Graphical representation of ribosomal RNA probe accessibility data using ARB software package

BACKGROUND: Taxon specific hybridization probes in combination with a variety of commonly used hybridization formats nowadays are standard tools in microbial identification. A frequently applied technology, fluorescence in situ hybridization (FISH), besides single cell identification, allows the localization and functional studies of the microbial community composition. Careful in silico design and evaluation of potential oligonucleotide probe targets is therefore crucial for performing successful hybridization experiments. RESULTS: The PROBE Design tools of the ARB software package take into consideration several criteria such as number, position and quality of diagnostic sequence differences while designing oligonucleotide probes. Additionally, new visualization tools were developed to enable the user to easily examine further sequence associated criteria such as higher order structure, conservation, G+C content, transition-transversion profiles and in situ target accessibility patterns. The different types of sequence associated information (SAI) can be visualized by user defined background colors within the ARB primary and secondary structure editors as well as in the PROBE Match tool. CONCLUSION: Using this tool, in silico probe design and evaluation can be performed with respect to in situ probe accessibility data. The evaluation of proposed probe targets with respect to higher-order rRNA structure is of importance for successful design and performance of in situ hybridization experiments. The entire ARB software package along with the probe accessibility data is available from the ARB home page

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

A new fluorescent oligonucleotide probe for in-situ identification of Microcystis aeruginosa in freshwater.

contaminated water bodies (freshwater, brackish and marine areas). Among 150 known cyanobacteria genera,>40 species are able to produce toxins, which are natural compounds that differ from both a chemical and toxicological point of view and are responsible for acute and chronic poisoning in animals and humans. Among the main classes of cyanotoxins, microcystins are frequently found in the environment. Fast and accurate methods for unequivocally identifying microcystin-producing cyanobacteria, such as Microcystis aeruginosa in water bodies, are necessary to distinguish them from other non-toxic cyanobacteria and to manage and monitor algal blooms. For this purpose, we designed, developed and validated an oligonucleotide probe for FISH (Fluorescence In Situ Hybridization) analysis to detect Microcystis aeruginosa at the species level even at relatively low concentrations in freshwater. The FISH probe, MicAerD03, was designed using the ARB software with the Silva database within the framework of the MicroCoKit project, also with the intention of adding it to the microarray from the EU project, μAQUA, for freshwater pathogens, which had only genus level probes for Microcystis. We tested various fixative methods to minimize the natural autofluorescence from chlorophyll-a and certain accessory pigments (viz., phycobilins and carotenoids). The FISH probe was tested on pure cultures of Microcystis aeruginosa, and then successfully applied to water samples collected from different sampling points of the Tiber River (Italy), using a laser confocal microscope. Subsequently, the probe was also conjugated at the 5′ end with horse-radish peroxidase (HRP-MicAerD03) to apply the CAtalysed Reported Deposition-FISH (CARD-FISH) for increasing the fluorescence signal of the mono-fluorescently labelled probe and make it possible to detect M. aeruginosa using an epifluorescence microscope. Samples taken within the EU MicroCokit project indicated thatmicroarray signals for Microcystis were coming from single cells and not colonial cells. We confirmed this with the CARD-FISH protocol used here to validate the microarray signals for Microcystis detected at the genus level in MicroCokit. This paper provides a new early warning tool for investigating M. aeruginosa at the species level even at low cell concentrations in surface water, which can be added to the μAqua microarray for all freshwater pathogens to complete the probe hierarchy for Microcystis aeruginosa

Mini Review: Molecular Techniques for Identification and Characterization of Marine Biodiversity

Molecular methods to detect organisms in the natural environment can be divided into whole cell and cell free formats. Whole cell methods are generally limited by the number of fluorochromes that can be detected, whereas cell free formats offer more possibilities for multiple species detection and multiple methods of detection. This mini review addresses the major tools applied in environmental studies using cell free methods. The methods reviewed include microarrays, biosensors, quantitative real-time PCR (qPCR), and next generation sequencing (NGS)

