Kristina Bayer

Physiology, phylogeny and in situ evidence for bacterial and archaeal nitrifiers in the marine sponge Aplysina aerophoba

The potential for nitrification in the Mediterranean sponge Aplysina aerophoba was assessed using a combined physiological and molecular approach. Nitrate excretion rates in whole sponges reached values of up to 344 nmol g(-1) dry weight (wt) h(-1) (unstimulated) and 1325 nmol g(-1) dry wt h(-1) (stimulated). Addition of nitrapyrin, a nitrification-specific inhibitor, effectively inhibited nitrate excretion. Ammonium was taken up by sponges in spring and excreted in fall, the sponges thus serving as either an ammonium sink or ammonium source. Nitrosospira cluster 1 and Crenarchaeota group I.1A 16S rRNA and amoA genes were recovered from A. aerophoba and other sponges from different worlds oceans. The archaeal 16S rRNA genes formed a sponge-specific subcluster, indicating that their representatives are members of the stable microbial community of sponges. On the other hand, clustering was not evident for Nitrosospira rRNA genes which is consistent with their presence in sediment and seawater samples. The presence of both Nitrosospira cluster 1 and crenarchaeal group 1 phylotypes in sponge tissue was confirmed using fluorescently labelled 16S rRNA gene probes. This study contributes to an ongoing effort to link microbial diversity with metabolic functions in the phylogenetically diverse, elusive and so far uncultivated microbial communities of marine sponges.

Diversity, abundance and natural products of marine sponge-associated actinomycetes

Actinomycetes are known for their unprecedented ability to produce novel lead compounds of clinical and pharmaceutical importance. This review focuses on the diversity, abundance and methodological approaches targeting marine sponge-associated actinomycetes. Additionally, novel qPCR data on actinomycete abundances in different sponge species and other environmental sources are presented. The natural products literature is covered, and we are here reporting on their chemical structures, their biological activities, as well as the source organisms from which they were isolated.

Specificity and transcriptional activity of microbiota associated with low and high microbial abundance sponges from the Red Sea

Marine sponges are generally classified as high microbial abundance (HMA) and low microbial abundance (LMA) species. Here, 16S rRNA amplicon sequencing was applied to investigate the diversity, specificity and transcriptional activity of microbes associated with an LMA sponge (Stylissa carteri), an HMA sponge (Xestospongia testudinaria) and sea water collected from the central Saudi Arabia coast of the Red Sea. Altogether, 887 068 denoised sequences were obtained, of which 806 661 sequences remained after quality control. This resulted in 1477 operational taxonomic units (OTUs) that were assigned to 27 microbial phyla. The microbial composition of S. carteri was more similar to that of sea water than to that of X. testudinaria, which is consistent with the observation that the sequence data set of S. carteri contained many more possibly sea water sequences (~24%) than the X. testudinaria data set (~6%). The most abundant OTUs were shared between all three sources (S. carteri, X. testudinaria, sea water), while rare OTUs were unique to any given source. Despite this high degree of overlap, each sponge species contained its own specific microbiota. The X. testudinaria‐specific bacterial taxa were similar to those already described for this species. A set of S. carteri‐specific bacterial taxa related to Proteobacteria and Nitrospira was identified, which are likely permanently associated with S. carteri. The transcriptional activity of sponge‐associated microorganisms correlated well with their abundance. Quantitative PCR revealed the presence of Poribacteria, representing typical sponge symbionts, in both sponge species and in sea water; however, low transcriptional activity in sea water suggested that Poribacteria are not active outside the host context.

Genomic Mining for Novel FADH2-Dependent Halogenases in Marine Sponge-Associated Microbial Consortia

Many marine sponges (Porifera) are known to contain large amounts of phylogenetically diverse microorganisms. Sponges are also known for their large arsenal of natural products, many of which are halogenated. In this study, 36 different FADH2-dependent halogenase gene fragments were amplified from various Caribbean and Mediterranean sponges using newly designed degenerate PCR primers. Four unique halogenase-positive fosmid clones, all containing the highly conserved amino acid motif “GxGxxG”, were identified in the microbial metagenome of Aplysina aerophoba. Sequence analysis of one halogenase-bearing fosmid revealed notably two open reading frames with high homologies to efflux and multidrug resistance proteins. Single cell genomic analysis allowed for a taxonomic assignment of the halogenase genes to specific symbiotic lineages. Specifically, the halogenase cluster S1 is predicted to be produced by a deltaproteobacterial symbiont and halogenase cluster S2 by a poribacterial sponge symbiont. An additional halogenase gene is possibly produced by an actinobacterial symbiont of marine sponges. The identification of three novel, phylogenetically, and possibly also functionally distinct halogenase gene clusters indicates that the microbial consortia of sponges are a valuable resource for novel enzymes involved in halogenation reactions.

