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Sponges (phylum Porifera) are evolutionary ancient, sessile filter-feeders that harbor a largely diverse microbial community within their internal mesohyl matrix. Throughout this thesis project, I aimed at exploring the adaptations of these symbionts to life within their sponge host by sequencing and analyzing the genomes of a variety of bacteria from the microbiome of the Mediterranean sponge Aplysina aerophoba. Employed methods were fluorescence-activated cell sorting with subsequent multiple displacement amplification and single-cell / ‘mini-metagenome’ sequencing, and metagenomic sequencing followed by differential coverage binning. These two main approaches both aimed at obtaining genome sequences of bacterial symbionts of A. aerophoba, that were then compared to each other and to references from other environments, to gain information on adaptations to the host sponge environment and on possible interactions with the host and within the microbial community.
Cyanobacteria are frequent members of the sponge microbial community. My ‘mini-metagenome’ sequencing project delivered three draft genomes of “Candidatus Synechococcus spongiarum,” the cyanobacterial symbiont of A. aerophoba and many more sponges inhabiting the photic zone. The most complete of these genomes was compared to other clades of this symbiont and to closely related free-living cyanobacterial references in a collaborative project published in Burgsdorf I*, Slaby BM* et al. (2015; *shared first authorship). Although the four clades of “Ca. Synechococcus spongiarum” from the four sponge species A. aerophoba, Ircinia variabilis, Theonella swinhoei, and Carteriospongia foliascens were approximately 99% identical on the level of 16S rRNA gene sequences, they greatly differed on the genomic level. Not only the genome sizes were different from clade to clade, but also the gene content and a number of features including proteins containing the eukaryotic-type domains leucine-rich repeats or tetratricopeptide repeats. On the other hand, the four clades shared a number of features such as ankyrin repeat domain-containing proteins that seemed to be conserved also among other microbial phyla in different sponge hosts and from different geographic locations. A possible novel mechanism for host phagocytosis evasion and phage resistance by means of an altered O antigen of the lipopolysaccharide was identified.
To test previous hypotheses on adaptations of sponge-associated bacteria on a broader spectrum of the microbiome of A. aerophoba while also taking a step forward in methodology, I developed a bioinformatic pipeline to combine metagenomic Illumina short-read sequencing data with PacBio long-read data. At the beginning of this project, no pipelines to combine short-read and long-read data for metagenomics were published, and at time of writing, there are still no projects published with a comparable aim of un-targeted assembly, binning and analysis of a metagenome. I tried a variety of assembly programs and settings on a simulated test dataset reflecting the properties of the real metagenomic data. The developed assembly pipeline improved not only the overall assembly statistics, but also the quality of the binned genomes, which was evaluated by comparison to the originally published genome assemblies.
The microbiome of A. aerophoba was studied from various angles in the recent years, but only genomes of the candidate phylum Poribacteria and the cyanobacterial sequences from my above-described project have been published to date. By applying my newly developed assembly pipeline to a metagenomic dataset of A. aerophoba consisting of a PacBio long-read dataset and six Illumina short-read datasets optimized for subsequent differential coverage binning, I aimed at sequencing a larger number and greater diversity of symbionts. The results of this project are currently in review by The ISME Journal. The complementation of Illumina short-read with PacBio long-read sequencing data for binning of this highly complex metagenome greatly improved the overall assembly statistics and improved the quality of the binned genomes. Thirty-seven genomes from 13 bacterial phyla and candidate phyla were binned representing the most prominent members of the microbiome of A. aerophoba. A statistical comparison revealed an enrichment of genes involved in restriction modification and toxin-antitoxin systems in most symbiont genomes over selected reference genomes. Both are defense features against incoming foreign DNA, which may be important for sponge symbionts due to the sponge’s filtration and phagocytosis activity that exposes the symbionts to high levels of free DNA. Also host colonization and matrix utilization features were significantly enriched. Due to the diversity of the binned symbiont genomes, a within-symbionts genome comparison was possible, that revealed three guilds of symbionts characterized by i) nutritional specialization on the metabolization of carnitine, ii) specialization on sulfated polysaccharides, and iii) apparent nutritional generalism. Both carnitine and sulfated polysaccharides are abundant in the sponge extracellular matrix and therefore available to the sponge symbionts as substrates. In summary, the genomes of the diverse community of symbionts in A. aerophoba were united in their defense features, but specialized regarding their nutritional preferences.
