SARS-CoV-2 reservoir in post-acute sequelae of COVID-19 (PASC).

Millions of people are suffering from Long COVID or post-acute sequelae of COVID-19 (PASC). Several biological factors have emerged as potential drivers of PASC pathology. Some individuals with PASC may not fully clear the coronavirus SARS-CoV-2 after acute infection. Instead, replicating virus and/or viral RNA-potentially capable of being translated to produce viral proteins-persist in tissue as a 'reservoir'. This reservoir could modulate host immune responses or release viral proteins into the circulation. Here we review studies that have identified SARS-CoV-2 RNA/protein or immune responses indicative of a SARS-CoV-2 reservoir in PASC samples. Mechanisms by which a SARS-CoV-2 reservoir may contribute to PASC pathology, including coagulation, microbiome and neuroimmune abnormalities, are delineated. We identify research priorities to guide the further study of a SARS-CoV-2 reservoir in PASC, with the goal that clinical trials of antivirals or other therapeutics with potential to clear a SARS-CoV-2 reservoir are accelerated.

Plastics select for distinct early colonizing microbial populations with reproducible traits across environmental gradients.

Little is known about early plastic biofilm assemblage dynamics and successional changes over time. By incubating virgin microplastics along oceanic transects and comparing adhered microbial communities with those of naturally occurring plastic litter at the same locations, we constructed gene catalogues to contrast the metabolic differences between early and mature biofilm communities. Early colonization incubations were reproducibly dominated by Alteromonadaceae and harboured significantly higher proportions of genes associated with adhesion, biofilm formation, chemotaxis, hydrocarbon degradation and motility. Comparative genomic analyses among the Alteromonadaceae metagenome assembled genomes (MAGs) highlighted the importance of the mannose-sensitive hemagglutinin (MSHA) operon, recognized as a key factor for intestinal colonization, for early colonization of hydrophobic plastic surfaces. Synteny alignments of MSHA also demonstrated positive selection for mshA alleles across all MAGs, suggesting that mshA provides a competitive advantage for surface colonization and nutrient acquisition. Large-scale genomic characteristics of early colonizers varied little, despite environmental variability. Mature plastic biofilms were composed of predominantly Rhodobacteraceae and displayed significantly higher proportions of carbohydrate hydrolysis enzymes and genes for photosynthesis and secondary metabolism. Our metagenomic analyses provide insight into early biofilm formation on plastics in the ocean and how early colonizers self-assemble, compared to mature, phylogenetically and metabolically diverse biofilms.

Publisher Correction: Genetic tool development in marine protists: emerging model organisms for experimental cell biology.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Genetic tool development in marine protists: emerging model organisms for experimental cell biology.

Diverse microbial ecosystems underpin life in the sea. Among these microbes are many unicellular eukaryotes that span the diversity of the eukaryotic tree of life. However, genetic tractability has been limited to a few species, which do not represent eukaryotic diversity or environmentally relevant taxa. Here, we report on the development of genetic tools in a range of protists primarily from marine environments. We present evidence for foreign DNA delivery and expression in 13 species never before transformed and for advancement of tools for eight other species, as well as potential reasons for why transformation of yet another 17 species tested was not achieved. Our resource in genetic manipulation will provide insights into the ancestral eukaryotic lifeforms, general eukaryote cell biology, protein diversification and the evolution of cellular pathways.

Evolutionary genomics of the cold-adapted diatom Fragilariopsis cylindrus.

The Southern Ocean houses a diverse and productive community of organisms. Unicellular eukaryotic diatoms are the main primary producers in this environment, where photosynthesis is limited by low concentrations of dissolved iron and large seasonal fluctuations in light, temperature and the extent of sea ice. How diatoms have adapted to this extreme environment is largely unknown. Here we present insights into the genome evolution of a cold-adapted diatom from the Southern Ocean, Fragilariopsis cylindrus, based on a comparison with temperate diatoms. We find that approximately 24.7 per cent of the diploid F. cylindrus genome consists of genetic loci with alleles that are highly divergent (15.1 megabases of the total genome size of 61.1 megabases). These divergent alleles were differentially expressed across environmental conditions, including darkness, low iron, freezing, elevated temperature and increased CO2. Alleles with the largest ratio of non-synonymous to synonymous nucleotide substitutions also show the most pronounced condition-dependent expression, suggesting a correlation between diversifying selection and allelic differentiation. Divergent alleles may be involved in adaptation to environmental fluctuations in the Southern Ocean.

