Refine
Has Fulltext
- yes (24)
Is part of the Bibliography
- yes (24)
Year of publication
Document Type
- Doctoral Thesis (21)
- Complete part of issue (2)
- Journal article (1)
Keywords
- Bakterien (24) (remove)
Institute
- Julius-von-Sachs-Institut für Biowissenschaften (6)
- Graduate School of Life Sciences (5)
- Institut für Molekulare Infektionsbiologie (5)
- Theodor-Boveri-Institut für Biowissenschaften (4)
- Institut für Hygiene und Mikrobiologie (2)
- Universität Würzburg (2)
- Institut für Organische Chemie (1)
- Institut für Pharmakologie und Toxikologie (1)
- Institut für Pharmazie und Lebensmittelchemie (1)
- Institut für Virologie und Immunbiologie (1)
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 sponges (phylum Porifera) are simple, sessile, filter-feeder animals. Microbial symbionts are commonly found in the sponge internal tissue, termed the mesohyl. With respect to the microbial content, sponges are classified as either low-microbial abundance sponges (LMA), or high-microbial abundance sponges (HMA). The HMA/LMA dichotomy was explored in this Thesis using the Red Sea sponges as experimental models. A range of methods encompassing transmission electron microscopy, 16S rRNA gene deep sequencing, and metatranscriptomics was employed towards this goal. Here, particular emphasis was placed on the functional analysis of sponge microbiomes.
The Red Sea sponges Stylissa carteri, Xestospongia testudinaria, Amphimedon ochracea, and Crella cyathophora were classified as HMA or LMA sponges using transmission electron microscopy. The diversity, specificity, and transcriptional activity of microbes associated with the sponges S. carteri (LMA) and X. testudinaria (HMA) and seawater were investigated using 16S rRNA amplicon pyrosequencing. The microbial composition of S. carteri was more similar to that of seawater than to that of X. testudinaria, which is consistent with the observation that the sequence data set of S. carteri contained many more possibly seawater sequences (~24%) than the X. testudinaria data set (~6%). The most abundant operational taxonomic units (OTUs) were shared between all three sources (S. carteri, X. testudinaria, seawater), while rare OTUs were unique to any given source. Despite this high degree of overlap, each sponge species contained its own specific microbiota. S. carteri microbiomes were enriched of Gammaproteobacteria and members of the genus Synechococcus and Nitrospira. Enriched members of X. testudinaria microbiomes included Chloroflexi, Deferribacteres, and Actinobacteria. The transcriptional activity of sponge-associated microorganisms was assessed by comparing 16S rRNA gene with transcript amplicons, which showed a good correlation.
The microbial functional gene repertoire of sponges and seawater from the Red Sea (X. testudinaria, S. carteri) and the Mediterranean (Aplysina aerophoba, Dysidea avara) were investigated with the environmental microarray GeoChip 4. Amplicon sequencing was performed alongside in order to assess microbial diversity. The typical microbial diversity patterns characteristic of HMA (abundance of Gammaproteobacteria, Chloroflexi, Acidobacteria, Deferribacteres, and others) and LMA sponges (abundance of Alpha-, Beta-, Gammaproteobacteria, Cyanobacteria, and Bacteroidetes) were confirmed. The HMA/LMA dichotomy was stronger than any possible geographic pattern based on microbial diversity (amplicon) and functional genes (GeoChip). However upon inspection of individual genes detected by GeoChip, very few specific differences were discernible, including differences related to microbial ammonia oxidation, ammonification (higher gene abundance in sponges over seawater) as well as denitrification (lower gene abundance). Furthermore, a higher abundance of a gene, pcc, representative of archaeal autotrophic carbon fixation was noted in sponges over seawater. Thirdly, stress-related genes, in particular those related to radiation, were found in lower abundances in sponge microbiomes than in seawater. With the exception of few documented specific differences, the functional gene repertoire between the different sources appeared largely similar.
