comparative plastome and mitogenome analyses indicate that the marine prasinophyte green algae Pycnococcus provasolii and Pseudoscourfieldia marina (Pseudoscourfieldiophyceae class nov., Chlorophyta) represent morphotypes of the same species.

The marine prasinophyte green algae Pycnococcus provasolii and Pseudoscourfieldia marina represent the only extant genera and known species of the Pycnococcaceae. However, their taxonomic status needs to be reassessed, owing to the very close relationship inferred from previous sequence comparisons of individual genes. Although Py. provasolii and Ps. marina are morphologically different, their plastid rbcL and nuclear small subunit rRNA genes were observed to be nearly or entirely identical in sequence, thus leading to the hypothesis that they represent distinct growth forms or alternate life-cycle stages of the same organism. To evaluate this hypothesis, we used organelle genomes as molecular markers. The plastome and mitogenome of Ps. marina UIO 007 were sequenced and compared with those available for two isolates of Py. provasolii (CCMP 1203 and CCAP 190/2). The Ps. marina organelle genomes proved to be almost identical in size and had the same gene content and gene order as their Py. provasolii counterparts. Single nucleotide substitutions and insertions/deletions were localized using genome-scale sequence alignments. Over 99.70% sequence identities were observed in all pairwise comparisons of plastomes and mitogenomes. Alignments of both organelle genomes revealed that Ps. marina UIO 007 is closer to Py. provasolii CCAP 190/2 than are the two Py. provasolii strains to one another. Therefore, our results are not consistent with the placement of Ps. marina and Py. provasolii strains into distinct genera. We propose a taxonomic revision of the Pycnococcaceae and the erection of a new class of Chlorophyta, the Pseudoscourfieldiophyceae.

SYNY: a pipeline to investigate and visualize collinearity between genomes.

Investigating collinearity between chromosomes is often used in comparative genomics to help identify gene orthologs, pinpoint genes that might have been overlooked as part of annotation processes and/or perform various evolutionary inferences. Collinear segments, also known as syntenic blocks, can be inferred from sequence alignments and/or from the identification of genes arrayed in the same order and relative orientations between investigated genomes. To help perform these analyses and assess their outcomes, we built a simple pipeline called SYNY (for synteny) that implements the two distinct approaches and produces different visualizations. The SYNY pipeline was built with ease of use in mind and runs on modest hardware. The pipeline is written in Perl and Python and is available on GitHub (https://github.com/PombertLab/SYNY) under the permissive MIT license.

Near chromosome-level genome assembly of the microsporidium Hamiltosporidium tvaerminnensis.

Microsporidia are intracellular parasitic fungi whose genomes rank among the smallest of all known eukaryotes. A number of outstanding questions remain concerning the evolution of their large-scale variation in genome architecture, responsible for genome size variation of more than an order of magnitude. This genome report presents the first near-chromosomal assembly of a large-genome microsporidium, Hamiltosporidium tvaerminnensis. Combined Oxford Nanopore, Pacific Biosciences (PacBio), and Illumina sequencing led to a genome assembly of 17 contigs, 11 of which represent complete chromosomes. Our assembly is 21.64 Mb in length, has an N50 of 1.44 Mb, and consists of 39.56% interspersed repeats. We introduce a novel approach in microsporidia, PacBio Iso-Seq, as part of a larger annotation pipeline for obtaining high-quality annotations of 3,573 protein-coding genes. Based on direct evidence from the full-length Iso-Seq transcripts, we present evidence for alternative polyadenylation and variation in splicing efficiency, which are potential regulation mechanisms for gene expression in microsporidia. The generated high-quality genome assembly is a necessary resource for comparative genomics that will help elucidate the evolution of genome architecture in response to intracellular parasitism.

Telomere-to-Telomere genome assemblies of human-infecting Encephalitozoon species.