Schwämme (Phylum Porifera) sind der älteste rezente Tierstamm der Erde. Insbesondere marine Vertreter dieser sessilen Invertebraten sind oftmals mit einem mikrobiellen Konsortium assoziiert, welches hochgradig wirtsspezifisch und phylogenetisch divers ist. Die Biomasse dieser Mikroflora kann dabei rund die Hälfte der Masse eines Schwamms ausmachen. Die Komplexität des Konsortiums sowie der Mangel an kultivierbaren Vertretern der Schwamm-spezifischen Kladen erschwert dabei eine gezielte funktionelle Charakterisierung. Von besonderem Interesse hierbei ist das exklusiv in marinen Schwämmen vorzufindende Candidatus Phylum Poribacteria, für das bislang kein kultivierter Vertreter vorliegt. Die metabolisch aktiven und hochabundanten Poribakterien liegen in der extrazellulären Matrix des Schwammes vor und zeichnen sich durch das Vorhandensein einer Nukleoid-ähnlichen intrazellulären Struktur aus. Ziel dieser Promotionsarbeit war es, neue Einzelzell-basierte Methoden auf das Gebiet der funktionellen Charakterisierung von Bakterien anzuwenden, welche spezifisch mit dem mediterranen Schwamm Aplysina aerophoba assoziiert sind. Dabei wurden sowohl kultivierungs-abhängige, als auch kultivierungs-unabhängige Versuchsansätze verfolgt. Das Hauptaugenmerk dieser Studien lag dabei auf dem Candidatus Phylum Poribacteria. Während auf dem ‚dilution-to-extinction‘-Prinzip beruhende Hochdurchsatz-Kultivierungen nicht zum Erhalt einer Schwammsymbionten-Reinkultur führten, konnten durch eine Kombination aus FACS-Vereinzelung von Schwamm-assoziierten Bakterien und anschließenden Einzel-Genom-Amplifizierungen (‚whole genome amplifications‘) umfassende Einblicke in die metabolischen Kapazitäten von Schwammsymbionten gewonnen werden. Ferner gelang durch die Anwendung dieser neuen kultivierungs-unabhängigen Methode eine spezifische Verknüpfung von Phylogenie und Funktion Schwamm-assoziierter, nicht-kultivierbarer Bakterien. So konnte im Rahmen dieser Dissertation eine neue nicht-ribosomale Peptidsynthetase (NRPS) einem Vertreter einer Schwamm-spezifischen Chloroflexi-Klade zugewiesen werden. Ferner gelang die Zuordnung einer exklusiv in marinen Schwämmen vorgefundenen Polyketidsynthase (Sup-PKS) zu den Poribacteria. Die Klonierung von hochmolekularer, Einzel-Genom-amplifizierter DNA in Cosmide gewährte zudem Einblicke in den genomischen Kontext dieser, mit dem bakteriellen Sekundärmetabolismus assoziierten Gene. Die Pyrosequenzierung eines amplifizierten, von einem einzelnen Poribakterium abstammenden Genoms führte zudem zum Erhalt von rund zwei Megabasen an genetischer Information über diese Schwammsymbionten. Dadurch wurden detaillierte Informationen über den poribakteriellen Primär- und Sekundärstoffwechsel gewonnen. Die Auswertung der automatisch annotierten 454-Daten erlaubte die Rekonstruktion von Stoffwechselwegen, so z.B. der Glykolyse oder des Citratzyklus und bestätigte das Vorhandensein eines Sup-PKS-Gens im poribakteriellen Genom. Ferner konnten Gemeinsamkeiten mit den Schwesterphyla Planctomycetes, Chlamydiae und Verrucomicrobia gefunden werden. Zudem zeigte die vergleichende Analyse mit einem poribakteriellen Referenzklon aus einer bestehenden Metagenombank die genomische Mikroheterogenität innerhalb dieses Phylums. Nicht zuletzt konnte die Auswertung der poribakteriellen 454-Sequenzierung eine Reihe von möglichen Symbiose-Determinanten aufdecken, die beispielsweise am Austausch von Metaboliten zwischen den Interaktionspartnern beteiligt sind. Die Ergebnisse dieser Dissertationsarbeit stellen die Basis für eine gezielte und detaillierte funktionelle Beschreibung einzelner Bakterien innerhalb komplexer mikrobieller Konsortien dar, wie sie in marinen Schwämmen vorzufinden sind. Dieser Studie gewährte erstmalig umfassende Einblicke in das genomische Potential der nicht-kultivierten, Schwamm-assoziierten Poribacteria. Weiterführende Einzelzell-basierte Experimente werden in Zukunft dazu beitragen, das Bild von der Interaktion zwischen Bakterien und eukaryontischen Wirten zu komplettieren.