Molecular epidemiology of Staphylococcus aureus in African children from rural and urban communities with atopic dermatitis.

BACKGROUND: Staphylococcus aureus has been associated with the exacerbation and severity of atopic dermatitis (AD). Studies have not investigated the colonisation dynamics of S. aureus lineages in African toddlers with AD. We determined the prevalence and population structure of S. aureus in toddlers with and without AD from rural and urban South African settings. METHODS: We conducted a study of AD-affected and non-atopic AmaXhosa toddlers from rural Umtata and urban Cape Town, South Africa. S. aureus was screened from skin and nasal specimens using established microbiological methods and clonal lineages were determined by spa typing. Logistic regression analyses were employed to assess risk factors associated with S. aureus colonisation. RESULTS: S. aureus colonisation was higher in cases compared to controls independent of geographic location (54% vs. 13%, p < 0.001 and 70% vs. 35%, p = 0.005 in Umtata [rural] and Cape Town [urban], respectively). Severe AD was associated with higher colonisation compared with moderate AD (86% vs. 52%, p = 0.015) among urban cases. Having AD was associated with colonisation in both rural (odds ratio [OR] 7.54, 95% CI 2.92-19.47) and urban (OR 4.2, 95% CI 1.57-11.2) toddlers. In rural toddlers, living in an electrified house that uses gas (OR 4.08, 95% CI 1.59-10.44) or utilises kerosene and paraffin (OR 2.88, 95% CI 1.22-6.77) for heating and cooking were associated with increased S. aureus colonisation. However, exposure to farm animals (OR 0.3, 95% CI 0.11-0.83) as well as living in a house that uses wood and coal (OR 0.14, 95% CI 0.04-0.49) or outdoor fire (OR 0.31, 95% CI 0.13-0.73) were protective. Spa types t174 and t1476, and t272 and t1476 were dominant among urban and rural cases, respectively, but no main spa type was observed among controls, independent of geographic location. In urban cases, spa type t002 and t442 isolates were only identified in severe AD, t174 was more frequent in moderate AD, and t1476 in severe AD. CONCLUSION: The strain genotype of S. aureus differed by AD phenotypes and rural-urban settings. Continued surveillance of colonising S. aureus lineages is key in understanding alterations in skin microbial composition associated with AD pathogenesis and exacerbation.

Genomic insights into waste valorized extracellular polymeric substances (EPS) produced by Bacillus sp. ISTL8.

The present study discusses the genomic analysis of Bacillus sp. ISTL8 along with the production of EPS (Extracellular polymeric substances) using carbofuran, a toxic carbamate pesticide. Bacillus strain was isolated from landfill soil and evaluated for high growth rates and EPS production. One strain, renamed ISTL8 grew on a broad range of carbon sources, including toxic carbofuran, while producing copious EPS. Growth assays verified the strain to be thermophilic, low salt tolerant, and with a preference for neutral pH. SEM (Scanning Electron Microscopy) was used for morphological characterization of the EPS while the monomeric composition, bonding patterns and functional groups were deduced by GC-MS (Gas Chromatography-Mass Spectrometry), 1H and 13C NMR (Nuclear Magnetic Resonance) and FTIR (Fourier Transform Infrared Spectroscopy). The production of EPS using carbofuran (carbamate pesticide) as a carbon source was found to be 6.20 ± 0.29 g L-1 containing 61.17% w/w carbohydrates, 29.72% w/w proteins and 6.11% w/w lipids (of dry EPS). The potential cytotoxicity of EPS was evaluated with 3- (4,5-dimethyl thiazol-2-Yl) -2,5-diphenyl tetrazolium bromide (MTT) assay and found non-toxic (2.25%). WGS (Whole genome sequencing) was performed for the strain Bacillus sp. ISTL8 producing EPS; an array of genes putatively involved in the EPS production were identified in several different genomic locations, guiding potential genetic manipulation studies in the future. The results highlight the potency of a bacterial isolate Bacillus sp. ISTL8 to produce non-cytotoxic EPS using carbofuran that can be further harnessed for environmental and commercial applications. Additionally, WGS revealed an array of EPS specific genes which can be effectively engineered for much enhanced production.

