Biology subjects

Karst, S. M.

Publications and source records attributed to Karst, S. M..

15 recordsLinked to original sources

bioRxiv2024

Microflora Danica: the atlas of Danish environmental microbiomes

The last 20 years have witnessed unprecedented advances in revealing the microbiomes underpinning important processes in natural and human associated environments. Recent large-scale metagenome surveys record the variety of microbial life in the oceans1, wastewater2, human gut3,4, and earth5,6, with compilations encompassing thousands of public datasets7-13. So far, large-scale microbiome studies either miss functional information or consistency in sample processing, and although they may cover thousands of locations, these are missing resolution, sparsely located, or lacking metadata. Here, we present Microflora Danica, an atlas of Danish environmental microbiomes, encompassing 10,686 shotgun metagenomes and 449 full-length 16S and 18S rRNA datasets linked to a detailed 5 level habitat classification scheme. We determine that while human-disturbed habitats have high alpha diversity, the same species reoccur, revealing hidden homogeneity and underlining the importance of natural systems for total species (gamma) diversity. In-depth studies of nitrifiers, a functional group closely linked to climate change, challenge existing perceptions regarding habitat preference and discover several novel nitrifiers as more abundant than canonical nitrifiers. Together, the Microflora Danica dataset provides an unprecedented resource and the foundation for answering fundamental questions underlying microbial ecology: what drives microbial diversity, distribution and function.

microbiology↗

bioRxiv2024

Metabolic immaturity of newborns and breast milk bile acids are the central determinants of heightened neonatal vulnerability to norovirus diarrhea

Noroviruses are the leading global cause of acute gastroenteritis, responsible for 685 million annual cases. While all age groups are susceptible to noroviruses, children are vulnerable to more severe infections than adults, underscored by 200 million pediatric cases and up to 200,000 deaths in children annually. Understanding the basis for the increased vulnerability of young hosts is critical to developing effective treatments. The pathogenic outcome of any enteric virus infection is governed by a complex interplay between the virus, intestinal microbiota, and host immune factors. A central mediator in these complex relationships are host- and microbiota-derived metabolites. Noroviruses bind a specific class of metabolites, bile acids, which are produced by the host and then modified by commensal bacterial enzymes. Paradoxically, bile acids can have both proviral and antiviral roles during norovirus infections. Considering these opposing effects, the microbiota-regulated balance of the bile acid pool may be a key determinant of the pathogenic outcome of a norovirus infection. The bile acid pool in newborns is unique due to immaturity of host metabolic pathways and developing gut microbiota, which could underlie the vulnerability of these hosts to severe norovirus infections. Supporting this concept, we demonstrate herein that microbiota and their bile acid metabolites protect from severe norovirus diarrhea whereas host-derived bile acids promote disease. Remarkably, we also report that maternal bile acid metabolism determines neonatal susceptibility to norovirus diarrhea during breastfeeding by delivering proviral bile acids to the newborn. Finally, directed targeting of maternal and neonatal bile acid metabolism can protect the neonatal host from norovirus disease. Altogether, these data support the conclusion that metabolic immaturity in newborns and ingestion of proviral maternal metabolites in breast milk are the central determinants of heightened neonatal vulnerability to norovirus disease.

microbiology↗

bioRxiv2023

High-throughput DNA extraction and cost-effective miniaturized metagenome and amplicon library preparation of soil samples for DNA sequencing

Reductions in sequencing costs have enabled widespread use of shotgun metagenomics and amplicon sequencing, which have drastically improved our understanding of the microbial world. However, large sequencing projects are now hampered by the cost of library preparation and low sample throughput. Here, we benchmarked three high-throughput DNA extraction methods: ZymoBIOMICS 96 MagBead DNA Kit, MP BiomedicalsTM FastDNATM-96 Soil Microbe DNA Kit, and DNeasy(R) 96 PowerSoil(R) Pro QIAcube(R) HT Kit. The DNA extractions were evaluated based on length, quality, quantity, and the observed microbial community across five diverse soil types. DNA extraction of all soil types was successful for all kits, however DNeasy(R) 96 PowerSoil(R) Pro QIAcube(R) HT Kit excelled across all performance parameters. We further used the nanoliter dispensing robot I.DOT One to miniaturize Illumina amplicon and metagenomic library preparation volumes by a factor of 5 and 10, respectively, with no significant impact on the observed microbial communities. With these protocols, DNA extraction, metagenomic library preparation, or amplicon library preparation for one 96-well plate are approx. 3, 5, and 6 hours, respectively. Furthermore, the miniaturization of amplicon and metagenome library preparation reduces the chemical and plastic costs from 5.0 to 3.6 and 59 to 7.3 USD pr. sample.