The gastrointestinal tract is abundantly colonized by microbes, yet the translocation of oral species to the intestine is considered a rare aberrant event, and a hallmark of disease. By studying salivary and fecal microbial strain populations of 310 species in 470 individuals from five countries, we found that transmission to, and subsequent colonization of, the large intestine by oral microbes is common and extensive among healthy individuals. We found evidence for a vast majority of oral species to be transferable, with increased levels of transmission in colorectal cancer and rheumatoid arthritis patients and, more generally, for species described as opportunistic pathogens. This establishes the oral cavity as an endogenous reservoir for gut microbial strains, and oral-fecal transmission as an important process that shapes the gastrointestinal microbiome in health and disease.
Diversity of Nonribosomal Peptide Synthetase Genes in the Microbial Metagenomes of Marine Sponges
(2012)
Genomic mining revealed one major nonribosomal peptide synthetase (NRPS) phylogenetic cluster in 12 marine sponge species, one ascidian, an actinobacterial isolate and seawater. Phylogenetic analysis predicts its taxonomic affiliation to the actinomycetes and hydroxy-phenyl-glycine as a likely substrate. Additionally, a phylogenetically distinct NRPS gene cluster was discovered in the microbial metagenome of the sponge Aplysina aerophoba, which shows highest similarities to NRPS genes that were previously assigned, by ways of single cell genomics, to a Chloroflexi sponge symbiont. Genomic mining studies such as the one presented here for NRPS genes, contribute to on-going efforts to characterize the genomic potential of sponge-associated microbiota for secondary metabolite biosynthesis.
Indoor house dust is a blend of organic and inorganic materials, upon which diverse microbial communities such as viruses, bacteria and fungi reside. Adequate moisture in the indoor environment helps microbial communities multiply fast. The outdoor air and materials that are brought into the buildings by airflow, sandstorms, animals pets and house occupants endow the indoor dust particles with extra features that impact human health. Assessment of the health effects of indoor dust particles, the type of indoor microbial inoculants and the secreted enzymes by indoor insects as allergens merit detailed investigation. Here, we discuss the applications of next generation sequencing (NGS) technology which is used to assess microbial diversity and abundance of the indoor dust environments. Likewise, the applications of NGS are discussed to monitor the gene expression profiles of indoor human occupants or their surrogate cellular models when exposed to aqueous solution of collected indoor dust samples. We also highlight the detection methods of dust allergens and analytical procedures that quantify the chemical nature of indoor particulate matter with a potential impact on human health. Our review is thus unique in advocating the applications of interdisciplinary approaches that comprehensively assess the health effects due to bad air quality in built environments.
