Evaluation of sequence alignments and oligonucleotide probes with respect to three-dimensional structure of ribosomal RNA using ARB software package

BACKGROUND: Availability of high-resolution RNA crystal structures for the 30S and 50S ribosomal subunits and the subsequent validation of comparative secondary structure models have prompted the biologists to use three-dimensional structure of ribosomal RNA (rRNA) for evaluating sequence alignments of rRNA genes. Furthermore, the secondary and tertiary structural features of rRNA are highly useful and successfully employed in designing rRNA targeted oligonucleotide probes intended for in situ hybridization experiments. RNA3D, a program to combine sequence alignment information with three-dimensional structure of rRNA was developed. Integration into ARB software package, which is used extensively by the scientific community for phylogenetic analysis and molecular probe designing, has substantially extended the functionality of ARB software suite with 3D environment. RESULTS: Three-dimensional structure of rRNA is visualized in OpenGL 3D environment with the abilities to change the display and overlay information onto the molecule, dynamically. Phylogenetic information derived from the multiple sequence alignments can be overlaid onto the molecule structure in a real time. Superimposition of both statistical and non-statistical sequence associated information onto the rRNA 3D structure can be done using customizable color scheme, which is also applied to a textual sequence alignment for reference. Oligonucleotide probes designed by ARB probe design tools can be mapped onto the 3D structure along with the probe accessibility models for evaluation with respect to secondary and tertiary structural conformations of rRNA. CONCLUSION: Visualization of three-dimensional structure of rRNA in an intuitive display provides the biologists with the greater possibilities to carry out structure based phylogenetic analysis. Coupled with secondary structure models of rRNA, RNA3D program aids in validating the sequence alignments of rRNA genes and evaluating probe target sites. Superimposition of the information derived from the multiple sequence alignment onto the molecule dynamically allows the researchers to observe any sequence inherited characteristics (phylogenetic information) in real-time environment. The extended ARB software package is made freely available for the scientific community via

probeCheck – a central resource for evaluating oligonucleotide probe coverage and specificity

The web server probeCheck, freely accessible at http://www.microbial-ecology.net/probecheck, provides a pivotal forum for rapid specificity and coverage evaluations of probes and primers against selected databases of phylogenetic and functional marker genes. Currently, 24 widely used sequence collections including the Ribosomal Database Project (RDP) II, Greengenes, SILVA and the Functional Gene Pipeline/Repository can be queried. For this purpose, probeCheck integrates a new online version of the popular ARB probe match tool with free energy (ΔG) calculations for each perfectly matched and mismatched probe-target hybrid, allowing assessment of the theoretical binding stabilities of oligo-target and non-target hybrids. For each output sequence, the accession number, the GenBank taxonomy and a link to the respective entry at GenBank, EMBL and, if applicable, the query database are displayed. Filtering options allow customizing results on the output page. In addition, probeCheck is linked with probe match tools of RDP II and Greengenes, NCBI blast, the Oligonucleotide Properties Calculator, the two-state folding tool of the DINAMelt server and the rRNA-targeted probe database probeBase. Taken together, these features provide a multifunctional platform with maximal flexibility for the user in the choice of databases and options for the evaluation of published and newly developed probes and primers

Dynamics of Symbiont Abundance in Bathymodiolin Deep-sea Symbioses

Deep-sea hydrothermal vents and cold seeps are widespread throughout the world's oceans and represent `oases of life' in a dark and hostile environment. These chemosynthetic habitats are often dominated by mussels of the genus Bathymodiolus. The bivalves harbor bacterial symbionts in their gills that use methane and reduced inorganic compounds such as sulfide and hydrogen as energy sources for chemosynthetic primary production. It is well known that the spatial and temporal gradients of these energy sources can be extremely steep at vents and seeps, and some studies have shown that differences in energy source availability affect symbiont abundance in Bathymodiolus mussels. However, in-depth analyses of physico-chemical gradients and their effect on symbiont abundance are lacking. One of the basic requirements for these analyses is a reliable quantification method for the symbionts. The goal of my thesis was therefore to develop an accurate and efficient protocol for determining symbiont abundance in bathymodiolin mussels

The Candidate phylum "Termite Group". Diversity, distribution, metabolism and evolution of representatives of an unexplored bacterial phylum