Pervasive iron limitation at subsurface chlorophyll maxima of the California Current.

Subsurface chlorophyll maximum layers (SCMLs) are nearly ubiquitous in stratified water columns and exist at horizontal scales ranging from the submesoscale to the extent of oligotrophic gyres. These layers of heightened chlorophyll and/or phytoplankton concentrations are generally thought to be a consequence of a balance between light energy from above and a limiting nutrient flux from below, typically nitrate (NO3). Here we present multiple lines of evidence demonstrating that iron (Fe) limits or with light colimits phytoplankton communities in SCMLs along a primary productivity gradient from coastal to oligotrophic offshore waters in the southern California Current ecosystem. SCML phytoplankton responded markedly to added Fe or Fe/light in experimental incubations and transcripts of diatom and picoeukaryote Fe stress genes were strikingly abundant in SCML metatranscriptomes. Using a biogeochemical proxy with data from a 40-y time series, we find that diatoms growing in California Current SCMLs are persistently Fe deficient during the spring and summer growing season. We also find that the spatial extent of Fe deficiency within California Current SCMLs has significantly increased over the last 25 y in line with a regional climate index. Finally, we show that diatom Fe deficiency may be common in the subsurface of major upwelling zones worldwide. Our results have important implications for our understanding of the biogeochemical consequences of marine SCML formation and maintenance.

METATRYP v 2.0: Metaproteomic Least Common Ancestor Analysis for Taxonomic Inference Using Specialized Sequence Assemblies-Standalone Software and Web Servers for Marine Microorganisms and Coronaviruses.

We present METATRYP version 2 software that identifies shared peptides across the predicted proteomes of organisms within environmental metaproteomics studies to enable accurate taxonomic attribution of peptides during protein inference. Improvements include ingestion of complex sequence assembly data categories (metagenomic and metatranscriptomic assemblies, single cell amplified genomes, and metagenome assembled genomes), prediction of the least common ancestor (LCA) for a peptide shared across multiple organisms, increased performance through updates to the backend architecture, and development of a web portal (https://metatryp.whoi.edu). Major expansion of the marine METATRYP database with predicted proteomes from environmental sequencing confirms a low occurrence of shared tryptic peptides among disparate marine microorganisms, implying tractability for targeted metaproteomics. METATRYP was designed to facilitate ocean metaproteomics and has been integrated into the Ocean Protein Portal (https://oceanproteinportal.org); however, it can be readily applied to other domains. We describe the rapid deployment of a coronavirus-specific web portal (https://metatryp-coronavirus.whoi.edu/) to aid in use of proteomics on coronavirus research during the ongoing pandemic. A coronavirus-focused METATRYP database identified potential SARS-CoV-2 peptide biomarkers and indicated very few shared tryptic peptides between SARS-CoV-2 and other disparate taxa analyzed, sharing <1% peptides with taxa outside of the betacoronavirus group, establishing that taxonomic specificity is achievable using tryptic peptide-based proteomic diagnostic approaches.

Lateral Gene Transfer Shapes the Distribution of RuBisCO among Candidate Phyla Radiation Bacteria and DPANN Archaea.

Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) is considered to be the most abundant enzyme on Earth. Despite this, its full diversity and distribution across the domains of life remain to be determined. Here, we leverage a large set of bacterial, archaeal, and viral genomes recovered from the environment to expand our understanding of existing RuBisCO diversity and the evolutionary processes responsible for its distribution. Specifically, we report a new type of RuBisCO present in Candidate Phyla Radiation (CPR) bacteria that is related to the archaeal Form III enzyme and contains the amino acid residues necessary for carboxylase activity. Genome-level metabolic analyses supported the inference that these RuBisCO function in a CO2-incorporating pathway that consumes nucleotides. Importantly, some Gottesmanbacteria (CPR) also encode a phosphoribulokinase that may augment carbon metabolism through a partial Calvin-Benson-Bassham cycle. Based on the scattered distribution of RuBisCO and its discordant evolutionary history, we conclude that this enzyme has been extensively laterally transferred across the CPR bacteria and DPANN archaea. We also report RuBisCO-like proteins in phage genomes from diverse environments. These sequences cluster with proteins in the Beckwithbacteria (CPR), implicating phage as a possible mechanism of RuBisCO transfer. Finally, we synthesize our metabolic and evolutionary analyses to suggest that lateral gene transfer of RuBisCO may have facilitated major shifts in carbon metabolism in several important bacterial and archaeal lineages.

