Würzburg, Univ., Diss., 2008 / Zsfassung in engl. Sprache

Würzburg, Univ., Diss., 2009. / Zsfassung in engl. Sprache.

University, Diss., 2004--Stuttgart.

Marine Schwämme der Gattung Aplysina besitzen Bromotyrosinealkaloide als typische Sekundärmetabolite. Diese Alkaloide werden in einer enzymatischen Abwehrreaktion zu biologisch aktiven Produkten abgebaut. In der vorliegenden Arbeit ist die Isolierung von 14 Schwamminhaltstsoffen aus dem Schwamm Aplysina insularis beschrieben. 14-oxo-Aerophobin-2 konnte als neues Bromoisoxazolinalkaloid beschrieben werden. Anhand des Schwammes Aplysina cauliformis wurde die Charakterisierung der enzymatischen Abwehrreaktion in Schwämmen der Gattung Aplysina vorgenommnen, die von zwei aufeinanderfolgenden Enzymen durchgeführt wird. Hierbei konnte erstmals eine Nitrilhydratase aus dem marinen Habitat beschrieben werden, die in der beschriebenen Abwehrreaktion als zweites Enzym beteiligt ist. / Marine sponges of the genus Aplysina posses bromotyrosine alkaloids as characteristic secondary metabolites. These alkaloids serve as substrates of a chemical defense mechanism in Aplysina sponges. In this dissertation the isolation of 14 constituents of the sponge Aplysina insularis is described. The bromoisoxazoline alkaloid 14-oxo-aerophobin-2 could be described as a new compound. The sponge Aplysina cauliformis was choosen for the characterization of the enzymatic defense mechanism in sponges of the genus Aplysina. In this two step mechanism a nitrilhydratase could be described for the first time. It is also the first time a nitrilhydratase could be described from a marine organism.