This thesis summarizes a series of studies of the phylum Elusimicrobia (formerly candidate phylum "Termite Group"). The environmental distribution of members of this phylum, and the genome sequence of the first and only cultivated representative, Elusimicrobium minutum, were the focus of these studies. In addition, a comparative genome analysis of the endosymbiotic representatives from this phylum was conducted. Intestinal tracts of insects harbor several novel deep-rooting lineages of hitherto uncultivated bacteria, whose physiology is obscure. One of these groups is the phylum Elusimicrobia. Originally, the Elusimicrobia were represented only by the so-called endomicrobia, which had been identified as endosymbionts of flagellate protists of lower termites and wood-feeding cockroaches, but public databases contain a growing number of distantly related 16S rRNA gene sequences that fall into the radiation of the Elusimicrobia phylum. In the first study, group-specific primers were used to demonstrate that members of the Elusimicrobia are widespread in the environment and, in addition to the endomicrobia, comprise numerous other monophyletic lineages occurring in various habitats. One of these lineages consisted of sequences obtained from various intestinal habitats. It comprised also the 16S rRNA gene of Elusimicrobium minutum, the first cultivated representative of the Elusimicrobia phylum, which was isolated from the gut of the scarab beetle larva Pachnoda ephippiata. This pure culture was the basis for the second study, which included the physiological and morphological characterization of E. minutum and the analysis of its genome. The genome allowed reconstruction of the metabolic pathways for the most important growth substrates (glucose, fructose, N-acetyl-glucosamine), revealing all genes required for uptake and fermentation of sugars via the Embden-Meyerhoff pathway and production of ethanol, acetate, H2, and CO2. Based on the genome, it could be predicted that a [NiFe] hydrogenase is responsible for the production of hydrogen, depending on the hydrogen partial pressure. This enzyme probably couples the production of hydrogen with the generation of a proton-motive force (energy-converting hydrogenase). At low hydrogen partial pressure, hydrogen production may shift to a [FeFe] hydrogenase that synergistically uses NADH and reduced ferredoxin to produce hydrogen. In addition the genome also encodes for an unusual peptide degradation pathway that comprises transamination reactions and leads to the formation of alanine from pyruvate, an explanation for why E. minutum excretes alanine in substantial amounts. The function of the exceptionally high number of pilE genes in the genome is still elusive, and since electron micrographs showed no cell appendages, their participation in pilus assembly is uncertain. The presence of a rubredoxin:oxygen oxidoreductase operon in the genome of E. minutum indicated that the strictly anaerobic bacterium may also be able to reduce small amounts of molecular oxygen, which is also in accordance with physiological observations. The phylogenetic analysis of 22 concatenated single-copy marker genes from the genome reinforced the phylum-level status of Elusimicrobia and confirmed the reproducible relationship between E. minutum and endomicrobia (represented by Candidatus "Endomicrobium trichonymphae" strain Rs-D17) as already predicted at the 16S rRNA level. This analysis was possible because of the recent publication of the genome of strain Rs-D17 from Trichonympha agilis. In parallel, a metagenome library from an enriched CET population derived from diverse flagellates of Zootermopsis nevadensis was prepared. The last part of this thesis consists of a comparative analysis of the large genome fragments from the metagenome library with the genome of strain Rs-D17. It revealed parallel evolution of endomicrobia strains in different termites and indicated the presence of genome rearrangements. The genome rearrangements suggest the ability for homologous recombination, which could be facilitated by the horizontal transfer of endosymbionts between Trichonympha species during occasional sexual reproduction. Together, these studies contribute fundamental insights into the diversity, distribution, metabolism, and evolution of representatives from the phylum Elusimicrobia. The genome revealed along with the metabolic capacities of E. minutum numerous genes with only hypothetical functions, e.g., polyketide synthesis, indicating the presence of hitherto undiscovered physiological traits which could be the subject for further investigations. The detailed annotation of E. minutum will also serve as reference for future annotations of other closely related members of this phylum. Together, these studies contribute fundamental insights into the diversity, distribution, metabolism and evolution of representatives from the phylum Elusimicrobia. The genome revealed along with the metabolic capacities of E. minutum numerous genes with only hypothetical functions, e.g., polyketide synthesis indicating the presence of hitherto undiscovered physiological traits which could be the subject for further investigation. The detailed annotation of E. minutum will also serve as reference for future annotations of other closely related members of this phylum

Struktur und räumliche Verteilung mikrobieller Gemeinschaften im Verdauungstrakt ausgewählter Boden-Invertebraten