The most actively expressed genes of S. carteri microbiomes were investigated with metatranscriptomics. Prokaryotic mRNA was enriched from sponge total RNA, sequenced using Illumina HiSeq technology, and annotated with the metagenomics Rapid Annotation using Subsystem Technology (MG-RAST) pipeline. High expression of archaeal ammonia oxidation and photosynthetic carbon fixation by members of the genus Synechococcus was detected. Functions related to stress response and membrane transporters were among the most highly expressed by S. carteri symbionts. Unexpectedly, gene functions related to methylotrophy were highly expressed by gammaproteobacterial symbionts. The presence of seawater-derived microbes is indicated by the phylogenetic proximity of organic carbon transporters to orthologs of members from the SAR11 clade. In summary, the most expressed functions of the S. carteri-associated microbial community were revealed and linked to the dominant taxonomic members of the microbiome.
In conclusion, HMA and LMA Red Sea sponges were used as models to gain insights into relevant themes in sponge microbiology, i.e. diversity, specificity, and functional activities. Overall, my Thesis contributes to a better understanding of sponge-associated microbial communities, and the implications of this association to marine ecology.
Host–microbe interactions are the key to understand why and how microbes inhabit specific environments. With the scientific fields of microbial genomics and metagenomics, evolving on an unprecedented scale, one is able to gain insights in these interactions on a molecular and ecological level. The goal of this PhD thesis was to make (meta–)genomic data accessible, integrate it in a comparative manner and to gain comprehensive taxonomic and functional insights into bacterial strains and communities derived from two different environments: the phyllosphere of Arabidopsis thaliana and the mesohyl interior of marine sponges.
This thesis focused first on the de novo assembly of bacterial genomes. A 5–step protocol was developed, each step including a quality control. The examination of different assembly software in a comparative way identified SPAdes as most suitable. The protocol enables the user to chose the best tailored assembly. Contamination issues were solved by an initial filtering of the data and methods normally used for the binning of metagenomic datasets. This step is missed in many published assembly pipelines. The described protocol offers assemblies of high quality ready for downstream analysis.
Subsequently, assemblies generated with the developed protocol were annotated and explored
in terms of their function. In a first study, the genome of a phyllosphere bacterium, Williamsia sp. ARP1, was analyzed, offering many adaptions to the leaf habitat: it can deal with temperature shifts, react to oxygen species, produces mycosporins as protection against UV–light, and is able to uptake photosynthates. Further, its taxonomic position within the Actinomycetales was infered from 16S rRNA and comparative genomics showing the close relation between the genera Williamsia and Gordonia.
In a second study, six sponge–derived actinomycete genomes were investigated for secondary metabolism. By use of state–of–the–art software, these strains exhibited numerous gene clusters, mostly linked to polykethide synthases, non–ribosomal peptide synthesis, terpenes, fatty acids and saccharides. Subsequent predictions on these clusters offered a great variety of possible produced compounds with antibiotic, antifungal or anti–cancer activity. These analysis highlight the potential for the synthesis of natural products and the use of genomic data as screening toolkit.
In a last study, three sponge–derived and one seawater metagenomes were functionally compared. Different signatures regarding the microbial composition and GC–distribution were observed between the two environments. With a focus on bacerial defense systems, the data indicates a pronounced repertoire of sponge associated bacteria for bacterial defense systems, in particular, Clustered Regularly Interspaced Short Palindromic Repeats, restriction modification system, DNA phosphorothioation and phage growth limitation. In addition, characterizing genes for secondary metabolite cluster differed between sponge and seawater microbiomes. Moreover, a variety of Type I polyketide synthases were only found within the sponge microbiomes. With that, metagenomics are shown to be a useful tool for the screening of secondary metabolite genes. Furthermore, enriched defense systems are highlighted as feature of sponge-associated microbes and marks them as a selective trait.
RNA sequencing (RNA-seq) has in recent years become the preferred method for gene expression analysis and whole transcriptome annotation. While initial RNA-seq experiments focused on eukaryotic messenger RNAs (mRNAs), which can be purified from the cellular ribonucleic acid (RNA) pool with relative ease, more advanced protocols had to be developed for sequencing of microbial transcriptomes. The resulting RNA-seq data revealed an unexpected complexity of bacterial transcriptomes and the requirement for specific analysis methods, which in many cases is not covered by tools developed for processing of eukaryotic data.