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 evolutionär alte, sessile Filtrierer, die eine äußerst vielfältige mikrobielle Gemeinschaft in ihrer internen Mesohylmatrix beherbergen. Das Ziel meiner Doktorarbeit war es, die Anpassungen dieser Symbionten an das Leben in ihrem Schwammwirt zu erforschen. Dazu habe ich die Genome einer Vielzahl von Bakterien aus dem Mikrobiom des Mittelmeer-Schwammes Aplysina aerophoba sequenziert und analysiert. Meine angewandten Methoden waren die fluoreszenzaktivierte Zellsortierung mit anschließender so genannter „multiple displacement amplification“ und Einzelzell- / „Mini-Metagenom“-Sequenzierung und metagenomischer Sequenzierung gefolgt von „differential coverage binning“. Diese beiden Ansätze zielten darauf ab, Genomsequenzen von bakteriellen Symbionten von A. aerophoba zu erhalten, die dann sowohl miteinander, als auch mit Referenzen aus anderen Habitaten verglichen wurden. So sollten Informationen gewonnen werden über Anpassungen an ein Leben im Wirtsschwamm und über mögliche Interaktionen mit dem Wirt und innerhalb der mikrobiellen Gemeinschaft. Cyanobakterien sind häufig Mitglieder der bakteriellen Gemeinschaft in Schwämmen. Mein "Mini-Metagenom"-Sequenzierprojekt lieferte drei Genom-Entwürfe von „Candidatus Synechococcus spongiarum,“ dem cyanobakteriellen Symbionten von A. aerophoba und vieler weiterer Schwämme, die die photische Zone bewohnen. Das vollständigste dieser Genome wurden mit anderen Kladen dieses Symbionten verglichen und mit nah verwandten, freien lebenden Cyanobakterien-Referenzen in Burgsdorf I *, Slaby BM * et al. (2015; * geteilte Erstautorenschaft). Obwohl die vier Kladen von „Ca. Synechococcus spongiarum“ aus den vier Schwammarten A. aerophoba, Ircinia variabilis, Theonella swinhoei und Carteriospongia foliascens auf der Ebene der 16S-rRNA-Gensequenzen zu etwa 99% identisch waren, unterschieden sie sich deutlich auf Genom-Ebene. Nicht nur die Genomgrößen waren von Klade zu Klade verschieden, sondern auch der Gengehalt und eine Reihe von Merkmalen, einschließlich Proteinen mit genannten „eukaryotic-like domains,“ leucinreiche „repeats“ oder Tetratricopeptid-„repeats“. Auf der anderen Seite teilten die vier Kladen eine Reihe von Merkmalen wie Ankyrin-„repeat“-Domänen-haltige Proteine, die auch in anderen Phyla von Schwammsymbionten in verschiedenen Wirtsschwämmen und aus verschiedenen geografischen Orten konserviert zu sein schienen. Ein möglicher neuartiger Mechanismus zur Phagozytose-Vermeidung und zur Phagenresistenz mittels eines veränderten O-Antigens des Lipopolysaccharids wurde identifiziert. Um vorherige Hypothesen über die Anpassung von Schwamm-assoziierten Bakterien auf ein breiteres Spektrum des Mikrobioms von A. aerophoba zu testen und gleichzeitig in der Methodik voran zu schreiten, entwickelte ich einen bioinformatischen Arbeitsablauf, um metagenomische Illumina-„short-read“-Sequenzdaten mit PacBio-„long-reads“ zu kombinieren. Zu Beginn dieses Projektes gab es keine veröffentlichte Methodik zur Verknüpfung von „short-reads“ und „long-reads“ für die Metagenomik, und auch jetzt gibt es keine veröffentlichten Projekte mit einem vergleichbaren Ziel von nicht-gezieltem „Assembly“, „Binning“ und Analyse eines Metagenoms. Ich habe eine Auswahl von „Assembly“-Programmen und Einstellungen auf einem simulierten Testdatensatz getestet, der die Eigenschaften der realen metagenomischen Daten widerspiegelt. Die entwickelte „Assembly“-Methode verbesserte nicht nur die Gesamtstatistik, sondern auch die Qualität der einzelnen, „gebinnten“ Genome, die durch Vergleich zu den ursprünglich veröffentlichten Genom-Sequenzen evaluiert wurde. Das Mikrobiom von A. aerophoba wurde in den letzten Jahren aus verschiedenen Blickwinkeln untersucht, aber nur Genome des Candidatus-Phylum Poribakterien und die Cyanobakteriensequenzen aus meinem oben beschriebenen Projekt wurden bisher veröffentlicht. Durch die Anwendung meiner neu entwickelten „Assembly“-Methodik auf einen metagenomischen Datensatz von A. aerophoba bestehend aus einem PacBio-„long-read“-Datensatz und sechs Illumina-„short-read“-Datensätzen, die für das anschließende „differential coverage binning“ optimiert waren, zielte ich darauf ab, eine größere Anzahl und Vielfalt von Symbionten zu sequenzieren. Die Ergebnisse dieses Projektes sind derzeit bei The ISME Journal in Review. Die Komplementierung von Illumina „short-read“ mit PacBio „long-read“-Sequenzdaten für das „binning“ dieses hochkomplexen Metagenoms hat die Gesamt-„assembly“-Statistik sowie die Qualität der „gebinnten“ Genome deutlich verbessert. Siebenunddreißig Genome aus 13 Bakterienphyla und Candidatus-Phyla wurden „gebinnt“, die die prominentesten Mitglieder des Mikrobioms von A. aerophoba darstellten. Ein statistischer Vergleich zeigte eine Anreicherung von Genen, die mit Restriktionsmodifikationen und Toxin-Antitoxin-Systemen zusammenhängen, in den meisten Symbionten-Genomen im Vergleich zu ausgewählten Referenzgenomen. Beides sind Mechanismen zur Verteidigung gegen eindringende Fremd-DNA, die für Schwamm-Symbionten aufgrund der Schwamm-Filtration und Phagozytose-Aktivität wichtig sein können, die die Symbionten hohen Konzentrationen von freier DNA aussetzen. Auch mögliche Wirtskolonisations- und Matrixnutzungsmechanismen waren signifikant angereichert. Wegen der Vielfalt der „gebinnten“ Symbionten-Genome war ein Genom-Vergleich innerhalb der Symbionten möglich, der drei Gilden von Symbionten zum Vorschein brachte, die gekennzeichnet waren durch i) Ernährungsspezialisierung auf die Metabolisierung von Carnitin, ii) Spezialisierung auf sulfatierte Polysaccharide und iii) scheinbaren Nahrungs-Generalismus. Sowohl Carnitin als auch sulfatierte Polysaccharide sind in der extrazellulären Schwammmatrix reichlich vorhanden und stehen so den Schwammsymbionten als Substrat zur Verfügung. Die Genome der diversen Symbionten-Gemeinschaft in A. aerophoba waren in ihren Verteidigungsmechanismen vereint, aber spezialisiert hinsichtlich ihrer Ernährung.