BACKGROUND: Microsporidia are diverse spore forming, fungal-related obligate intracellular pathogens infecting a wide range of hosts. This diversity is reflected at the genome level with sizes varying by an order of magnitude, ranging from less than 3 Mb in Encephalitozoon species (the smallest known in eukaryotes) to more than 50 Mb in Edhazardia spp. As a paradigm of genome reduction in eukaryotes, the small Encephalitozoon genomes have attracted much attention with investigations revealing gene dense, repeat- and intron-poor genomes characterized by a thorough pruning of molecular functions no longer relevant to their obligate intracellular lifestyle. However, because no Encephalitozoon genome has been sequenced from telomere-to-telomere and since no methylation data is available for these species, our understanding of their overall genetic and epigenetic architectures is incomplete. METHODS: In this study, we sequenced the complete genomes from telomere-to-telomere of three human-infecting Encephalitozoon spp. -E. intestinalis ATCC 50506, E. hellem ATCC 50604 and E. cuniculi ATCC 50602- using short and long read platforms and leveraged the data generated as part of the sequencing process to investigate the presence of epigenetic markers in these genomes. We also used a mixture of sequence- and structure-based computational approaches, including protein structure prediction, to help identify which Encephalitozoon proteins are involved in telomere maintenance, epigenetic regulation, and heterochromatin formation. RESULTS: The Encephalitozoon chromosomes were found capped by TTAGG 5-mer telomeric repeats followed by telomere associated repeat elements (TAREs) flanking hypermethylated ribosomal RNA (rRNA) gene loci featuring 5-methylcytosines (5mC) and 5-hemimethylcytosines (5hmC), themselves followed by lesser methylated subtelomeres and hypomethylated chromosome cores. Strong nucleotide biases were identified between the telomeres/subtelomeres and chromosome cores with significant changes in GC/AT, GT/AC and GA/CT contents. The presence of several genes coding for proteins essential to telomere maintenance, epigenetic regulation, and heterochromatin formation was further confirmed in the Encephalitozoon genomes. CONCLUSION: Altogether, our results strongly support the subtelomeres as sites of heterochromatin formation in Encephalitozoon genomes and further suggest that these species might shutdown their energy-consuming ribosomal machinery while dormant as spores by silencing of the rRNA genes using both 5mC/5hmC methylation and facultative heterochromatin formation at these loci.

The Rad9-Rad1-Hus1 DNA Repair Clamp is Found in Microsporidia.

DNA repair is an important component of genome integrity and organisms with reduced repair capabilities tend to accumulate mutations at elevated rates. Microsporidia are intracellular parasites exhibiting high levels of genetic divergence postulated to originate from the lack of several proteins, including the heterotrimeric Rad9-Rad1-Hus1 DNA repair clamp. Microsporidian species from the Encephalitozoonidae have undergone severe streamlining with small genomes coding for about 2,000 proteins. The highly divergent sequences found in Microsporidia render functional inferences difficult such that roughly half of these 2,000 proteins have no known function. Using a structural homology-based annotation approach combining protein structure prediction and tridimensional similarity searches, we found that the Rad9-Rad1-Hus1 DNA clamp is present in Microsporidia, together with many other components of the DNA repair machinery previously thought to be missing from these organisms. Altogether, our results indicate that the DNA repair machinery is present and likely functional in Microsporidia.

A new microsporidian parasite, Ordospora pajunii sp. nov (Ordosporidae), of Daphnia longispina highlights the value of genomic data for delineating species boundaries.

