Exploring Symbioses by Single-Cell Genomics

Single-cell genomics has advanced the field of microbiology from the analysis of microbial metagenomes where information is “drowning in a sea of sequences,” to recognizing each microbial cell as a separate and unique entity. Single-cell genomics employs Phi29 polymerase-mediated whole-genome amplification to yield microgram-range genomic DNA from single microbial cells. This method has now been applied to a handful of symbiotic systems, including bacterial symbionts of marine sponges, insects (grasshoppers, termites), and vertebrates (mouse, human). In each case, novel insights were obtained into the functional genomic repertoire of the bacterial partner, which, in turn, led to an improved understanding of the corresponding host. Single-cell genomics is particularly valuable when dealing with uncultivated microorganisms, as is still the case for many bacterial symbionts. In this review, we explore the power of single-cell genomics for symbiosis research and highlight recent insights into the symbiotic systems that were obtained by this approach

Bacterial community profiles in low microbial abundance sponges

It has long been recognized that sponges differ in the abundance of associated microorganisms, and they are therefore termed either 'low microbial abundance' (LMA) or 'high microbial abundance' (HMA) sponges. Many previous studies concentrated on the dense microbial communities in HMA sponges, whereas little is known about microorganisms in LMA sponges. Here, two LMA sponges from the Red Sea, two from the Caribbean and one from the South Pacific were investigated. With up to only five bacterial phyla per sponge, all LMA sponges showed lower phylum-level diversity than typical HMA sponges. Interestingly, each LMA sponge was dominated by a large clade within either Cyanobacteria or different classes of Proteobacteria. The overall similarity of bacterial communities among LMA sponges determined by operational taxonomic unit and UniFrac analysis was low. Also the number of sponge-specific clusters, which indicate bacteria specifically associated with sponges and which are numerous in HMA sponges, was low. A biogeographical or host-dependent distribution pattern was not observed. In conclusion, bacterial community profiles of LMA sponges are clearly different from profiles of HMA sponges and, remarkably, each LMA sponge seems to harbour its own unique bacterial communit

The pathology of sponge orange band disease affecting the Caribbean barrel sponge Xestospongia muta

The aim of this study was to examine sponge orange band (SOB) disease affecting the prominent Caribbean sponge Xestospongia muta. Scanning and transmission electron microscopy revealed that SOB is accompanied by the massive destruction of the pinacoderm. Chlorophyll a content and the main secondary metabolites, tetrahydrofurans, characteristic of X. muta, were significantly lower in bleached than in healthy tissues. Denaturing gradient gel electrophoresis using cyanobacteria-specific 16S rRNA gene primers revealed a distinct shift from the Synechococcus/Prochlorococcus clade of sponge symbionts towards several clades of unspecific cyanobacteria, including lineages associated with coral disease (i.e. Leptolyngbya sp.). Underwater infection experiments were conducted by transplanting bleached cores into healthy individuals, but revealed no signs of SOB development. This study provided no evidence for the involvement of a specific microbial pathogen as an etiologic agent of disease; hence, the cause of SOB disease in X. muta remains unidentified

Methods for the extraction, storage, amplification and sequencing of DNA from environmental samples

Advances in the sequencing of DNA extracted from media such as soil and water offer huge opportunities for biodiversity monitoring and assessment, particularly where the collection or identification of whole organisms is impractical. However, there are myriad methods for the extraction, storage, amplification and sequencing of DNA from environmental samples. To help overcome potential biases that may impede the effective comparison of biodiversity data collected by different researchers, we propose a standardised set of procedures for use on different taxa and sample media, largely based on recent trends in their use. Our recommendations describe important steps for sample pre-processing and include the use of (a) Qiagen DNeasy PowerSoil® and PowerMax® kits for extraction of DNA from soil, sediment, faeces and leaf litter; (b) DNeasy PowerSoil® for extraction of DNA from plant tissue; (c) DNeasy Blood and Tissue kits for extraction of DNA from animal tissue; (d) DNeasy Blood and Tissue kits for extraction of DNA from macroorganisms in water and ice; and (e) DNeasy PowerWater® kits for extraction of DNA from microorganisms in water and ice. Based on key parameters, including the specificity and inclusivity of the primers for the target sequence, we recommend the use of the following primer pairs to amplify DNA for analysis by Illumina MiSeq DNA sequencing: (a) 515f and 806RB to target bacterial 16S rRNA genes (including regions V3 and V4); (b) #3 and #5RC to target eukaryote 18S rRNA genes (including regions V7 and V8); (c) #3 and #5RC are also recommended for the routine analysis of protist community DNA; (d) ITS6F and ITS7R to target the chromistan ITS1 internal transcribed spacer region; (e) S2F and S3R to target the ITS2 internal transcribed spacer in terrestrial plants; (f) fITS7 or gITS7, and ITS4 to target the fungal ITS2 region; (g) NS31 and AML2 to target glomeromycota 18S rRNA genes; and (h) mICOIintF and jgHCO2198 to target cytochrome c oxidase subunit I (COI) genes in animals. More research is currently required to confirm primers suitable for the selective amplification of DNA from specific vertebrate taxa such as fish. Combined, these recommendations represent a framework for efficient, comprehensive and robust DNA-based investigations of biodiversity, applicable to most taxa and ecosystems. The adoption of standardised protocols for biodiversity assessment and monitoring using DNA extracted from environmental samples will enable more informative comparisons among datasets, generating significant benefits for ecological science and biosecurity applications