Esponjas do gênero Aplysina (Ordem: VERONGIDA) são conhecidas por apresentarem em seu metabolismo secundário compostos derivados da dibromotirosina, os quais são um importante aliado para o processo de identificação dessas esponjas. Este trabalho descreve o estudo do perfil químico de 14 espécimens de esponjas do gênero Aplysina, coletadas na Bahia de Todos os Santos-BA, no ano de 2007. Também foi desenvolvida uma metodologia de desreplicação, utilizando-se da técnica hifenada HPLC-PDA-MS(ESI), busca na literatura (MarinLit) e comparação com padrões. Os resultados obtidos demonstram claramente que essas esponjas apresentam os derivados da dibromotirosina e que a metodologia de desreplicação permitiu confirmar e/ou prever possíveis estruturas desses compostos com eficiência e sem a necessidade do seu isolamento. Entretanto, não foi possível distinguir as espécies dos organismos estudados através do seu perfil químico, uma vez que eles apresentaram praticamente os mesmos metabólitos em todas as frações analisadas. Isto demonstra que a classificação taxonômica em nível de espécie requer, necessariamente, a análise do esqueleto (espículas) das esponjas e possivelmente análises genômicas. A análise das frações aquosas permitiu apenas confirmar a presença desses derivados através da análise das absorções típicas no espectro de UV. Também, este é o primeiro trabalho sobre o estudo do perfil químico dessas esponjas com a utilização de um sistema de desreplicação que inclui a técnica hifenada HPLC-PDA-MS(ESI). / Sponges of the genus Aplysina (Order: VERONGIDA) are commonly recognized by the presence of dibromotyrisine derivates at their secondary metabolites, compounds which are an important tool for their taxonomic identification. The present study describes the chemical profile of 14 sponges of Aplysina sp., collected in Bahia de Todos os Santos-BA. We developed a dereplication methodology using the hyphenated technique HPLC-PDA-MS(ESI), a database research (MarinLit) and comparison with chemical standards. The results showed that the 14 sponges analysed present dibromotyrisine derivates and that the dereplication methodology enabled the prediction and/or confirmation of such structures without their previous isolation. However, the taxonomic identification of the 14 individual sponges at the species level was impossible due to the fact that chemical profile was of the sponges too similar. This result demonstrates that the taxonomic identification Aplysina sponges at the species level also requires the skeleton analysis and possibly genomic studies. Analyses of aqueous fraction confirmed the presence of dibromotyrisine derivates. Furthermore, this is the first study of the chemical profile of Aplysina sponges that utilizes a dereplication methodology which includes the hyphenated technique HPLC-PDA-MS(ESI).