Speciation is a complex and continuous process that makes the delineation of species boundaries a challenging task in particular in species with little morphological differentiation, such as parasites. In this case, the use of genomic data is often necessary, such as for the intracellular Microsporidian parasites. Here, we characterize the genome of a gut parasite of the cladoceran Daphnia longispina (isolate FI-F-10), which we propose as a new species within the genus Ordospora: Ordospora pajunii sp. nov (Ordosporidae). FI-F-10 closest relative, Ordospora colligata is only found in D. magna. Both microsporidian species share several morphological features. Although it is not possible to estimate divergence times for Microsporidia due to the lack of fossil records and accelerated evolutionary rates, we base our proposal on the phylogenomic and genomic distances between both microsporidian lineages. The phylogenomic reconstruction shows that FI-F-10 forms an early diverging branch basal to the cluster that contains all known O. colligata strains. Whole-genome comparisons show that FI-F-10 presents a greater divergence at the sequence level than observed among O. colligata strains, and its genomic average nucleotide identity (ANI) values against O. colligata are beyond the intraspecific range previously established for yeast and prokaryotes. Our data confirm that the ANI metrics are useful for fine genetic divergence calibration across Microsporidia taxa. In combination with phylogenetic and ecological data, genome-based metrics provide a powerful approach to delimitate species boundaries.

3DFI: a pipeline to infer protein function using structural homology.

Motivation: Inferring protein function is an integral part of genome annotation and analysis. This process is usually performed in silico, and most in silico inferences are based on sequence homology approaches, which can fail when in presence of divergent sequences. However, because protein structures and their biological roles are intertwined, protein function can also be inferred by searching for structural homology. Many excellent tools have been released in recent years with regards to protein structure prediction, structural homology searches and protein visualization. Unfortunately, these tools are disconnected from each other and often use a web server-based approach that is ill-suited to high-throughput genome-wide analyses. To help assist genome annotation, we built a structural homology-based pipeline called 3DFI (for tridimensional functional inference) leveraging some of the best structural homology tools. This pipeline was built with simplicity of use in mind and enables genome-wide structural homology inferences. Availability and implementation: 3DFI is available on GitHub https://github.com/PombertLab/3DFI under the permissive MIT license. The pipeline is written in Perl and Python.

Microsporidia with Vertical Transmission Were Likely Shaped by Nonadaptive Processes.

Microsporidia have the leanest genomes among eukaryotes, and their physiological and genomic simplicity has been attributed to their intracellular, obligate parasitic life-style. However, not all microsporidia genomes are small or lean, with the largest dwarfing the smallest ones by at least an order of magnitude. To better understand the evolutionary mechanisms behind this genomic diversification, we explore here two clades of microsporidia with distinct life histories, Ordospora and Hamiltosporidium, parasitizing the same host species, Daphnia magna. Based on seven newly assembled genomes, we show that mixed-mode transmission (the combination of horizontal and vertical transmission), which occurs in Hamiltosporidium, is found to be associated with larger and AT-biased genomes, more genes, and longer intergenic regions, as compared with the exclusively horizontally transmitted Ordospora. Furthermore, the Hamiltosporidium genome assemblies contain a variety of repetitive elements and long segmental duplications. We show that there is an excess of nonsynonymous substitutions in the microsporidia with mixed-mode transmission, which cannot be solely attributed to the lack of recombination, suggesting that bursts of genome size in these microsporidia result primarily from genetic drift. Overall, these findings suggest that the switch from a horizontal-only to a mixed mode of transmission likely produces population bottlenecks in Hamiltosporidium species, therefore reducing the effectiveness of natural selection, and allowing their genomic features to be largely shaped by nonadaptive processes.

A streamlined and predominantly diploid genome in the tiny marine green alga Chloropicon primus.

Tiny marine green algae issued from two deep branches of the Chlorophyta, the Mamiellophyceae and Chloropicophyceae, dominate different regions of the oceans and play key roles in planktonic communities. Considering that the Mamiellophyceae is the sole lineage of prasinophyte algae that has been intensively investigated, the extent to which these two algal groups differ in their metabolic capacities and cellular processes is currently unknown. To address this gap of knowledge, we investigate here the nuclear genome sequence of a member of the Chloropicophyceae, Chloropicon primus. Among the main biological insights that emerge from this 17.4 Mb genome, we find an unexpected diploid structure for most chromosomes and a propionate detoxification pathway in green algae. Our results support the notion that separate events of genome minimization, which entailed differential losses of genes/pathways, have occurred in the Chloropicophyceae and Mamiellophyceae, suggesting different strategies of adaptation to oceanic environments.