Boden-Makroinvertebraten sind entscheidend an der Transformation organischer Substanz beteiligt, die für viele Schlüsselfunktionen des Bodens verantwortlich ist. An den Transformationsprozessen, die während der Darmpassage ingestierter organischer Substanz ablaufen, sind intestinale Mikroorganismen beteiligt, die auch für die Ernährung ihrer Wirte von entscheidender Bedeutung sind. Über die Zusammensetzung der mikrobiellen Gemeinschaften im Verdauungstrakt der meisten Boden-Invertebraten sowie ihre räumliche Verteilung innerhalb verschiedener Darmabschnitte ist allerdings nur wenig bekannt. Gerade die Topologie mikrobieller Gemeinschaften wird aber als eine wichtige Voraussetzung zum tieferen Verständnis ihrer Funktion in Verdauungstrakten von Invertebraten angesehen, die von ausgeprägten axialen und radialen Gradienten physiko-chemischer Parameter geprägt sind. Im Rahmen dieser Arbeit wurden daher Struktur und räumliche Verteilung mikrobieller Gemeinschaften im Verdauungstrakt der Larven zweier Scarabaeiden (Pachnoda ephippiata [Kongo-Rosenkäfer] und Melolontha melolontha [Feldmaikäfer]) und von Regenwürmern (Lumbricus terrestris) mit Methoden der molekularen mikrobiellen Ökologie untersucht. Während der Untersuchungen wurde zudem ein neuartiger PCR-Artefakt, die Bildung sog. pseudo-T-RFs, mit bedeutenden Auswirkungen für die T-RFLP-Analyse mikrobieller Gemeinschaften entdeckt, beschrieben und mögliche Ansätze zu seiner Vermeidung aufgezeigt. In einer T-RFLP-Studie mit Regenwürmern wurde gezeigt, dass sich die Darmmikrobiota dieser Tiere aus der Nahrung rekrutiert, d.h. ihnen im Gegensatz zu vielen anderen Boden-Invertebraten eine spezifische Darmflora fehlt. Es wurde deutlich, dass die mit dem gefressenen Boden aufgenommene mikrobielle Gemeinschaft während der Darmpassage signifikante Veränderungen ihrer relativen Zusammensetzung erfährt und dass die Unterschiede zwischen den mikrobiellen Gemeinschaften von Futter, Darm und Losung stark von der Diät der Regenwürmer beeinflusst werden. Die hier präsentierten Ergebnisse zur Intestinalmikrobiologie von Scarabaeidenlarven stellen die ersten ihrer Art für Käferlarven und mit die ersten für andere Boden-Arthropoden als Termiten dar. Es konnte gezeigt werden, dass sich die ausgeprägten Unterschiede physiko-chemischer Parameter (pH-Wert, Redoxpotential, Fettsäurespektren), die zwischen den Haupt-Darmabschnitten (Mittel- und Enddarm) der Larven herrschen, in einer deutlich unter-schiedlichen Besiedlung mit Mikroorganismen widerspiegeln. Im Gegensatz zu den untersuchten Regenwürmern ist die Darmmikrobiota der Scarabaeidenlarven als spezifische Darmflora anzusehen, da sie deutlich verschieden zur Mikrobiota der aufgenommenen Nahrung war. Bei beiden Larven war der Mitteldarmabschnitt weniger dicht besiedelt als der als Gärkammer angesehene Enddarm. Die Methanogenese war stets auf den Enddarm beschränkt; bei den Maikäferlarven wurden Methanobrevibacter-Arten, bei den Rosenkäferlarven zusätzlich Methanomicrococcus-Arten als verantwortliche Methanogene identifiziert. Im Vergleich zu den Bacteria war aber sowohl die Diversität als auch die relative Häufigkeit der Archaea sehr gering. Die phylogenetische Analyse der Bakteriengemeinschaften zeigte eine sehr große Diversität auf, die offensichtlich viele bislang unkultivierte Arten umfasst. Die überwiegende Mehrheit aller Sequenzen ließ sich den Actinobacteria, Bacillales, Bacteroidetes, Clostridiales, Lactobacillales und Proteobacteria zuordnen; viele Klone gruppierten mit Klonen und Isolaten aus anderen Intestinalsystemen, ein weiterer Beleg für die Darmspezifität der Scarabaeiden-Mikrobiota. Die Verwandtschaft vieler Klone zu hydrolytischen, cellulolytischen und gärenden Isolaten stand in Einklang mit den Fettsäureprofilen der Darmabschnitte (v.a. Acetat und Lactat) und deutet die Beteiligung von Mikroorganismen an der Transformation organischer Substanz nach dem Modell einer anaeroben Nahrungskette zumindest im Enddarm an. Ob dies auch für den Mitteldarmabschnitt gilt, ist noch unklar, da bei den Maikäferlarven keine stabile Darmmikrobiota in diesem Kompartiment nachgewiesen werden konnte. Am Enddarm der Maikäferlarven wurde erstmals für Arthropoden eine umfassende Analyse der mikrobiellen Gemeinschaften in den Unterfraktionen Wand und Lumen eines Darmabschnittes durchgeführt. Hierbei wurden ausgeprägte Unterschiede in der Besiedlung dieser beiden Fraktionen festgestellt, die als Anpassungen an morphologische (Chitinbäumchen an der Enddarmwand) und eventuell auch physiko-chemische Unterschiede (Gradient eindringenden Sauerstoffs) zwischen Darmwand und -lumen interpretiert werden können. Der auffälligste Unterschied war eine hohe Abundanz (10 - 15% aller Bakterien) Desulfovibrio-verwandter Bakterien an der Enddarmwand, die sowohl mit PCR-abhängigen als auch PCR-unabhängigen Methoden abgesichert werden konnte. In seiner Eindeutigkeit ist dieser Befund für Arthropoden bislang einmalig