Estudos realizados em 1979 por Kelecom e Kanengiesser mostraram que extratos brutos obtidos a partir de amostras da esponja marinha Aplysina fulva não possuíam derivados bromados. Estes foram resultados inesperados, uma vez que esponjas pertencentes à ordem Verongida são conhecidos produtores de metabólitos derivados da dibromotirosina. O presente projeto teve como meta a investigação química de extratos obtidos de duas amostras de A. fulva, sendo uma coletada em São Sebastião (SP) e a outra em Angra dos Reis (RJ), objetivando verificar a ocorrência de derivados da bromotirosina e uma possível variabilidade química dependendo da distribuição geográfica das esponjas. Sete derivados da dibromotirosina foram isolados das duas amostras de Aplysina fulva: quatro compostos da amostra de Angra dos Reis e três da de São Sebastião. As estruturas dos compostos foram identificadas por análises espectroscópicas (como IV, UV, RMN 1H, RMN 13C, HMQC, COSY, HMBC) e de espectrometria de massas, e ainda por comparação com dados da literatura. Os resultados obtidos confirmaram que os derivados da dibromotirosina são bons marcadores quimiotaxonômicos para as esponjas da Ordem Verongida. Além disso, a variabilidade química observada para a A. fulva parece ser influência de fatores abióticos e bióticos como sazonalidade, disponibilidade de nutrientes, ou associação com diferentes micro-organismos. / Studies developed in 1979 by Kelecom and Kanengiesser showed that crude extracts obtained from samples of the marine sponge Aplysina fulva were devoid of bromotyrosine derivatives. These results were rather unexpected, since sponges belonging to the Order Verongida are well-known producers of bromotyrosine-derived metabolites. The present project aimed the chemical investigation of crude extracts obtainded from two samples of A. fulva, one collected at São Sebastião (SP) and the second from Angra dos Reis (RJ), in order to verify the occurrence of bromotyrosine derivatives and a possible chemical variability depending on the geographical distribution of the sponge. Seven bromoyrosine derivatives have been isolated from the two samples of A. fulva: four compounds from the Angra dos Reis sample and three from the São Sebastião sample. The structures of the compounds have been established by spectroscopic analysis, (including IV, UV, RMN 1H, RMN 13C, HMQC, COSY, HMBC) and mass spectrometry, as well as by comparison with literature data. The results obtained confirmed that bromotyrosine derivatives are good chemotaxonomic markers for sponges of the Order Verongida. Moreover, it appears that chemical variability of A. fulva may be influenced by abiotic and biotic factors, such as seasonality, nutrients availability, or association with distinct micro-organisms.

Estudos realizados em 1979 por Kelecom e Kanengiesser mostraram que extratos brutos obtidos a partir de amostras da esponja marinha Aplysina fulva não possuíam derivados bromados. Estes foram resultados inesperados, uma vez que esponjas pertencentes à ordem Verongida são conhecidos produtores de metabólitos derivados da dibromotirosina. O presente projeto teve como meta a investigação química de extratos obtidos de duas amostras de A. fulva, sendo uma coletada em São Sebastião (SP) e a outra em Angra dos Reis (RJ), objetivando verificar a ocorrência de derivados da bromotirosina e uma possível variabilidade química dependendo da distribuição geográfica das esponjas. Sete derivados da dibromotirosina foram isolados das duas amostras de Aplysina fulva: quatro compostos da amostra de Angra dos Reis e três da de São Sebastião. As estruturas dos compostos foram identificadas por análises espectroscópicas (como IV, UV, RMN 1H, RMN 13C, HMQC, COSY, HMBC) e de espectrometria de massas, e ainda por comparação com dados da literatura. Os resultados obtidos confirmaram que os derivados da dibromotirosina são bons marcadores quimiotaxonômicos para as esponjas da Ordem Verongida. Além disso, a variabilidade química observada para a A. fulva parece ser influência de fatores abióticos e bióticos como sazonalidade, disponibilidade de nutrientes, ou associação com diferentes micro-organismos. / Studies developed in 1979 by Kelecom and Kanengiesser showed that crude extracts obtained from samples of the marine sponge Aplysina fulva were devoid of bromotyrosine derivatives. These results were rather unexpected, since sponges belonging to the Order Verongida are well-known producers of bromotyrosine-derived metabolites. The present project aimed the chemical investigation of crude extracts obtainded from two samples of A. fulva, one collected at São Sebastião (SP) and the second from Angra dos Reis (RJ), in order to verify the occurrence of bromotyrosine derivatives and a possible chemical variability depending on the geographical distribution of the sponge. Seven bromoyrosine derivatives have been isolated from the two samples of A. fulva: four compounds from the Angra dos Reis sample and three from the São Sebastião sample. The structures of the compounds have been established by spectroscopic analysis, (including IV, UV, RMN 1H, RMN 13C, HMQC, COSY, HMBC) and mass spectrometry, as well as by comparison with literature data. The results obtained confirmed that bromotyrosine derivatives are good chemotaxonomic markers for sponges of the Order Verongida. Moreover, it appears that chemical variability of A. fulva may be influenced by abiotic and biotic factors, such as seasonality, nutrients availability, or association with distinct micro-organisms.