Nitrate Reductase Knockout Uncouples Nitrate Transport from Nitrate Assimilation and Drives Repartitioning of Carbon Flux in a Model Pennate Diatom.

The ecological prominence of diatoms in the ocean environment largely results from their superior competitive ability for dissolved nitrate (NO3-). To investigate the cellular and genetic basis of diatom NO3- assimilation, we generated a knockout in the nitrate reductase gene (NR-KO) of the model pennate diatom Phaeodactylum tricornutum In NR-KO cells, N-assimilation was abolished although NO3- transport remained intact. Unassimilated NO3- accumulated in NR-KO cells, resulting in swelling and associated changes in biochemical composition and physiology. Elevated expression of genes encoding putative vacuolar NO3- chloride channel transporters plus electron micrographs indicating enlarged vacuoles suggested vacuolar storage of NO3- Triacylglycerol concentrations in the NR-KO cells increased immediately following the addition of NO3-, and these increases coincided with elevated gene expression of key triacylglycerol biosynthesis components. Simultaneously, induction of transcripts encoding proteins involved in thylakoid membrane lipid recycling suggested more abrupt repartitioning of carbon resources in NR-KO cells compared with the wild type. Conversely, ribosomal structure and photosystem genes were immediately deactivated in NR-KO cells following NO3- addition, followed within hours by deactivation of genes encoding enzymes for chlorophyll biosynthesis and carbon fixation and metabolism. N-assimilation pathway genes respond uniquely, apparently induced simultaneously by both NO3- replete and deplete conditions.

Diatom centromeres suggest a mechanism for nuclear DNA acquisition.

Centromeres are essential for cell division and growth in all eukaryotes, and knowledge of their sequence and structure guides the development of artificial chromosomes for functional cellular biology studies. Centromeric proteins are conserved among eukaryotes; however, centromeric DNA sequences are highly variable. We combined forward and reverse genetic approaches with chromatin immunoprecipitation to identify centromeres of the model diatom Phaeodactylum tricornutum We observed 25 unique centromere sequences typically occurring once per chromosome, a finding that helps to resolve nuclear genome organization and indicates monocentric regional centromeres. Diatom centromere sequences contain low-GC content regions but lack repeats or other conserved sequence features. Native and foreign sequences with similar GC content to P. tricornutum centromeres can maintain episomes and recruit the diatom centromeric histone protein CENH3, suggesting nonnative sequences can also function as diatom centromeres. Thus, simple sequence requirements may enable DNA from foreign sources to persist in the nucleus as extrachromosomal episomes, revealing a potential mechanism for organellar and foreign DNA acquisition.

Genomic Changes Associated with the Evolutionary Transitions of Nostoc to a Plant Symbiont.

Cyanobacteria belonging to the genus Nostoc comprise free-living strains and also facultative plant symbionts. Symbiotic strains can enter into symbiosis with taxonomically diverse range of host plants. Little is known about genomic changes associated with evolutionary transition of Nostoc from free-living to plant symbiont. Here, we compared the genomes derived from 11 symbiotic Nostoc strains isolated from different host plants and infer phylogenetic relationships between strains. Phylogenetic reconstructions of 89 Nostocales showed that symbiotic Nostoc strains with a broad host range, entering epiphytic and intracellular or extracellular endophytic interactions, form a monophyletic clade indicating a common evolutionary history. A polyphyletic origin was found for Nostoc strains which enter only extracellular symbioses, and inference of transfer events implied that this trait was likely acquired several times in the evolution of the Nostocales. Symbiotic Nostoc strains showed enriched functions in transport and metabolism of organic sulfur, chemotaxis and motility, as well as the uptake of phosphate, branched-chain amino acids, and ammonium. The genomes of the intracellular clade differ from that of other Nostoc strains, with a gain/enrichment of genes encoding proteins to generate l-methionine from sulfite and pathways for the degradation of the plant metabolites vanillin and vanillate, and of the macromolecule xylan present in plant cell walls. These compounds could function as C-sources for members of the intracellular clade. Molecular clock analysis indicated that the intracellular clade emerged ca. 600 Ma, suggesting that intracellular Nostoc symbioses predate the origin of land plants and the emergence of their extant hosts.