Outdoor dust covers a shattered range of microbial agents from land over transportation, human microbial flora, which includes pathogen and commensals, and airborne from the environment. Dust aerosols are rich in bacterial communities that have a major impact on human health and living environments. In this study, outdoor samples from roadside barricades, safety walls, and fences (18 samples) were collected from Abu Dhabi, UAE and bacterial diversity was assessed through a 16S rRNA amplicon next generation sequencing approach. Clean data from HiSeq produced 1,099,892 total reads pairs for 18 samples. For all samples, taxonomic classifications were assigned to the OTUs (operational taxonomic units) representative sequence using the Ribosomal Database Project database. Analysis such as alpha diversity, beta diversity, differential species analysis, and species relative abundance were performed in the clustering of samples and a functional profile heat map was obtained from the OTUs by using bioinformatics tools. A total of 2814 OTUs were identified from those samples with a coverage of more than 99%. In the phylum, all 18 samples had most of the bacterial groups such as Actinobacteria, Proteobacteria, Firmicutes, and Bacteroidetes. Twelve samples had Propionibacteria acnes and were mainly found in RD16 and RD3. Major bacteria species such as Propionibacteria acnes, Bacillus persicus, and Staphylococcus captis were found in all samples. Most of the samples had Streptococcus mitis, Staphylococcus capitis. and Nafulsella turpanensis and Enhydrobacter aerosaccus was part of the normal microbes of the skin. Salinimicrobium sp., Bacillus alkalisediminis, and Bacillus persicus are halophilic bacteria found in sediments. The heat map clustered the samples and species in vertical and horizontal classification, which represents the relationship between the samples and bacterial diversity. The heat map for the functional profile had high properties of amino acids, carbohydrate, and cofactor and vitamin metabolisms of all bacterial species from all samples. Taken together, our analyses are very relevant from the perspective of out-door air quality, airborne diseases, and epidemics, with broader implications for health safety and monitoring.
The biosphere harbors a large quantity and diversity of microbial organisms that can thrive in all environments. Estimates of the total number of microbial species reach up to 1012, of which less than 15,000 have been characterized to date. It has been challenging to delineate phenotypically, evolutionary and ecologically meaningful lineages such as for example, species, subspecies and strains. Even within recognized species, gene content can vary considerably between sublineages (for example strains), a problem that can be addressed by analyzing pangenomes, defined as the non-redundant set of genes within a phylogenetic clade, as evolutionary units.
Species considered to be ecologically and evolutionary coherent units, however to date it is still not fully understood what are primary habitats and ecological niches of many prokaryotic species and how environmental preferences drive their genomic diversity. Majority of comparative genomics studies focused on a single prokaryotic species in context of clinical relevance and ecology. With accumulation of sequencing data due to genomics and metagenomics, it is now possible to investigate trends across many species, which will facilitate understanding of pangenome evolution, species and subspecies delineation.
The major aims of this thesis were 1) to annotate habitat preferences of prokaryotic species and strains; 2) investigate to what extent these environmental preferences drive genomic diversity of prokaryotes and to what extent phylogenetic constraints limit this diversification; 3) explore natural nucleotide identity thresholds to delineate species in bacteria in metagenomics gene catalogs; 4) explore species delineation for applications in subspecies and strain delineation in metagenomics.
The first part of the thesis describes methods to infer environmental preferences of microbial species. This data is a prerequisite for the analyses performed in the second part of the thesis which explores how the structure of bacterial pangenomes is predetermined by past evolutionary history and how is it linked to environmental preferences of the species. The main finding in this subchapter that habitat preferences explained up to 49% of the variance for pangenome structure, compared to 18% by phylogenetic inertia. In general, this trend indicates that phylogenetic inertia does not limit evolution of pangenome size and diversity, but that convergent evolution may overcome phylogenetic constraints. In this project we show that core genome size is associated with higher environmental ubiquity of species. It is likely this is due to the fact that species need to have more versatile genomes and most necessary genes need to be present in majority of genomes of that species to be highly prevalent. Taken together these findings may be useful for future predictive analyses of ecological niches in newly discovered species.