Einzelzell-genomische Analysen des Candidatus Phylums Poribacteria

Marine sponges are the most ancient metazoans and of large ecological importance as drivers of water and nutrient flows in benthic habitats. Furthermore marine sponges are well known for their association with highly abundant and diverse microbial consortia. Microorganisms inhabit the extracellular matrix of marine sponges where they can make up to 35% of the sponge’s biomass. Many microbial symbionts of marine sponges are highly host specific and cannot, or only in very rare abundances, be found outside of their host environment. Of special interest is the candidate phylum Poribacteria that was first discovered in marine sponges and still remains almost exclusive to their hosts. Phylogenetically Poribacteria were placed into the Planctomycetes, Verrucomicrobia, Chlamydiae superphylum and similarly to many members of this superphylum cell compartmentation has been proposed to occur in members of the Poribacteria. The status as a candidate phylum implies that no member of Poribacteria has been obtained in culture yet. This restricts the investigations of Poribacteria and their interactions with marine sponges to culture independent methods and makes functional characterisation a difficult task. In this PhD thesis I used the novel method of single-cell genomics to investigate the genomic potential of the candidate phylum Poribacteria. Single-cell genomics enables whole genome sequencing of uncultivated microorganisms by singularising cells from the environment, subsequent cell lysis and multiple displacement amplification of the total genomic DNA. This process yields sufficient amounts of DNA for whole genome sequencing and genome analysis. This technique and its relevance for symbiosis studies are discussed in this PhD thesis. Through the application of single-cell genomics it was possible to increase the number of single-amplified genomes of the candidate phylum Poribacteria from initially one to a total of six. Analyses of these datasets made it possible to enhance our understanding of the metabolism, taxonomy, and phylum diversity of Poribacteria and thus made these one of the best-characterised sponge symbionts today. The poribacterial genomes represented three phylotypes within the candidate phylum of which one appeared dominant. Phylogenetic and phylogenomic analyses revealed a novel phylogenetic positioning of Poribacteria distinctly outside of the Planctomycete, Verrucomicorbia, Chlamydiae superphylum. The occurrence of cell compartmentation in Poribacteria was also revisited based on the obtained genome sequences and revealed evidence for bacterial microcompartments instead of the previously suggested nucleotide-like structures. An extensive genomic repertoire of glycoside hydrolases, glycotransferases, and other carbohydrate active enzymes was found to be the central shared feature between all poribacterial genomes and showed that Poribacteria are among those marine bacteria with the largest genomic repertoire for carbohydrate degradation. Detailed analysis of the carbohydrate metabolism revealed that Poribacteria have the genomic potential for degradation of a variety of polymers, di- and monosaccharaides that allow these symbionts to feed various nutrient sources accessible through the filter-feeding activities of the sponge host. Furthermore the poribacterial glycobiome appeared to enable degradation of glycosaminoglycan chains, one of the main building blocks of extracellular matrix of marine sponges. Different lifestyles resulting from the poribacterial carbohydrate degradation potential are discussed including the influence of nutrient cycling in sponges, nutrient recycling and scavenging. The findings of this thesis emphasise the long overlooked importance of heterotrophic symbionts such as Poribacteria for the interactions with marine sponges and represent a solid basis for future studies of the influence heterotrophic symbionts have on their sponge hosts.Marine Schwämme sind die ältesten rezenten Vertreter der Metazoen. Durch ihre Lebensweise als Nahrungsfiltrierer und den damit verbundenen Einfluss auf Nährstoffzyklen sind sie von großer ökologischer Relevanz. Des Weiteren zeichnen sich marine Schwämme durch das Zusammenleben mit hoch abundanten und diversen mikrobiellen Konsortien aus. Diese Mikroorganismen finden sich meist in der extrazellulären Matrix des Schwamms und können mehr als 35% der Biomasse ihres Wirtes ausmachen. Viele mikrobielle Symbionten mariner Schwämme sind hochgradig Wirts-spezifisch und können außerhalb des Schwamms, wenn überhaupt, nur in sehr geringer Anzahl gefunden werden. Von besonderem Interesse ist das Candidatus Phylum Poribacteria, dessen Vertreter erstmals in Schwämmen detektiert wurden, und bis heute fast ausschließlich in Schwämmen zu finden sind. Phylogenetisch wurden die Poribacteria dem Planctomycetes, Verrucomicrobia, Chlamydiae (PVC) Superphylum zugeordnet. Einige Vertreter dieses Superphylums zeigen einen kompartimentierten Zellplan auf, eine Eigenschaft, die auch für Poribacteria vermutet wird. Der Status der Poribacteria als Candidatus Phylum zeigt das Fehlen von Vertretern dieses Phylums in Reinkultur an. Dies beschränkt die Untersuchung von Poribacteria auf kultivierungs-unabhängige Methoden, was die funktionelle Charakterisierung dieser Symbionten erheblich erschwert. In dieser Doktorarbeit wurde das Candidatus Phylum Poribacteria mit Hilfe der Einzelzellgenomik untersucht. Diese Methode ermöglicht es aus vereinzelten mikrobiellen Zellen genomische Komplett-DNA zu gewinnen und diese, mit Hilfe der so genannten „multiple displacement amplification“ so hochgradig anzureichern, dass eine Sequenzierung und anschließende Analyse erfolgen kann. Die Anwendung dieser Methode im Allgemeinen und in der Symbiose Forschung wird in dieser Doktorarbeit diskutiert. Die Einzelzellgenomik ermöglichte die Anzahl poribakterieller Datensätze, von zunächst einem auf sechs Genome zu erhöhen. Die Analyse dieser Genome konnte unser Verständnis vom metabolischen Potential, der Taxonomie und der Diversität innerhalb dieses Phylums deutlich zu verbessern. Die poribakteriellen Genome beschrieben drei Phylotypen, von denen einer deutlich dominierte. Die phylogenetische Position des Phylums Poribacteria wurde außerdem anhand von phylogenetischen und phylogenomischen Berechnungen neu zugeordnet, und resultierte in einer deutlichen Positionierung außerhalb des PVC Superphylums. Weiterhin wurden genomische Hinweise auf einen kompartimentierten Zellplan in Poribacteria gefunden. Diese deuten aber nicht, wie vorher vermutet, auf eine Zellkern-ähnliche Struktur hin, sondern auf bakterielle Mikrokompartimente mit noch ungeklärter Funktion. Die Analysen des genomischen Potentials zeigte in allen Datensätzen eine hohe Frequenz von Genen, die für Glycosidasen, Glycosyltransferasen und weiteren Proteinen der so-genannten „carbohydrate active enzymes“ kodieren, was ein ausgeprägtes Vermögen zum Kohlehydratabbau aufzeigt. Das genomische Potential von Poribacteria zum Abbau von Kohlehydratpolymeren, Di- und Monosacchariden konnte durch detaillierte Analyse des Kohlehydratmetabolismus genau beschrieben werden. Außerdem schienen Poribacteria Glycosaminoglycanketten, die zentrale Bausteine der extrazellulären Matrix des Schwammes sind, abbauen zu können. Die aus dem poribakteriellen Glycobiome resultierenden möglichen Lebensweisen als Wiederverwerter von Nährstoffen, Mitesser, oder auch der Einfluss von Poribacteria auf Nährstoffzyklen im Schwamm werden in dieser Doktorarbeit diskutiert. Die Ergebnisse dieser Doktorarbeit machen Poribacteria zu den genomische am besten beschriebenen Schwammsymbionten und zeigen die lange übersehene Relevanz heterotropher Symbionten in marinen Schwämmen