microbiology↗

bioRxiv2022

Greengenes2 enables a shared data universe for microbiome studies

16S rRNA and shotgun metagenomics studies typically yield different results, usually attributed to biases in PCR amplification of 16S rRNA genes. Here, we introduce Greengenes2 and show that differences in reference phylogeny are more important. By inserting sequences into a whole-genome phylogeny, we show that 16S rRNA and shotgun metagenomic data generated from the same samples agree in principal coordinates space, taxonomy, and in phenotype effect size when analyzed with the same tree.

bioinformatics↗

bioRxiv2021

Assessing long-read sequencing with Nanopore R9, R10, and PacBio CCS to obtain high-quality metagenome assembled genomes from complex microbial communities

Long-read Oxford Nanopore sequencing has democratized microbial genome sequencing and enables the recovery of highly contiguous microbial genomes from isolates or metagenomes. However, to obtain near-perfect genomes it has been necessary to include short-read polishing to correct insertions and deletions derived from homopolymer regions. Here, we show that Oxford Nanopore R10.4 can be used to generate near-perfect microbial genomes from isolates or metagenomes without shortread or reference polishing.

microbiology↗

bioRxiv2021

Multi-omics profiling of Earth's biomes reveals that microbial and metabolite composition are shaped by the environment

As our understanding of the structure and diversity of the microbial world grows, interpreting its function is of critical interest for understanding and managing the many systems microbes influence. Despite advances in sequencing, lack of standardization challenges comparisons among studies that could provide insight into the structure and function of microbial communities across multiple habitats on a planetary scale. Technical variation among distinct studies without proper standardization of approaches prevents robust meta-analysis. Here, we present a multi-omics, meta-analysis of a novel, diverse set of microbial community samples collected for the Earth Microbiome Project. We include amplicon (16S, 18S, ITS) and shotgun metagenomic sequence data, and untargeted metabolomics data (liquid chromatography-tandem mass spectrometry and gas chromatography mass spectrometry), centering our description on relationships and co-occurrences of microbially-related metabolites and microbial taxa across environments. Standardized protocols and analytical methods for characterizing microbial communities, including assessment of molecular diversity using untargeted metabolomics, facilitate identification of shared microbial and metabolite features, permitting us to explore diversity at extraordinary scale. In addition to a reference database for metagenomic and metabolomic data, we provide a framework for incorporating additional studies, enabling the expansion of existing knowledge in the form of a community resource that will become more valuable with time. To provide examples of applying this database, we outline important ecological questions that can be addressed, and test the hypotheses that every microbe and metabolite is everywhere, but the environment selects. Our results show that metabolite diversity exhibits turnover and nestedness related to both microbial communities and the environment. The relative abundances of microbially-related metabolites vary and co-occur with specific microbial consortia in a habitat-specific manner, and highlight the power of certain chemistry - in particular terpenoids - in distinguishing Earths environments.

ecology↗

bioRxiv2021

Genome-scale CRISPR Screens Identify Host Factors that Promote Human Coronavirus Infection

The COVID-19 pandemic has resulted in 153 million infections and 3.2 million deaths as of May 2021. While effective vaccines are being administered globally, there is still a great need for antiviral therapies as potentially antigenically distinct SARS-CoV-2 variants continue to emerge across the globe. Viruses require host factors at every step in their life cycle, representing a rich pool of candidate targets for antiviral drug design. To identify host factors that promote SARS-CoV-2 infection with potential for broad-spectrum activity across the coronavirus family, we performed genome-scale CRISPR knockout screens in two cell lines (Vero E6 and HEK293T ectopically expressing ACE2) with SARS-CoV-2 and the common cold-causing human coronavirus OC43. While we identified multiple genes and functional pathways that have been previously reported to promote human coronavirus replication, we also identified a substantial number of novel genes and pathways. Of note, host factors involved in cell cycle regulation were enriched in our screens as were several key components of the programmed mRNA decay pathway. Finally, we identified novel candidate antiviral compounds targeting a number of factors revealed by our screens. Overall, our studies substantiate and expand the growing body of literature focused on understanding key human coronavirus-host cell interactions and exploit that knowledge for rational antiviral drug development. One Sentence SummaryGenome-wide CRISPR screens identified host factors that promote human coronavirus infection, revealing novel antiviral drug targets.