Characterization of Listeria monocytogenes enhanced cold-tolerance variants isolated during prolonged cold storage.

In this study, we show that growth and prolonged storage of Listeria monocytogenes at 4 °C can promote the selection of variants with enhanced cold and heat tolerance. Enhanced cold-tolerance (ECT) variants (n = 12) were successfully isolated from a strain with impaired cold growth abilities following 84 days of storage at 4 °C in brain heart infusion broth (BHIB). Whole genome sequencing, membrane fatty acid analysis, and stress tolerance profiling were performed on the parent strain and two ECT variants: one displaying regular-sized colonies and the other displaying small colonies when grown at 37 °C on BHI agar. Under cold stress conditions, the parent strain exhibited an impaired ability to produce branched-chain fatty acids which are known to be important for cold adaptation in L.monocytogenes. The ECT variants were able to overcome this limitation, a finding which is hypothesized to be associated with the identification of two independent single-nucleotide polymorphisms in genes encoding subunits of acetyl-coA carboxylase, an enzyme critical for fatty acid biosynthesis. While the ECT phenotype was not found to be associated with improved salt (BHIB + 6% NaCl, 25 °C), acid (BHIB pH 5, 25 °C) or desiccation (33% RH, 20 °C) tolerance, the small-colony variant exhibited significantly (p < 0.05) enhanced heat tolerance at 52 °C in buffered peptone water compared to the parent strain and the other variant. The results from this study demonstrate that the continuous use of refrigeration along the food-supply chain has the potential to select for L.monocytogenes variants with enhanced cold and heat tolerance, highlighting the impact that microbial intervention strategies can have on the evolution of bacterial strains and likewise, food safety.

The bacteriocin from the prophylactic candidate Streptococcus suis 90-1330 is widely distributed across S. suis isolates and appears encoded in an integrative and conjugative element.

The Gram-positive α-hemolytic Streptococcus suis is a major pathogen in the swine industry and an emerging zoonotic agent that can cause several systemic issues in both pigs and humans. A total of 35 S. suis serotypes (SS) have been identified and genotyped into > 700 sequence types (ST) by multilocus sequence typing (MLST). Eurasian ST1 isolates are the most virulent of all S. suis SS2 strains while North American ST25 and ST28 strains display moderate to low/no virulence phenotypes, respectively. Notably, S. suis 90-1330 is an avirulent Canadian SS2-ST28 isolate producing a lantibiotic bacteriocin with potential prophylactic applications. To investigate the suitability of this strain for such purposes, we sequenced its complete genome using the Illumina and PacBio platforms. The S. suis 90-1330 bacteriocin was found encoded in a locus cargoed in what appears to be an integrative and conjugative element (ICE). This bacteriocin locus was also found to be widely distributed across several streptococcal species and in a few Staphylococcus aureus strains. Because the locus also confers protection from the bacteriocin, the potential prophylactic benefits of using this strain may prove limited due to the spread of the resistance to its effects. Furthermore, the S. suis 90-1330 genome was found to code for genes involved in blood survival, suggesting that strain may not be a benign as previously thought.

Complete Genome Sequence of Vitreoscilla sp. Strain C1, Source of the First Bacterial Hemoglobin.

Vitreoscilla sp. strain C1 is of historical importance as the source of the first prokaryotic hemoglobin identified. Vitreoscilla spp. rely on their hemoglobin and cytochrome oxidase to grow in microaerobic environments despite their aerobic nature. To help characterize this historically relevant strain, we sequenced the complete Vitreoscilla sp. strain C1 genome.

Complete Genome Sequences of the Potential Zoonotic Pathogens Staphylococcus felis and Staphylococcus kloosii.

