Improving Single Cell ‘Omics Methods for Investigating Microbial Dark Matter

Die überwiegende Mehrheit des mikrobiellen Lebens ist unentdeckt und wenig erforscht, da sie bisher noch nicht erfolgreich kultiviert werden konnten. Wir bezeichnen sie daher als mikrobielle dunkle Materie (microbial dark matter, MDM). MDM hat hohes biotechnologisches Potential, z.B. für die Nutzung nachhaltiger Energiequellen, zur biologischen Sanierung kontaminierter Böden, oder für medizinische Anwendungen. Der Einsatz kulturunabhängiger Methoden zur Untersuchung von Mikroorganismen in der Natur, die Metagenomik und Metatranskriptomik, hat unser Verständnis von MDM erheblich verbessert. Allerdings ist es mit diesen Methoden immer noch schwierig, einzelne Spezies bioinformatisch zu analysieren, insbesondere von Organismen mit geringer Häufigkeit in komplexen Habitaten. Stammvariationen, die falsche Zuordnung von Sequenzen, insbesondere mobiler genetische Elemente sowie sich stark wiederholende Sequenzregionen sind nur einige der Probleme, mit denen z.B. die Metagenomik konfrontiert ist. Auch bei der Metatranskriptomik führen die phänotypische Heterogenität der Zellen und die Diversität der mikrobiellen Gemeinschaften zu komplexen Transkriptionsprofilen, die nicht vollständig zugeordnet werden können. Daher wurden die Einzelzellgenomik (single cell genomics, SCG) und die Einzelzelltranskriptomik (single cell transcriptomics, SCT), die zusammen als Einzelzell-\u27omics (SC \u27omics) bezeichnet werden, entwickelt, um die Nachteile der Metagenomik und Metatranskriptomik zu überwinden. Die Anwendung von SCG hat sich zu einem wichtigen Instrument für die Erweiterung unseres Wissens über MDM entwickelt, beispielsweise durch die jüngste Entdeckung mehrerer neuer Phyla, von denen es derzeit nur sogenannte single amplified genomes (SAGs) gibt. Vollständige SAGs von vielen Mikroorganismen, insbesondere von solchen mit geringer Abundanz, sind jedoch aufgrund der vielen technischen Herausforderungen und der hohen Kosten selten. Auch die SCT ist mit den vielen Herausforderungen der Arbeit mit RNA konfrontiert, wie z. B. der kurzen Halbwertszeit von mRNA und geringen Genexpression, weshalb sie in der Mikrobiologie noch nicht häufig angewendet wird. Daher haben sich die hohen Erwartungen an mikrobielle SC \u27omics noch nicht vollständig erfüllen können. In einem typischen SCG-Arbeitsablauf können die Zellen nach der Probenentnahme vor der Einzelzellisolierung mit Fluoreszenzfarbstoffen markiert werden. Nach der Isolierung werden die Zellen lysiert und das Genom anschließend amplifiziert, gefolgt von der Sequenzierung und bioinformatischen Datenanalyse. In dieser Arbeit wurden die Schritte der Zellmarkierung, Isolierung, Lyse und Ganzgenom-Amplifikation (whole genome amplifikation, WGA) verbessert, um die Methodik zu verbessern. Zunächst wurde ein Ansatz zur gezielten Zellmarkierung entwickelt, der die Anreicherung von Mikroorganismen mit geringer Häufigkeit aus Umweltproben ermöglichte. Dieser Ansatz half bei der Entdeckung neuer Phylogenien und Stoffwechseln von Mikroorganismen die in geringer Abundanz vorkommen und die andernfalls durch konventionelle Metagenomik übersehen worden wären. Darüber hinaus trägt dieser Ansatz dazu bei, die Kosten für SCG zu senken, da nun nicht mehr Zehntausende von Einzelzellen sequenziert werden müssen, um seltene Mikroorganismen zu analysieren. Als nächstes wurden die Schritte der Zellisolierung und Zelllyse verbessert, um sowohl physische Zellschäden als auch den DNA-Abbau zu minimieren, was den Erfolg des nachgeschalteten Genom-Amplifikationsschritts erhöht. Für den WGA-Schritt wurde ein Ansatz zur Volumenreduzierung systematisch getestet und etabliert, um die Homogenität und Vollständigkeit der Genomabdeckung deutlich zu verbessern. Dies Ergebnisse der Versuche zeigen, dass eine weitere Volumenreduzierung in den nL oder pL Bereich nicht erforderlich. Die Kosten der WGA konnten um 97,5 % gesenkt werden konnten, was den Durchsatz von SCG erhöhen und die Verwendung dieses Ansatzes in weiteren Forschungsgruppen positiv beeinflussen dürfte. Da SCG allein nur Informationen über die Phylogenie, genetische Struktur und das Stoffwechselpotenzial, nicht aber über die tatsächliche Aktivität einer Zelle liefert, wurde in dieser Arbeit eine mikrobielle SCT-Pipeline entwickelt, um die individuellen Funktionen der Zelle in einer Gemeinschaft besser zu verstehen. Derzeit gibt es nur sehr wenige Methoden für mikrobielle SCT, und die, die es gibt, bleiben aufgrund ihrer schwierigen Anwendung und geringen Zugänglichkeit außerhalb ihrer jeweiligen Arbeitsgruppen weitgehend ungenutzt. Daher wurden in dieser Studie Änderungen und Verbesserungen an einer eukaryotischen Einzelzell-RNA-Sequenzierungsmethode (RNA-seq) vorgenommen, um ihre Anwendung bei Prokaryoten zu ermöglichen. Es wurde festgestellt, dass der Zusatz von Dithiothreitol (DTT) im Lysepuffer wahrscheinlich die DNase I hemmt, was zu einer DNA Kontamination führt. Die hier vorgestellten Einzelzell-RNA-seq-Ergebnisse zeigten zuverlässige Transkriptionsprofile im Vergleich zu RNA-seq-Ergebnissen aus der gesamten Probe. Dies wurde auch durch ein Proof-of-Principle-Experiment bestätigt, bei dem hitzeschockbehandelte und unbehandelte Escherichia coli Zellen verglichen wurden. Darüber hinaus wurden in den Einzelzelldaten im Vergleich zur Populations-Analyse Hinweise auf einzigartige Reaktionen bei der Synthese von Sekundärmetaboliten und der CRISPR-Cas-Editierung gefunden, was die Bedeutung der Untersuchung der Heterogenität seltener funktioneller Subpopulationen auf Einzelzellebene unterstreicht. Insgesamt wird erwartet, dass die verbesserten SCG- und SCT-Methoden, die in dieser Arbeit etabliert wurden, eine breitere Anwendung für ein besseres Verständnis der MDM-Diversität und -Funktion in der Umwelt ermöglichen