microbiology↗

bioRxiv2021

Human norovirus infection of primary B cells triggers immune activation in vitro

Human norovirus (HNoV) is a global health and socio-economic burden, estimated to infect every individual at least five times during their lifetime. The underlying mechanism for the potential lack of long-term immune protection from HNoV infections is not understood and prompted us to investigate HNoV susceptibility of primary human B cells and its functional impact. Primary B cells isolated from whole-blood were infected with HNoV-positive stool samples and harvested 3 days post infection (dpi) to assess viral RNA yield by RT-qPCR. A 3-18 fold increase in HNoV RNA yield was observed in 50-60% donors. Infection was further confirmed in B cells derived from splenic and lymph node biopsies. Next, we characterized infection of whole-blood derived B cells by flow cytometry in specific functional B cell subsets (naive CD27-IgD+, memory switched CD27+IgD-, memory unswitched CD27+IgD+ and double-negative CD27-IgD-). While susceptibility of subsets was similar, we observed changes in B cell subsets distribution upon infection that were recapitulated after treatment with HNoV virus-like particles and mRNA encoding for HNoV NS1-2 protein. Importantly, treatment of immortalized BJAB B cell lines with the predicted recombinant NS1 protein triggered cell proliferation, increased ATP production, and induced metabolic changes, as detected by means of CFSE/Ki67 staining, seahorse analysis and metabolomics, respectively. These data demonstrate the susceptibility of primary B cells to HNoV infection and suggest that the secreted NS1 protein affects B cell function, proliferation and metabolism in vitro, which could have implications for viral pathogenesis and immune response in vivo. ImportanceHuman norovirus (HNoV) is the most prevalent causative agent of gastroenteritis worldwide. Infection results in a self-limiting disease that can become chronic and severe in the immunocompromised, elderly and infants. There are currently no approved therapeutic and preventative strategies to limit the health and socio-economic burden associated with HNoV infections. Moreover, HNoV does not elicit life-long immunity as repeat infections are common, presenting a challenge for vaccine development. Given the importance of B cells for humoral immunity, we investigated susceptibility and impact of HNoV infection on human B cells. We found that HNoV replicates in human primary B cells derived from blood, spleen and lymph nodes specimens and induces functional changes in B cells, mediated in part by the non-structural protein NS1. Because of the secreted nature of NS1, we put forward the hypothesis that HNoV infection can modulate bystander B cell function with potential implications in systemic immune response.

microbiology↗

bioRxiv2020

Connecting structure to function with the recovery of over 1000 high-quality activated sludge metagenome-assembled genomes encoding full-length rRNA genes using long-read sequencing

Microorganisms are critical to water recycling, pollution removal and resource recovery processes in the wastewater industry. While the structure of this complex community is increasingly understood based on 16S rRNA gene studies, this structure cannot currently be linked to functional potential due to the absence of high-quality metagenome-assembled genomes (MAGs) with full-length rRNA genes for nearly all species. Here, we sequence 23 Danish full-scale wastewater treatment plant metagenomes, producing >1 Tbp of long-read and >0.9 Tbp of short-read data. We recovered 1083 high-quality MAGs, including 57 closed circular genomes. The MAGs accounted for ~30% of the community, and meet the stringent MIMAG high-quality draft requirements including full-length rRNA genes. We show how novel high-quality MAGs in combination with >13 years of amplicon data, Raman microspectroscopy and fluorescence in situ hybridisation can be used to uncover abundant undescribed lineages belonging to important functional groups.

microbiology↗

bioRxiv2020

A Novel Small Animal Model of Norovirus Diarrhea

Human noroviruses are the leading cause of severe childhood diarrhea worldwide yet we know very little about their pathogenic mechanisms. Murine noroviruses cause diarrhea in interferon-deficient adult mice but these hosts also develop systemic pathology and lethality, reducing confidence in the translatability of findings to human norovirus disease. Herein we report that a murine norovirus causes self-resolving diarrhea in the absence of systemic disease in wild-type neonatal mice, thus mirroring the key features of human norovirus disease and representing a robust norovirus small animal disease model. Intriguingly, lymphocytes are critical for controlling acute norovirus replication while simultaneously contributing to disease severity, likely reflecting their dual role as targets of viral infection and key components of the host response.

microbiology↗

bioRxiv2020

Exploring the upper pH limits of nitrite oxidation: diversity, ecophysiology, and adaptive traits of haloalkalitolerant Nitrospira