Esponjas do gênero Aplysina (Ordem: VERONGIDA) são conhecidas por apresentarem em seu metabolismo secundário compostos derivados da dibromotirosina, os quais são um importante aliado para o processo de identificação dessas esponjas. Este trabalho descreve o estudo do perfil químico de 14 espécimens de esponjas do gênero Aplysina, coletadas na Bahia de Todos os Santos-BA, no ano de 2007. Também foi desenvolvida uma metodologia de desreplicação, utilizando-se da técnica hifenada HPLC-PDA-MS(ESI), busca na literatura (MarinLit) e comparação com padrões. Os resultados obtidos demonstram claramente que essas esponjas apresentam os derivados da dibromotirosina e que a metodologia de desreplicação permitiu confirmar e/ou prever possíveis estruturas desses compostos com eficiência e sem a necessidade do seu isolamento. Entretanto, não foi possível distinguir as espécies dos organismos estudados através do seu perfil químico, uma vez que eles apresentaram praticamente os mesmos metabólitos em todas as frações analisadas. Isto demonstra que a classificação taxonômica em nível de espécie requer, necessariamente, a análise do esqueleto (espículas) das esponjas e possivelmente análises genômicas. A análise das frações aquosas permitiu apenas confirmar a presença desses derivados através da análise das absorções típicas no espectro de UV. Também, este é o primeiro trabalho sobre o estudo do perfil químico dessas esponjas com a utilização de um sistema de desreplicação que inclui a técnica hifenada HPLC-PDA-MS(ESI). / Sponges of the genus Aplysina (Order: VERONGIDA) are commonly recognized by the presence of dibromotyrisine derivates at their secondary metabolites, compounds which are an important tool for their taxonomic identification. The present study describes the chemical profile of 14 sponges of Aplysina sp., collected in Bahia de Todos os Santos-BA. We developed a dereplication methodology using the hyphenated technique HPLC-PDA-MS(ESI), a database research (MarinLit) and comparison with chemical standards. The results showed that the 14 sponges analysed present dibromotyrisine derivates and that the dereplication methodology enabled the prediction and/or confirmation of such structures without their previous isolation. However, the taxonomic identification of the 14 individual sponges at the species level was impossible due to the fact that chemical profile was of the sponges too similar. This result demonstrates that the taxonomic identification Aplysina sponges at the species level also requires the skeleton analysis and possibly genomic studies. Analyses of aqueous fraction confirmed the presence of dibromotyrisine derivates. Furthermore, this is the first study of the chemical profile of Aplysina sponges that utilizes a dereplication methodology which includes the hyphenated technique HPLC-PDA-MS(ESI).

Zugl.: Karlsruhe, Univ., Diss., 2008 / Hergestellt on demand