Diversity, specificity and evolutionary history of marine invertebrate symbioses and functions of the sulfur-oxidizing symbionts

Many marine invertebrates have established symbioses with chemosynthetic bacteria that metabolize reduced sulfur compounds and provide nutrition to their host. Two key questions in the field of symbiosis are: (1) How specific and evolutionarily stable are these symbioses? Chapters II, III and IV of this thesis contribute to a more comprehensive understanding of this question by investigating the diversity, specificity and evolutionary history of three sulfur-oxidizing symbioses: deep-sea vestimentiferan tubeworm endosymbioses, shallow water gutless phallodriline oligochaete endo- and stilbonematine nematode ectosymbioses. The studies emphasize the power of molecular analyses to uncover hidden symbiont diversity and highlight the remarkably stable and specific stilbonematine ectosymbioses. (2) What are the benefits for the symbiotic partners? Chapter V strengthens the hypothesis of stilbonematine ectosymbionts' nutritional role for their host and the genomic study in Chapter VI discusses potential additional functions of the ectosymbionts for their nematode host

Diversity studies and molecular analyses with single cells and filaments of large, colorless sulfur bacteria

Large sulfur bacteria feature conspicuous morphologies that are usually visible with the naked eye. Most representatives were already described in the 19th and early 20th century and it needed nearly another 100 years until a new morphotype of large sulfur bacteria was discovered. This discovery encouraged a search for other yet unknown types in this group of bacteria and indeed led to the finding of a new type in two marine seep settings. This novel morphotype is presented in Chapter 2 and is the first non-filamentous member in the family Beggiatoaceae that shows a dimorphic life cycle, exhibiting alternation between sessile and free-living forms. In Chapter 3, another three novel morphotypes are presented, which were discovered in Namibian sediments. The detection of these novel morphotypes of large sulfur bacteria led to the necessity for an improved phylogenetic classification of the entire group. Earlier attempts to sequence the 16S rRNA genes of the large sulfur bacteria resulted in only few nearly full-length sequences, whereas some genera were even represented by only partial sequences. Accordingly, differentiation of genera in this group is still based on morphological features. Now, Chapter 3 presents the sequencing of 16S rRNA genes and ITS regions of more than 100 individual cells and filaments of large sulfur bacteria, revealing a major insight into the phylogeny of these extraordinary bacteria. It is demonstrated that the traditional, morphology-based classification does not correlate with the phylogeny derived from 16S rRNA gene sequences. Consequently, a reclassification of the entire family is proposed, being completely independent from former morphological categories. Sequencing single cells and filaments of large sulfur bacteria furthermore revealed that they represent the first group of bacteria, which commonly contain large and numerous introns in their 16S rRNA genes. In Chapter 4, it is demonstrated that the introns are removed efficiently from the rRNA precursor, thereby ligating the two exons, and are not present in the native ribosome. Furthermore, it was experimentally verified that a commonly applied PCR approach introduces a length heterogeneity bias and systematically discriminates against enlarged genes and favors the amplification of shorter homologues. Consequently, this fact questions the universality of 16S rRNA-based clone libraries for diversity studies. At least the group of large sulfur bacteria is systematically discriminated against in such universal PCR approaches and it can be assumed that this PCR bias also affects a yet unknown amount of other microorganisms

Dynamics of methane oxidation and composition of methanotrophic community in planted rice microcosms

Thesis about dynamics of methane oxidation and composition of methanotrophic community in planted rice microcosms