Patterns of thaumarchaeal gene expression in culture and diverse marine environments.

Thaumarchaea are ubiquitous in marine habitats where they participate in carbon and nitrogen cycling. Although metatranscriptomes suggest thaumarchaea are active microbes in marine waters, we understand little about how thaumarchaeal gene expression patterns relate to substrate utilization and activity. Here, we report the global transcriptional response of the marine ammonia-oxidizing thaumarchaeon 'Candidatus Nitrosopelagicus brevis' str. CN25 to ammonia limitation using RNA-Seq. We further describe the genome and transcriptome of Ca. N. brevis str. U25, a new strain capable of urea utilization. Ammonia limitation in CN25 resulted in reduced expression of transcripts coding for ammonia oxidation proteins, and increased expression of a gene coding an Hsp20-like chaperone. Despite significantly different transcript abundances across treatments, two ammonia monooxygenase subunits (amoAB), a nitrite reductase (nirK) and both ammonium transporter genes were always among the most abundant transcripts, regardless of growth state. Ca. N. brevis str. U25 cells expressed a urea transporter 139-fold more than the urease catalytic subunit ureC. Gene coexpression networks derived from culture transcriptomes and 10 thaumarchaea-enriched metatranscriptomes revealed a high degree of correlated gene expression across disparate environmental conditions and identified a module of coexpressed genes, including amoABC and nirK, that we hypothesize to represent the core ammonia oxidation machinery.

Genomic and proteomic characterization of "Candidatus Nitrosopelagicus brevis": an ammonia-oxidizing archaeon from the open ocean.

Thaumarchaeota are among the most abundant microbial cells in the ocean, but difficulty in cultivating marine Thaumarchaeota has hindered investigation into the physiological and evolutionary basis of their success. We report here a closed genome assembled from a highly enriched culture of the ammonia-oxidizing pelagic thaumarchaeon CN25, originating from the open ocean. The CN25 genome exhibits strong evidence of genome streamlining, including a 1.23-Mbp genome, a high coding density, and a low number of paralogous genes. Proteomic analysis recovered nearly 70% of the predicted proteins encoded by the genome, demonstrating that a high fraction of the genome is translated. In contrast to other minimal marine microbes that acquire, rather than synthesize, cofactors, CN25 encodes and expresses near-complete biosynthetic pathways for multiple vitamins. Metagenomic fragment recruitment indicated the presence of DNA sequences >90% identical to the CN25 genome throughout the oligotrophic ocean. We propose the provisional name "Candidatus Nitrosopelagicus brevis" str. CN25 for this minimalist marine thaumarchaeon and suggest it as a potential model system for understanding archaeal adaptation to the open ocean.

The diversity of CO2-concentrating mechanisms in marine diatoms as inferred from their genetic content.

Marine diatoms are one of the most ecologically significant primary producers in the ocean. Most diatoms use a CO2-concentrating mechanism (CCM) to overcome the scarcity of CO2 in the ocean and limitations of the carbon-fixing enzyme Rubisco. However, the CCMs in model diatoms differ substantially in their genetic make-up and structural organization. To assess the extent of CCM diversity in marine diatoms more generally, we analyzed genome and transcriptome data from 31 diatom strains to identify putative CCM genes, examine the overall CCM architecture, and study CCM development in the context of the evolutionary history of these diatoms. Key CCM genes [carbonic anhydrases (CAs) and solute carrier 4 (SLC4) bicarbonate transporters] identified in the diatoms were placed into groups of likely orthologs by sequence similarity (OrthoMCL) and phylogenetic methods. These analyses indicated that diatoms seem to share similar HCO3- transporters, but possess a variety of CAs that have either undergone extensive diversification within the diatom lineage or have been acquired through horizontal gene transfer. Hierarchical clustering of the diatom species based on their CCM gene content suggests that CCM development is largely congruent with evolution of diatom species, despite some notable differences in CCM genes even among closely related species.

Evolution and metabolic significance of the urea cycle in photosynthetic diatoms.