Back to Basics: A Simplified Improvement to Multiple Displacement Amplification for Microbial Single-Cell Genomics

Microbial single-cell genomics (SCG) provides access to the genomes of rare and uncultured microorganisms and is a complementary method to metagenomics. Due to the femtogram-levels of DNA in a single microbial cell, sequencing the genome requires whole genome amplification (WGA) as a preliminary step. However, the most common WGA method, multiple displacement amplification (MDA), is known to be costly and biased against specific genomic regions, preventing high-throughput applications and resulting in uneven genome coverage. Thus, obtaining high-quality genomes from many taxa, especially minority members of microbial communities, becomes difficult. Here, we present a volume reduction approach that significantly reduces costs while improving genome coverage and uniformity of DNA amplification products in standard 384-well plates. Our results demonstrate that further volume reduction in specialized and complex setups (e.g., microfluidic chips) is likely unnecessary to obtain higher-quality microbial genomes. This volume reduction method makes SCG more feasible for future studies, thus helping to broaden our knowledge on the diversity and function of understudied and uncharacterized microorganisms in the environment

Genome characterization of two novel deep-sea sediment fungi, Penicillium pacificagyrus sp. nov. and Penicillium pacificasedimenti sp. nov., from South Pacific Gyre subseafloor sediments, highlights survivability

