Ecological and biotechnological aspects of Aplysina-associated microorganisms

The research described in this thesis had the goal to increase our understanding of marine sponge microbial ecology by integrating both cultivation-dependent and –independent approaches. Simultaneously, steps towards accessing the biotechnological potential of sponge-associated microbes are presented in this work. Thus, the insights presented here will deepen our understanding of sponge microbial ecology as well as provide directions for further bioprospecting efforts targeting marine sponges, especially Aplysina species. Sponges harbor dense and diverse microbial communities, and are key members of marine ecosystems. Chapter 1 introduced the sponge, provided an overview of the importance of these animals in their environment and summarized the current knowledge on functional aspects of their associated microbiomes. This chapter furthermore outlined the biotechnological potential inherent to sponge-associated microorganisms, such as the production of secondary metabolites with antibiotic, antiviral and anticancer properties. Furthermore, a brief introduction of microbial cultivation was given, and previous efforts on obtaining sponge-associated microbes in culture were highlighted. In many cases, the microbes inhabiting sponges have been demonstrated to be the actual producers of often halogenated bioactive secondary metabolites. Microorganisms attach halogen atoms such as chlorine or bromine to organic scaffolds using specialized enzymes, including halogenases. Such enzymes are of major biotechnological interest for the production of pharmaceutical or agrochemical compounds, since they halogenate regioselectively and under mild reaction conditions. In Chapter 2, six sponge species from the genus Aplysina were screened for flavin-dependent tryptophan halogenase sequence variants as well as the composition and structure of their bacterial communities using a PCR-based approach. In these sponge species from the Mediterranean and Caribbean seas we detected four phylogenetically diverse clades of putative tryptophan halogenase protein sequences, of which most were only distantly related to previously reported halogenases. The Mediterranean A. aerophoba harbored unique halogenase sequences, whereas the Caribbean species shared numerous sequence variants. By correlating the relative abundances of halogenases with those of bacterial taxa, we could identify prominent sponge-associated taxa belonging to Chloroflexi and Acidobacteria as putative owners of corresponding halogenase-encoding genes and therefore likely to be involved in the production of halogenated secondary metabolites in Aplysina spp. Certain microorganisms have been found to be highly specific in their association to marine sponges and are rarely detected in other habitats. As such, members of the candidate phylum ‘Poribacteria’ are considered promising model microorganisms for studying the origin of host-microbe interactions in sponges. In Chapter 3, we investigated the global diversity and phylogenetic distribution of poribacteria among different sponge hosts. By generating a phylogenetic network, we could decipher the genetic distances between poribacterial phylotypes and visualize their distribution amongst numerous sponge species. In total, 361 poribacterial 16S rRNA gene sequences were examined, and neither co-speciation with the host, nor biogeographical correlations could be detected. However, analyses resulted in the discovery of a novel phylogenetic clade of Poribacteria, which might represent a link between the previously established clades. We expanded the number of Sanger-sequenced poribacterial 16S rRNA genes by approximately one third and could thus contribute to mapping the global diversity and distribution of this sponge-associated bacterial candidate phylum. Chapter 4 describes several approaches to increase the cultivability of bacteria associated to the sponge Aplysina aerophoba. Alternative cultivation setups such as a Winogradsky-column approach, a liquid-solid media approach as well as media based on multi-omic-derived information on the metabolism of Poribacteria were applied. We found that most bacteria remained viable after cryo-preservation, however, only 2% of the initial diversity detected in A. aerophoba could be recovered through cultivation. We observed that medium dilution, rather than carbon source, most strongly affected the composition of cultivated microbial communities. Furthermore, the sponge-derived antibiotic aeroplysinin-1 negatively affected microbial growth, and selectively inhibited several taxa such as Flavobacteriaceae. The Winogradsky-column approach led to enrichment of distinct microbial communities at different locations in the columns, which included members of the class Clostridia and OTUs that were distantly related to the Planctomycetes. Pseudovibrio and Ruegeria spp. were obtained under almost all cultivation conditions applied, while other taxa such as Bacteroidetes were more specific to certain media types. Even though the predominant sponge-associated microorganisms remained uncultured, we could enrich 256 OTUs encompassing seven microbial phyla. Microbial cultivation is often a tedious game of trial-and-error, however, pure or defined cultures are needed to decipher microbial physiology, curate and improve gene- and protein database annotations and realize novel biotechnological applications. Chapter 5 discusses a novel approach to microbial cultivation: Multi-omics-derived information has accumulated exponentially over the last decade and provides a plethora of information awaiting integration into the development of novel cultivation strategies. This review summarizes ground-breaking studies that translated information derived from multi-omics into successful isolation strategies for previously unculturable microorganisms. Such strategies include specific media formulations, screening techniques and selective enrichments. Inspired by the microbial complexity of the environment, we integrated these inventive methods and concluded with proposing a workflow for future omics-aided cultivation experiments: Initial diversity estimation results in deciding the method for obtaining the genome of the targeted organism. Based on the resulting metabolic model, media can be formulated, while environmental parameters are included into defining cultivation conditions. Molecular probes can assist targeted screening strategies of novel high-throughput cultivation methods. This multi-omics integration should increase the chances of isolating novel microbial species. Finally, Chapter 6 integrated the findings from the previous chapters and portrayed them in the light of recent work from within the field of sponge microbiology and ecology. Using Poribacteria as an example, hypotheses on the origin of sponge-microbe symbiosis were combined and discussed. In short, mixed vertical and horizontal symbiont transmission resulted in a lack in host-species specificity and might indicate that sponge-poribacterial symbiosis originated in the Precambrian, before the phylum Porifera diverged into different classes. Additionally, aspects such as methods for assessing sponge-associated communities, or factors influencing microbial cultivability were elaborated on in more detail. Furthermore, this chapter summarized the successes, as well as failures, of isolating sponge-associated microorganisms and highlighted future avenues for bringing these fastidious organisms into culture. In this thesis, we aimed to create a synergy between cultivation-dependent and cultivation-independent methods, by incorporating genomic predictions on carbohydrate metabolism as well as micro- and macro-environmental parameters into defining novel cultivation strategies for sponge-associated microorganisms. However, the inherent search space remains far from exhausted: Future efforts should integrate additional multi-omics-predicted metabolic capabilities such as anaerobic metabolic pathways or autotrophy into cultivation strategies, since cultivated representatives of sponge-associated microbes will reveal novel ways to tap into their biotechnological potential.