The aim of this thesis was the development and application of specific data analysis methods for different RNA-seq-based approaches used to gain insights into transcription and gene regulatory processes in prokaryotes.
The differential RNA sequencing (dRNA-seq) approach allows for transcriptional start site (TSS) annotation by differentiating between primary transcripts with a 5’-triphosphate (5’-PPP) and processed transcripts with a 5’-monophosphate (5’-P). This method was applied in combination with an automated TSS annotation tool to generate global trancriptome maps for Escherichia coli (E. coli) and Helicobacter pylori (H. pylori).
In the E. coli study we conducted different downstream analyses to gain a deeper understanding of the nature and properties of transcripts in our TSS map. Here, we focused especially on putative antisense RNAs (asRNAs), an RNA class transcribed from the opposite strand of known protein-coding genes with the potential to regulate corresponding sense transcripts. Besides providing a set of putative asRNAs and experimental validation of candidates via Northern analysis, we analyzed and discussed different sources of variation in RNA-seq data.
The aim of the H. pylori study was to provide a detailed description of the dRNA-seq approach and its application to a bacterial model organism. It includes information on experimental protocols and requirements for data analysis to generate a genome-wide TSS map. We show how the included TSS can be used to identify and analyze transcriptome and regulatory features and discuss challenges in terms oflibrary preparation protocols, sequencing platforms, and data analysis including manual and automated TSS annotation.
The TSS maps and associated transcriptome data from both H. pylori and E. coli were made available for visualization in an easily accessible online browser.
Furthermore, a modified version of dRNA-seq was used to identify transcriptome targets of the RNA pyrophosphohydrolase (RppH) in H. pylori. RppH initiates 5’-end-dependent degradation of transcripts by converting the 5’-PPP of primary transcripts to a 5’-P. I developed an analysis method, which uses data from complementary DNA (cDNA) libraries specific for transcripts carrying a 5’-PPP, 5’-P or both, to specifically identify transcripts modified by RppH. For this, the method assessed the 5’-phosphorylation state and cellular concentration of transcripts in rppH deletion in comparison to strains with the intact gene. Several of the identified potential RppH targets were further validated via half-life measurements and quantification of their 5’-phosphorylation state in wild-type and mutant cells. Our findings suggest an important role for RppH in post-transcriptional gene regulationin H. pylori and related organisms.
In addition, we applied two RNA-seq -based approaches, RNA immunoprecipitation followed by sequencing (RIP-seq) and cross-linking immunoprecipitation followed by sequencing (CLIP-seq), to identify transcripts bound by Hfq and CsrA, two RNA-binding proteins (RBPs) with an important role in post-transcriptional regulation.
For RIP-seq -based identification of CsrA binding regions in Campylobacter jejuni(C. jejuni), we used annotation-based analysis and, in addition, a self-developed peak calling method based on a sliding window approach. Both methods revealed flaA mRNA, encoding the major flagellin, as the main target and functional analysis of identified targets showed a significant enrichment of genes involved in flagella biosynthesis. Further experimental analysis revealed the role of flaA mRNA in post-transcriptional regulation. In comparison to RIP-seq, CLIP-seq allows mapping of RBP binding sites with a higher resolution. To identify these sites an approach called “block-based peak calling” was developed and resulting peaks were used to identify sequence and structural constraints required for interaction of Hfq and CsrA with Salmonella transcripts.
Overall, the different RNA-seq-based approaches described in this thesis together with their associated analyis pipelines extended our knowledge on the transcriptional repertoire and modes of post-transcriptional regulation in bacteria. The global TSS maps, including further characterized asRNA candidates, putative RppH targets, and identified RBP interactomes will likely trigger similar global studies in the same or different organisms or will be used as a resource for closer examination of these features.