Background Marine deep subsurface sediments were once thought to be devoid of eukaryotic life, but advances in molecular technology have unlocked the presence and activity of well-known closely related terrestrial and marine fungi. Commonly detected fungi in deep marine sediment environments includes Penicillium, Aspergillus, Cladosporium, Fusarium, and Schizophyllum, which could have important implications in carbon and nitrogen cycling in this isolated environment. In order to determine the diversity and unknown metabolic capabilities of fungi in deep-sea sediments, their genomes need to be fully analyzed. In this study, two Penicillium species were isolated from South Pacific Gyre sediment enrichments during Integrated Ocean Drilling Program Expedition 329. The inner gyre has very limited productivity, organic carbon, and nutrients. Results Here, we present high-quality genomes of two proposed novel Penicillium species using Illumina HiSeq and PacBio sequencing technologies. Single-copy homologues within the genomes were compared to other closely related genomes using OrthoMCL and maximum-likelihood estimation, which showed that these genomes were novel species within the genus Penicillium. We propose to name isolate SPG-F1 as Penicillium pacificasedimenti sp. nov. and SPG-F15 as Penicillium pacificagyrus sp. nov. The resulting genome sizes were 32.6 Mbp and 36.4 Mbp, respectively, and both genomes were greater than 98% complete as determined by the presence of complete single-copy orthologs. The transposable elements for each genome were 4.87% for P. pacificasedimenti and 10.68% for P. pacificagyrus. A total of 12,271 genes were predicted in the P. pacificasedimenti genome and 12,568 genes in P. pacificagyrus. Both isolates contained genes known to be involved in the degradation of recalcitrant carbon, amino acids, and lignin-derived carbon. Conclusions Our results provide the first constructed genomes of novel Penicillium isolates from deep marine sediments, which will be useful for future studies of marine subsurface fungal diversity and function. Furthermore, these genomes shed light on the potential impact fungi in marine sediments and the subseafloor could have on global carbon and nitrogen biogeochemical cycles and how they may be persisting in the most energy-limited sedimentary biosphere

Besser und günstiger: Volumenreduktion in der Einzelzellgenomik

Single cell genomics (SCG) is a is a complementary method to metagenomics for exploring the genomes of uncultivated microorganisms. However, due to the minute amounts of DNA in the individual microbial cell, an amplification step is required before sequencing. Unfortunately, this reaction is notoriously costly and does not amplify all genomic regions equally well, preventing high-throughput applications and leading to incomplete and biased genomes. Here, we show a simple volume reduction approach to make SCG more feasible

A three-colour stress biosensor reveals multimodal response in single cells and spatiotemporal dynamics of biofilms

The plethora of stress factors that can damage microbial cells has evolved sophisticated stress response mechanisms. While existing bioreporters can monitor individual responses, sensors for detecting multimodal stress responses in living microorganisms are still lacking. Orthogonally detectable red, green, and blue fluorescent proteins combined in a single plasmid, dubbed RGB-S reporter, enable simultaneous, independent, and real-time analysis of the transcriptional response of Escherichia coli using three promoters which report physiological stress (PosmY for RpoS), genotoxicity (PsulA for SOS), and cytotoxicity (PgrpE for RpoH). The bioreporter is compatible with standard analysis and Fluorescent Activated Cell Sorting (FACS) combined with subsequent transcriptome analysis. Various stressors, including the biotechnologically relevant 2-propanol, activate one, two, or all three stress responses, which can significantly impact non-stress-related metabolic pathways. Implemented in microfluidic cultivation with confocal fluorescence microscopy imaging, the RGB-S reporter enabled spatiotemporal analysis of live biofilms revealing stratified subpopulations of bacteria with heterogeneous stress responses

Taxonomic Re-Classification and Expansion of the Phylum Chloroflexota Based on over 5000 Genomes and Metagenome-Assembled Genomes

The phylum Chloroflexota (formerly Chloroflexi) encompasses metabolically diverse bacteria that often have high prevalence in terrestrial and aquatic habitats, some even with biotechnological application. However, there is substantial disagreement in public databases which lineage should be considered a member of the phylum and at what taxonomic level. Here, we addressed these issues through extensive phylogenomic analyses. The analyses were based on a collection of >5000 Chloroflexota genomes and metagenome-assembled genomes (MAGs) from public databases, novel environmental sites, as well as newly generated MAGs from publicly available sequence reads via an improved binning approach incorporating covariance information. Based on calculated relative evolutionary divergence, we propose that Candidatus Dormibacterota should be listed as a class (i.e., Ca. Dormibacteria) within Chloroflexota together with the classes Anaerolineae, Chloroflexia, Dehalococcoidia, Ktedonobacteria, Ca. Limnocylindria, Thermomicrobia, and two other classes containing only uncultured members. All other Chloroflexota lineages previously listed at the class rank appear to be rather orders or families in the Anaerolineae and Dehalococcoidia, which contain the vast majority of genomes and exhibited the strongest phylogenetic radiation within the phylum. Furthermore, the study suggests that a common ecophysiological capability of members of the phylum is to successfully cope with low energy fluxes