Nitrite-oxidizing bacteria of the genus Nitrospira are key players of the biogeochemical nitrogen cycle. However, little is known about their occurrence and survival strategies in extreme pH environments. Here, we report on the discovery of physiologically versatile, haloalkalitolerant Nitrospira that drive nitrite oxidation at exceptionally high pH. Nitrospira distribution, diversity, and ecophysiology were studied in hypo- and subsaline (1.3-12.8 g salt/l), highly alkaline (pH 8.9-10.3) lakes by amplicon sequencing, metagenomics, and cultivation-based approaches. Surprisingly, not only were Nitrospira populations detected, but they were also considerably diverse with presence of members of Nitrospira lineages I, II and IV. Furthermore, the ability of Nitrospira enrichment cultures to oxidize nitrite at neutral to highly alkaline pH of 10.5 was demonstrated. Metagenomic analysis of a newly enriched Nitrospira lineage IV species, "Candidatus Nitrospira alkalitolerans", revealed numerous adaptive features of this organism to its extreme environment. Among them were a sodium-dependent N-type ATPase and NADH:quinone oxidoreductase next to the proton-driven forms usually found in Nitrospira. Other functions aid in pH and cation homeostasis and osmotic stress defense. "Ca. Nitrospira alkalitolerans" also possesses group 2a and 3b [NiFe] hydrogenases, suggesting it can use hydrogen as alternative energy source. These results reveal how Nitrospira cope with strongly fluctuating pH and salinity conditions and expand our knowledge of nitrogen cycling in extreme habitats.

microbiology↗

bioRxiv2019

Enabling high-accuracy long-read amplicon sequences using unique molecular identifiers and Nanopore sequencing

High-throughput amplicon sequencing of large genomic regions remains challenging for short-read technologies. Here, we report a high-throughput amplicon sequencing approach combining unique molecular identifiers (UMIs) with Oxford Nanopore Technologies or Pacific Biosciences CCS sequencing, yielding high accuracy single-molecule consensus sequences of large genomic regions. Our approach generates amplicon and genomic sequences of >10,000 bp in length with a mean error-rate of 0.0049-0.0006% and chimera rate <0.022%.

molecular biology↗

bioRxiv2018

ampvis2: an R package to analyse and visualise 16S rRNA amplicon data

SummaryMicrobial community analysis using 16S rRNA gene amplicon sequencing is the backbone of many microbial ecology studies. Several approaches and pipelines exist for processing the raw data generated through DNA sequencing and convert the data into OTU-tables. Here we present ampvis2, an R package designed for analysis of microbial community data in OTU-table format with focus on simplicity, reproducibility, and sample metadata integration, with a minimal set of intuitive commands. Unique features include flexible heatmaps and simplified ordination. By generating plots using the ggplot2 package, ampvis2 produces publication-ready figures that can be easily customised. Furthermore, ampvis2 includes features for interactive visualisation, which can be convenient for larger, more complex data.\n\nAvailabilityampvis2 is implemented in the R statistical language and is released under the GNU A-GPL license. Documentation website and source code is maintained at: https://github.com/MadsAlbertsen/ampvis2\n\nContactMads Albertsen (ma@bio.aau.dk)

bioinformatics↗

bioRxiv2017

Novel prosthecate bacteria from the candidate phylum Acetothermia revealed by culture-independent genomics and advanced microscopy

Members of the candidate phylum Acetothermia are globally distributed and detected in various habitats. However, little is known about their physiology and ecological importance. In this study, an OTU belonging to Acetothermia was detected at high abundance in two full-scale anaerobic digesters. The first closed genome from this phylum was obtained by differential coverage binning of metagenomes and scaffolding with nanopore data. Genome annotation and metabolic reconstruction suggested an anaerobic chemoheterotrophic lifestyle in which the bacterium obtain energy and carbon via fermentation of peptides, amino acids, and simple sugars to acetate, formate, and hydrogen. The morphology was unusual and composed of a central rod-shaped cell with bipolar prosthecae as revealed by fluorescence in situ hybridization combined with confocal laser scanning microscopy, Raman microspectroscopy and atomic force microscopy. We hypothesize that these prosthecae allow for increased nutrient uptake by greatly expanding the cell surface area, providing a competitive advantage under nutrient-limited conditions.

microbiology↗

bioRxiv2017

Identifying the abundant and active microorganisms common to full scale anaerobic digesters

Anaerobic digestion is widely applied to treat organic waste at wastewater treatment plants. Characterisation of the underlying microbiology represents a source of information to develop strategies for improved operation. To this end, we investigated the microbial community composition of thirty-two full-scale digesters over a six-year period using 16S rRNA gene amplicon sequencing. Sampling of the sludge fed into these systems revealed that several of the most abundant populations were likely inactive and immigrating with the influent. This observation indicates that a failure to consider immigration will interfere with correlation analysis and give an inaccurate picture of the active microbial community. Furthermore, several abundant OTUs could not be classified to genus level with commonly applied taxonomies, making inference of their function unreliable. As such, the existing MiDAS taxonomy was updated to include these abundant phylotypes. The communities of individual plants surveyed were remarkably similar - with only 300 OTUs representing 80% of the total reads across all plants, and 15% of these identified as likely inactive immigrating microbes. By identifying the abundant and active taxa in anaerobic digestion, this study paves the way for targeted characterisation of the process important organisms towards an in-depth understanding of the microbial ecology of these biotechnologically important systems.

microbiology↗