Quantification of bacterial and archaeal symbionts in high and low microbial abundance sponges using real-time PCR

In spite of considerable insights into the microbial diversity of marine sponges, quantitative information on microbial abundances and community composition remains scarce. Here, we established qPCR assays for the specific quantification of four bacterial phyla of representative sponge symbionts as well as the kingdoms Eubacteria and Archaea. We could show that the 16S rRNA gene numbers of Archaea, Chloroflexi, and the candidate phylum Poribacteria were 4–6 orders of magnitude higher in high microbial abundance (HMA) than in low microbial abundance (LMA) sponges and that actinobacterial 16S rRNA gene numbers were 1–2 orders higher in HMA over LMA sponges, while those for Cyanobacteria were stable between HMA and LMA sponges. Fluorescence in situ hybridization of Aplysina aerophoba tissue sections confirmed the numerical dominance of Chloroflexi, which was followed by Poribacteria. Archaeal and actinobacterial cells were detected in much lower numbers. By use of fluorescence-activated cell sorting as a primer- and probe-independent approach, the dominance of Chloroflexi, Proteobacteria, and Poribacteria in A. aerophoba was confirmed. Our study provides new quantitative insights into the microbiology of sponges and contributes to a better understanding of the HMA/LMA dichotomy

16S rRNA gene based maximum likelihood tree illustrating the phylogenetic position of the candidate phylum <i>Poribacteria</i>.

<p>Bootstrap values (500 resamplings) ≥50% are shown on tree nodes. Numbers of sequences included per group is shown in group boxes. Outgroup consists of 80 sequences belonging to the <i>Bacteroidetes</i>. Scale bar represents 10% sequences divergence.</p

Phylogenomic tree based on a concatenated alignment of up to 83 genes illustrating the phylogenetic position of the candidate phylum <i>Poribacteria</i>.

<p>Bootstrap value (100 resamplings) are shown on tree nodes where support ≥50%. Number of genomes per group is displayed in group boxes. Outgroup consists of several species of <i>Spirochaetes</i> and <i>Gammaproteobacteria</i>. The scale bar represents 10% sequence divergence.</p