Drug‐Induced Differential Gene Expression Analysis on Nanoliter Droplet Microarrays: Enabling Tool for Functional Precision Oncology

Drug-induced differential gene expression analysis (DGEA) is essential for uncovering the molecular basis of cell phenotypic changes and understanding individual tumor responses to anticancer drugs. Performing high throughput DGEA is challenging due to the high cost and labor-intensive multi-step sample preparation protocols. In particular, performing drug-induced DGEA on cancer cells derived from patient biopsies is even more challenging due to the scarcity of available cells. A novel, miniaturized, nanoliter-scale method for drug-induced DGEA is introduced, enabling high-throughput and parallel analysis of patient-derived cell drug responses, overcoming the limitations and laborious nature of traditional protocols. The method is based on the Droplet Microarray (DMA), a microscope glass slide with hydrophilic spots on a superhydrophobic background, facilitating droplet formation for cell testing. DMA allows microscopy-based phenotypic analysis, cDNA extraction, and DGEA. The procedure includes cell lysis for mRNA isolation and cDNA conversion followed by droplet pooling for qPCR analysis. In this study, the drug-induced DGEA protocol on the DMA platform is demonstrated using patient-derived chronic lymphocytic leukemia (CLL) cells. This methodology is critical for DGEA with limited cell numbers and promise for applications in functional precision oncology. This method enables molecular profiling of patient-derived samples after drug treatment, crucial for understanding individual tumor responses to anticancer drugs

A coupled channel analysis of the centrally produced K+K- and pi+pi- final states in pp interactions at 450 GeV/c

A coupled channel analysis of the centrally produced K+K- and pi+pi- final states has been performed in pp collisions at an incident beam momentum of 450 GeV/c. The pole positions and branching ratios to pipi and KK of the f0(980), f0(1370), f0(1500) and f0(1710) have been determined. A systematic study of the production properties of all the resonances observed in the pi+pi- and K+K- channels has been performed.Comment: 16 pages, Latex, 5 Figure

Corporate reputation past and future: a review and integration of existing literature and a framework for future research

The concept of corporate reputation is steadily growing in interest among management researchers and practitioners. In this article, we trace key milestones in the development of reputation literature over the past six decades to suggest important research gaps as well as to provide contextual background for a subsequent integration of approaches and future outlook. In particular we explore the need for better categorised outcomes; a wider range of causes; and a deeper understanding of contingencies and moderators to advance the field beyond its current state while also taking account of developments in the macro business environment. The article concludes by presenting a novel reputation framework that integrates insights from reputation theory and studies, outlines gaps in knowledge and offers directions for future research

Global patient outcomes after elective surgery: prospective cohort study in 27 low-, middle- and high-income countries.

BACKGROUND: As global initiatives increase patient access to surgical treatments, there remains a need to understand the adverse effects of surgery and define appropriate levels of perioperative care. METHODS: We designed a prospective international 7-day cohort study of outcomes following elective adult inpatient surgery in 27 countries. The primary outcome was in-hospital complications. Secondary outcomes were death following a complication (failure to rescue) and death in hospital. Process measures were admission to critical care immediately after surgery or to treat a complication and duration of hospital stay. A single definition of critical care was used for all countries. RESULTS: A total of 474 hospitals in 19 high-, 7 middle- and 1 low-income country were included in the primary analysis. Data included 44 814 patients with a median hospital stay of 4 (range 2-7) days. A total of 7508 patients (16.8%) developed one or more postoperative complication and 207 died (0.5%). The overall mortality among patients who developed complications was 2.8%. Mortality following complications ranged from 2.4% for pulmonary embolism to 43.9% for cardiac arrest. A total of 4360 (9.7%) patients were admitted to a critical care unit as routine immediately after surgery, of whom 2198 (50.4%) developed a complication, with 105 (2.4%) deaths. A total of 1233 patients (16.4%) were admitted to a critical care unit to treat complications, with 119 (9.7%) deaths. Despite lower baseline risk, outcomes were similar in low- and middle-income compared with high-income countries. CONCLUSIONS: Poor patient outcomes are common after inpatient surgery. Global initiatives to increase access to surgical treatments should also address the need for safe perioperative care. STUDY REGISTRATION: ISRCTN5181700