Coagulase-negative staphylococci (CoNS) are opportunistic pathogens frequently encountered in nosocomial infections. Animal-associated CoNS pose a zoonotic risk and constitute a potential reservoir for virulence and antimicrobial resistance genes. To improve our knowledge of animal-associated CoNS, we sequenced the complete genomes of Staphylococcus felis (ATCC 49168) and Staphylococcus kloosii (ATCC 43959).

Complete Genome Sequences of Two Human Oral Microbiome Commensals, Streptococcus salivarius ATCC 25975 and S. salivarius ATCC 27945.

Streptococcus salivarius strains are significant contributors to the human oral microbiome. Some possess unique fimbriae that give them the ability to coaggregate and colonize particular oral structures. We present here the complete genomes of Streptococcus salivarius Lancefield K-/K+ strains ATCC 25975 and ATCC 27945, which can and cannot, respectively, produce fimbriae.

Complete Genome Sequence of Staphylococcus lutrae ATCC 700373, a Potential Pathogen Isolated from Deceased Otters.

Despite their relevance to human health, not all staphylococcal species have been characterized. As such, the potential zoonotic threats posed by uninvestigated species and their contribution to the staphylococcal pangenome are unclear. Here, we report the complete genome sequence of Staphylococcus lutrae ATCC 700373, a coagulase-positive species isolated from deceased otters.

Genetic Characterization of the Exceptionally High Heat Resistance of the Non-toxic Surrogate Clostridium sporogenes PA 3679.

Clostridium sporogenes PA 3679 is a non-toxic endospore former that is widely used as a surrogate for Clostridium botulinum by the food processing industry to validate thermal processing strategies. PA 3679 produces spores of exceptionally high heat resistance without botulinum neurotoxins, permitting the use of PA 3679 in inoculated pack studies while ensuring the safety of food processing facilities. To identify genes associated with this heat resistance, the genomes of C. sporogenes PA 3679 isolates were compared to several other C. sporogenes strains. The most significant difference was the acquisition of a second spoVA operon, spoVA2, which is responsible for transport of dipicolinic acid into the spore core during sporulation. Interestingly, spoVA2 was also found in some C. botulinum species which phylogenetically cluster with PA 3679. Most other C. sporogenes strains examined both lack the spoVA2 locus and are phylogenetically distant within the group I Clostridium, adding to the understanding that C. sporogenes are dispersed C. botulinum strains which lack toxin genes. C. sporogenes strains are thus a very eclectic group, and few strains possess the characteristic heat resistance of PA 3679.

Comparative genomics of microsporidian genomes reveals a minimal non-coding RNA set and new insights for transcription in minimal eukaryotic genomes.

Microsporidia are ubiquitous intracellular pathogens whose opportunistic nature led to their increased recognition with the rise of the AIDS pandemic. As the RNA world was largely unexplored in this parasitic lineage, we developed a dedicated in silico methodology to carry out exhaustive identification of ncRNAs across the Encephalitozoon and Nosema genera. Thus, the previously missing U1 small nuclear RNA (snRNA) and small nucleolar RNAs (snoRNAs) targeting only the LSU rRNA were highlighted and were further validated using 5' and 3'RACE-PCR experiments. Overall, the 15 ncRNAs that were found shared between Encephalitozoon and Nosema spp. may represent the minimal core set required for parasitic life. Interestingly, the systematic presence of a CCC- or GGG-like motif in 5' of all ncRNA and mRNA gene transcripts regardless of the RNA polymerase involved suggests that the RNA polymerase machineries in microsporidia species could use common factors. Our data provide additional insights in accordance with the simplification processes observed in these reduce genomes and underline the usefulness of sequencing closely related species to help identify highly divergent ncRNAs in these parasites.

Enhancement of Microbial Biodesulfurization via Genetic Engineering and Adaptive Evolution.