The third part of the thesis explores data-driven, operational species boundaries. I show that homologous genes from the same species from different genomes tend to share at least 95% of nucleotide identity, while different species within the same genus have lower nucleotide identity. This is in line with other studies showing that genome-wide natural species boundary might be in range of 90-95% of nucleotide identity. Finally, the fourth part of the thesis discusses how challenges in species delineation are relevant for the identification of meaningful within-species groups, followed by a discussion on how advancements in species delineation can be applied for classification of within-species genomic diversity in the age of metagenomics.
Microalga are of high relevance for the global carbon cycling and it is well-known that they are associated with a microbiota. However, it remains unclear, if the associated microbiota, often found in phycosphere biofilms, is specific for the microalga strains and which role individual bacterial taxa play. Here we provide experimental evidence that \(Chlorella\) \(saccharophila\), \(Scenedesmus\) \(quadricauda\), and \(Micrasterias\) \(crux-melitensis\), maintained in strain collections, are associated with unique and specific microbial populations. Deep metagenome sequencing, binning approaches, secretome analyses in combination with RNA-Seq data implied fundamental differences in the gene expression profiles of the microbiota associated with the different microalga. Our metatranscriptome analyses indicates that the transcriptionally most active bacteria with respect to key genes commonly involved in plant–microbe interactions in the Chlorella (Trebouxiophyceae) and Scenedesmus (Chlorophyceae) strains belong to the phylum of the α-Proteobacteria. In contrast, in the Micrasterias (Zygnematophyceae) phycosphere biofilm bacteria affiliated with the phylum of the Bacteroidetes showed the highest gene expression rates. We furthermore show that effector molecules known from plant-microbe interactions as inducers for the innate immunity are already of relevance at this evolutionary early plant-microbiome level.
With the technological advances of the last decade, it is now feasible to analyze microbiome samples, such as human stool specimens, using multi-omic techniques. Given the inherent sample complexity, there exists a need for sample methods which preserve as much information as possible about the biological system at the time of sampling. Here, we analyzed human stool samples preserved and stored using different methods, applying metagenomics as well as metaproteomics. Our results demonstrate that sample preservation and storage have a significant effect on the taxonomic composition of identified proteins. The overall identification rates, as well as the proportion of proteins from Actinobacteria were much higher when samples were flash frozen. Preservation in RNAlater overall led to fewer protein identifications and a considerable increase in the share of Bacteroidetes, as well as Proteobacteria. Additionally, a decrease in the share of metabolism-related proteins and an increase of the relative amount of proteins involved in the processing of genetic information was observed for RNAlater-stored samples. This suggests that great care should be taken in choosing methods for the preservation and storage of microbiome samples, as well as in comparing the results of analyses using different sampling and storage methods. Flash freezing and subsequent storage at −80 °C should be chosen wherever possible.