Diatoms dominate the biomass of phytoplankton in nutrient-rich conditions and form the basis of some of the world's most productive marine food webs. The diatom nuclear genome contains genes with bacterial and plastid origins as well as genes of the secondary endosymbiotic host (the exosymbiont), yet little is known about the relative contribution of each gene group to diatom metabolism. Here we show that the exosymbiont-derived ornithine-urea cycle, which is similar to that of metazoans but is absent in green algae and plants, facilitates rapid recovery from prolonged nitrogen limitation. RNA-interference-mediated knockdown of a mitochondrial carbamoyl phosphate synthase impairs the response of nitrogen-limited diatoms to nitrogen addition. Metabolomic analyses indicate that intermediates in the ornithine-urea cycle are particularly depleted and that both the tricarboxylic acid cycle and the glutamine synthetase/glutamate synthase cycles are linked directly with the ornithine-urea cycle. Several other depleted metabolites are generated from ornithine-urea cycle intermediates by the products of genes laterally acquired from bacteria. This metabolic coupling of bacterial- and exosymbiont-derived proteins seems to be fundamental to diatom physiology because the compounds affected include the major diatom osmolyte proline and the precursors for long-chain polyamines required for silica precipitation during cell wall formation. So far, the ornithine-urea cycle is only known for its essential role in the removal of fixed nitrogen in metazoans. In diatoms, this cycle serves as a distribution and repackaging hub for inorganic carbon and nitrogen and contributes significantly to the metabolic response of diatoms to episodic nitrogen availability. The diatom ornithine-urea cycle therefore represents a key pathway for anaplerotic carbon fixation into nitrogenous compounds that are essential for diatom growth and for the contribution of diatoms to marine productivity.

Distribution and expression of microbial rhodopsins in the Baltic Sea and adjacent waters.

Rhodopsins are light-driven ion-pumping membrane proteins found in many organisms and are proposed to be of global importance for oceanic microbial energy generation. Several studies have focused on marine environments, with less exploration of rhodopsins in brackish waters. We investigated microbial rhodopsins in the Baltic Sea using size-fractionated metagenomic and metatranscriptomic datasets collected along a salinity gradient spanning from ∼0 to 35 PSU. The normalised genomic abundance of rhodopsins in Bacteria, as well as rhodopsin gene expression, was highest in the smallest size fraction (0.1-0.8 µm), relative to the medium (0.8-3.0 µm) and large (>3.0 µm) size fractions. The abundance of rhodopsins in the two smaller size fractions displayed a positive correlation with salinity. Proteobacteria and Bacteroidetes rhodopsins were the most abundant while Actinobacteria rhodopsins, or actinorhodopsins, were common at lower salinities. Phylogenetic analysis indicated that rhodopsins have adapted independently to the marine-brackish transition on multiple occasions, giving rise to green light-adapted variants from ancestral blue light-adapted ones. A notable diversity of viral-like rhodopsins was also detected in the dataset and potentially linked with eukaryotic phytoplankton blooms. Finally, a new clade of likely proton-pumping rhodopsin with non-canonical amino acids in the spectral tuning and proton accepting site was identified.

Genomic and functional adaptation in surface ocean planktonic prokaryotes.

The understanding of marine microbial ecology and metabolism has been hampered by the paucity of sequenced reference genomes. To this end, we report the sequencing of 137 diverse marine isolates collected from around the world. We analysed these sequences, along with previously published marine prokaryotic genomes, in the context of marine metagenomic data, to gain insights into the ecology of the surface ocean prokaryotic picoplankton (0.1-3.0 µm size range). The results suggest that the sequenced genomes define two microbial groups: one composed of only a few taxa that are nearly always abundant in picoplanktonic communities, and the other consisting of many microbial taxa that are rarely abundant. The genomic content of the second group suggests that these microbes are capable of slow growth and survival in energy-limited environments, and rapid growth in energy-rich environments. By contrast, the abundant and cosmopolitan picoplanktonic prokaryotes for which there is genomic representation have smaller genomes, are probably capable of only slow growth and seem to be relatively unable to sense or rapidly acclimate to energy-rich conditions. Their genomic features also lead us to propose that one method used to avoid predation by viruses and/or bacterivores is by means of slow growth and the maintenance of low biomass.