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.

An Enrichment of CRISPR and Other Defense-Related Features in Marine Sponge-Associated Microbial Metagenomes

Many marine sponges are populated by dense and taxonomically diverse microbial consortia. We employed a metagenomics approach to unravel the differences in the functional gene repertoire among three Mediterranean sponge species, Petrosia ficiformis, Sarcotragus foetidus, Aplysina aerophoba and seawater. Different signatures were observed between sponge and seawater metagenomes with regard to microbial community composition, GC content, and estimated bacterial genome size. Our analysis showed further a pronounced repertoire for defense systems in sponge metagenomes. Specifically, clustered regularly interspaced short palindromic repeats, restriction modification, DNA phosphorothioation and phage growth limitation systems were enriched in sponge metagenomes. These data suggest that defense is an important functional trait for an existence within sponges that requires mechanisms to defend against foreign DNA from microorganisms and viruses. This study contributes to an understanding of the evolutionary arms race between viruses/phages and bacterial genomes and it sheds light on the bacterial defenses that have evolved in the context of the sponge holobiont.

Metagenomic binning of a marine sponge microbiome reveals unity in defense but metabolic specialization

Marine sponges are ancient metazoans that are populated by distinct and highly diverse microbial communities. In order to obtain deeper insights into the functional gene repertoire of the Mediterranean sponge Aplysina aerophoba, we combined Illumina short-read and PacBio long-read sequencing followed by un-targeted metagenomic binning. We identified a total of 37 high-quality bins representing 11 bacterial phyla and two candidate phyla. Statistical comparison of symbiont genomes with selected reference genomes revealed a significant enrichment of genes related to bacterial defense (restriction-modification systems, toxin-antitoxin systems) as well as genes involved in host colonization and extracellular matrix utilization in sponge symbionts. A within-symbionts genome comparison revealed a nutritional specialization of at least two symbiont guilds, where one appears to metabolize carnitine and the other sulfated polysaccharides, both of which are abundant molecules in the sponge extracellular matrix. A third guild of symbionts may be viewed as nutritional generalists that perform largely the same metabolic pathways but lack such extraordinary numbers of the relevant genes. This study characterizes the genomic repertoire of sponge symbionts at an unprecedented resolution and it provides greater insights into the molecular mechanisms underlying microbial-sponge symbiosis.

Einzelzell-Genomik: das Bakterium als Individuum

The concept of single-cell genomics lies in the recognition of each bacterial cell as a unique and distinct entity. Single-cell genomics employ phi29 polymerase and random hexamer primers to yield microgram-range genomic DNA from single microbial cells. The unprecedented insights obtained from single-cell genome analyses have allowed major advances in the fields of environmental microbiology, symbiosis, evolution, and bioprospecting.

Marine sponges as Chloroflexi hot-spots: Genomic insights and high resolution visualization of an abundant and diverse symbiotic clade

Chloroflexi represent a widespread, yet enigmatic bacterial phylum. Meta-and single cell genomics were performed to shed light on the functional gene repertoire of Chloroflexi symbionts from the HMA sponge Aplysina aerophoba. Eighteen draft genomes were reconstructed and placed into phylogenetic context of which six were investigated in detail. Common genomic features of Chloroflexi sponge symbionts were related to central energy and carbon converting pathways, amino acid and fatty acid metabolism and respiration. Clade specific metabolic features included a massively expanded genomic repertoire for carbohydrate degradation in Anaerolineae and Caldilineae genomes, and amino acid utilization as nutrient source by SAR202. While Anaerolineae and Caldilineae import cofactors and vitamins, SAR202 genomes harbor genes encoding for co-factor biosynthesis. A number of features relevant to symbiosis were further identified, including CRISPRs-Cas systems, eukaryote-like repeat proteins and secondary metabolite gene clusters. Chloroflexi symbionts were visualized in the sponge extracellular matrix at ultrastructural resolution by FISH-CLEM method. Chloroflexi cells were generally rod-shaped and about 1 μm in length, albeit displayed different and characteristic cellular morphotypes per each class. The extensive potential for carbohydrate degradation has been reported previously for Ca. Poribacteria and SAUL, typical symbionts of HMA sponges, and we propose here that HMA sponge symbionts collectively engage in degradation of dissolved organic matter, both labile and recalcitrant. Thus sponge microbes may not only provide nutrients to the sponge host, but also contribute to DOM re-cycling and primary productivity in reef ecosystems via a pathway termed the “sponge loop”.

Den Unkultivierbaren auf der Spur

Single cell genomics has advanced the field of microbiology to recognizing each microbial cell as a separate and unique entity. The combination of new high-throughput sequencing technologies, constantly improving bioinformatic tools, and single cell genomic/metagenomic workflows has greatly enhanced our understanding of the uncultivated microbial world.