In previous work from our laboratories a synthetic gene encoding a peptide ("Sulpeptide 1" or "S1") with a high proportion of methionine and cysteine residues had been designed to act as a sulfur sink and was inserted into the dsz (desulfurization) operon of Rhodococcus erythropolis IGTS8. In the work described here this construct (dszAS1BC) and the intact dsz operon (dszABC) cloned into vector pRESX under control of the (Rhodococcus) kstD promoter were transformed into the desulfurization-negative strain CW25 of Rhodococcus qingshengii. The resulting strains (CW25[pRESX-dszABC] and CW25[pRESX-dszAS1BC]) were subjected to adaptive selection by repeated passages at log phase (up to 100 times) in minimal medium with dibenzothiophene (DBT) as sole sulfur source. For both strains DBT metabolism peaked early in the selection process and then decreased, eventually averaging four times that of the initial transformed cells; the maximum specific activity achieved by CW25[pRESX-dszAS1BC] exceeded that of CW25[pRESX-dszABC]. Growth rates increased by 7-fold (CW25[pRESX-dszABC]) and 13-fold (CW25[pRESX-dszAS1BC]) and these increases were stable. The adaptations of CW25[pRESX-dszAS1BC] were correlated with a 3-5X increase in plasmid copy numbers from those of the initial transformed cells; whole genome sequencing indicated that during its selection processes no mutations occurred to any of the dsz, S1, or other genes and promoters involved in sulfur metabolism, stress response, or DNA methylation, and that the effect of the sulfur sink produced by S1 is likely very small compared to the cells' overall cysteine and methionine requirements. Nevertheless, a combination of genetic engineering using sulfur sinks and increasing Dsz capability with adaptive selection may be a viable strategy to increase biodesulfurization ability.

The 'other' coral symbiont: Ostreobium diversity and distribution.

Ostreobium is an endolithic algal genus thought to be an early-diverging lineage of the Bryopsidales (Ulvophyceae, Chlorophyta). Ostreobium can live in low-light conditions on calcium carbonate substrata in tropical conditions. It is best known as a symbiont of corals, where it lives deep within the animal skeleton and exchanges nitrogen and carbon, as well as providing nutrients and photoassimilates. In contrast to the relatively well-studied role of the photosynthetic zooxanthellae symbionts in coral (Symbiodinium), Ostreobium phylogeny, diversity and distribution are all poorly understood. Here, we describe the phylogenetic position and diversity of Ostreobium based on plastid 16S ribosomal DNA (rDNA), 18S rDNA and rbcL genes from a nuclear genome survey and complete plastid genome, and determined its environmental diversity and distribution by screening the publicly available environmental data for those genes. The results shed light on the phylogeny and the ecology of the 'other' coral symbiont.

Moramonas marocensis gen. nov., sp. nov.: a jakobid flagellate isolated from desert soil with a bacteria-like, but bloated mitochondrial genome.

A new jakobid genus has been isolated from Moroccan desert soil. The cyst-forming protist Moramonas marocensis gen. nov., sp. nov. has two anteriorly inserted flagella of which one points to the posterior cell pole accompanying the ventral feeding groove and is equipped with a dorsal vane-a feature typical for the Jakobida. It further shows a flagellar root system consisting of singlet microtubular root, left root (R1), right root (R2) and typical fibres associated with R1 and R2. The affiliation of M. marocensis to the Jakobida was confirmed by molecular phylogenetic analyses of the SSU rRNA gene, five nuclear genes and 66 mitochondrial protein-coding genes. The mitochondrial genome has the high number of genes typical for jakobids, and bacterial features, such as the four-subunit RNA polymerase and Shine-Dalgarno sequences upstream of the coding regions of several genes. The M. marocensis mitochondrial genome encodes a similar number of genes as other jakobids, but is unique in its very large genome size (greater than 264 kbp), which is three to four times higher than that of any other jakobid species investigated yet. This increase seems to be due to a massive expansion in non-coding DNA, creating a bloated genome like those of plant mitochondria.