Marine Schwämme (Porifera) sind sessile Invertebraten, deren Biomasse bis zu 60% von assoziierten Mikroorganismen gebildet werden kann. Dieses mikrobielle Konsortium ist phylogenetisch komplex, die monophyletischen Abstammungslinien sind hochgradig wirtsspezifisch und bisher konnte kein Vertreter dieser Mikroflora kultiviert werden. In seiner Zusammensetzung unterscheidet sich dieses Konsortium sowohl von der Mikroflora mariner Sedimente, als auch vom marinen Bakterioplankton. Durch 16S rRNA Sequenzanalysen und Fluoreszenz in situ Hybridisierung (FISH) konnte während dieser Arbeit das neue Candidatus Phylum Poribacteria kultivierungsunabhängig identifiziert werden. Poribacteria bilden definitionsgemäß ein unabhängiges Candidatus Phylum, da sie weniger als 75% Sequenzhomologie innerhalb der 16S rRNA zu anderen prokaryontischen Phyla zeigen. Sie sind verwandt mit Planctomycetes. Der Name „Poribacteria“ wurde gewählt, da diese Organismen spezifisch mit marinen Porifera assoziiert zu sein scheinen. Bisher konnten Poribacteria in Porifera der Ordnungen Verongida, Haplosclerida und Lithistida nachgewiesen werden, während sie in den Ordnungen Poecilosclerida, Agelasida, Halichondrida und Hadromerida nicht nachweisbar waren. Im marinen Sediment und im Bakterioplankton wurden Poribacteria ebenfalls nicht detektiert. Durch FISH Analysen wurde deutlich, dass Poribacteria in A. aerophoba (Verongida) eine abundante Fraktion der assoziierten Mikroflora bilden. Da Vertreter des mikrobiellen Konsortiums mariner Schwämme bisher nicht kultiviert werden konnten, wurde das „Metagenom“ dieser Mikroorganismen durch die ex situ Isolierung hoch molekularer DNA direkt kloniert. Eine Charakterisierung von Metagenomen erlaubt unabhängig von der Kultivierbarkeit der entsprechenden Organismen direkte Einblicke in deren Genotyp und liefert so eine erste Verbindung zwischen phylogenetischer Diversität und physiologischen Eigenschaften. Für die Erstellung der Metagenombank wurde mikrobielle Biomasse aus A. aerophoba vom Mesohyl getrennt und lysiert und die gereinigte DNA in Fosmid Vektoren in E. coli kloniert. Die resultierende Metagenombank APAE02 umfasst ca. 1,1 Gb hoch molekularer prokaryontischer genomischer DNA. Eine Bestimmung der in dieser Metagenombank archivierten mikrobiellen Diversität lieferte zusätzlich zu bekannten 16S rRNA kodierenden Loci aus Cyanobacteria, Chloroflexi, Acidobacteria und Gammaproteobacteria einen 16S rRNA kodierenden poribakteriellen Fosmidklon. Die Annotation der flankierenden genomischen Regionen des 16S rRNA Gens führte zur Detektion eines unterbrochenen rrn Operons, eines wahrscheinlich neuen Transporters, einer neuen Molybdän enthaltenen Oxidoreduktase und orthologer „open reading frames“ (ORFs) aus Rhodopirellula baltica (Planctomycetes) in Poribacteria. Die Charakterisierung dieses 38,7 kb DNA Fragmentes stellt die Basis für weitere genomische Untersuchungen an Poribacteria dar. Metagenombanken repräsentieren eine reichhaltige Quelle zum Nachweis neuer Enzyme oder Biosyntheseoperons. Somit konnten in der Metagenombank APAE02 neuartige Typ I Polyketidsynthasen (PKS) nachgewiesen werden. Phylogenetische Analysen der Ketosynthasedomäne zeigten, dass diese Systeme nicht herkömmlichen Typ I cis-AT bzw. trans-AT (Acyltransferase) PKS Systemen zugeordnet werden können. Die kodierenden Bereiche der PKS Systeme sind mit nur ca. 10 kb relativ klein. Im Gegensatz zu der Organisation sich wiederholender multipler Module herkömmlicher PKS Typ I Systeme bestehen sie nur aus einem einzigen Modul und könnten vermutlich bei der Synthese von Fettsäuren beteiligt sein. Die Struktur und Funktion der Produkte ist bisher unbekannt. Generell ist durch in silico Analysen eine Abbildung des „funktionellen Repertoires“ unkultivierter Mikroorganismen möglich. Es wäre denkbar, dass durch weitere Studien fundierte Einblicke in den Genpool der Poribacteria und anderer Organismen des mikrobiellen Konsortiums aus Poriferen eröffnet werden, um metabolische Eigenschaften zu rekonstruieren und die Mechanismen zur Interaktion mit dem Wirt verstehen zu können.