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

(Acido)bacterial diversity in space and time

Recent technological achievements enabled microbiologists to fully grasp the vast diversity of microbial life that is resident in soils, highly complex matrices of alternating micro-habitats on very small scales. Since then, microbial community composition has been catalogued for many different terrestrial habitats. This triggered the investigation and definition of processes which shape these communities. In most cases, the environment determines community composition, and similar habitats may feature similar microbial communities despite being far apart. However, some habitats have been described as subjected to pronounced neutral processes, which are dispersal, ecological drift or speciation. The balance between these process types is now the subject of many studies looking at microbial communities. It is also clear that these processes need to be monitored on both temporal and spatial scales, as the two dimensions are inseparably interlinked. However, most microbial studies deal with only one aspect, but do not control for the other. In this work, the outcome of a highly sophisticated plot scale experiment is presented encompassing 358 sampling locations distributed between six intra-annual sampling points on a 10 m x 10 m unfertilized grassland site in the Swabian Alb. RNA was extracted from the A-horizon of each soil and the hypervariable region 3 of the ribosomal small subunit was amplified and sequenced with barcoded Illumina sequencing. Roughly 400 million eubacterial reads were obtained. The dataset was used to assess the population dynamics of Acidobacteria, as well as the spatio-temporal co-occurenze of functionally depending microorganism. Additionally, preliminary results motivated the assessment of common methods for the examination of rhizospheric communities. In combination, the diversity of bacterial communities in space and time was tested from different angles, reflecting different research question, and they all revealed a far more complex reality than previously thought

Endosymbionts of two species of mediterranean lucinid clams

Symbiosen stellen, wie anhand einiger Invertebraten belegt, eine potentiell wichtige Überlebensstrategie vieler Tiere in unwirtlichen Lebensräumen dar. Solche Lebensräume können etwa schwefelhaltige Sedimentschichten sein. Um mit den lebensfeindlichen Bedingungen hier zu Recht zu kommen, besitzen Muscheln der Familie Lucinidae (dt. Mondmuscheln) Sulfid-oxidierende Endosymbionten. Außerdem schließen die Symbionten durch den chemischen Prozess die energiereichen reduzierten Schwefelverbindungen als zusätzliche Nahrungsquelle auf. Da bislang vorwiegend tropische Vertreter der Familie untersucht wurden, beschäftigt sich meine Diplomarbeit mit den kleineren Mittelmeerarten Loripes lacteus (Linneaus, 1758) und Anodontia (Loripinus) fragilis (Philippi, 1836). Ziel der Arbeit war eine Beschreibung der Endosymbionten beider Muschelarten. Die analysierten Tiere stammten aus Sulfid-reichen Sedimentschichten unterhalb einer Cymodocea nodosa-Seegraswiese. Sequenzanalysen hatten das 16S rRNA Gen des bakteriellen Symibonten zum Ziel. Niedrige Sequenzähnlichkeiten, basierend auf der Berechnung von paarweisen Distanzen (P-distances), gaben einen ersten Hinweis, dass die Endosymbionten von L. lacteus und A. fragilis zwei unterschiedlichen Bakterienarten angehören. Basierend auf der Sequenz wurden klonspezifische Sonden für die Muschelsymbionten entwickelt und mittels Fluoreszenz in situ Hybridisierung (FISH) die Spezifität geprüft. Außerdem konnte mittels confocaler Laser-Scannung Mikroskopie eine gleichmäßige Verteilung des fluoreszierenden Symbiontensignals in der gesamten Lateralzone der Kiemenfilamente beobachtet werden. Die Untersuchung ergab, dass L. lacteus und A. fragilis eine einzige, spezifische Endosymbiontenpopulation aus der Gruppe der γ-Proteobacteria beherbergen, die über drei Monate unveränderlich blieb. Transmissionselektronenmikroskopische Aufnahmen zeigen einzelne Endosymbionten eingeschlossen in Vakuolen, die innerhalb großer Bacteriocyten über die gesamte Lateralzone verteilt sind. Außerdem variieren die zusätzlichen Zelltypen in den untersuchten Bivalven, was sich vermutlich auf unterschiedliche physiologische Anpassungen an die Symbiose zurückführen lässt. Im Bakterioplasma der Endosymbionten von L. lacteus befinden sich zahlreiche Vakuolen, die möglicherweise der Sulfidspeicherung dienen. Phylogenetische Analysen unterstützen eine Monophylie von Endosymbionten der Familie Lucinidae nicht. Stattdessen zeigen symbiontische Sequenzen basierend auf Kiemen-Extrakten von L. lacteus eine nahe Verwandtschaft mit Symbionten von Lucina floridana und Codakia costata. 16S rRNA Sequenzen aus A. fragilis bilden dagegen eine Schwesterngruppe mit A. phillipiana und Solemya terraeregina. Diese Gruppe clustert innerhalb eines allgemeinen Symbiontenclades, das neben der Endosymbiontensequenz der Lucinidae Phacoides (Lucina) pectinata auch die Symbionten der Bivalvenfamilien Solemyidae und Thyasiridae, sowie solche von vestimentiferen Röhrenwürmern enthält. Damit ist durch die phylogenetische Analyse die Existenz zweier unterschiedlicher endosymbiontischer Arten in L. lacteus und A. fragilis untermauert.Symbioses are a potent survival solution for organisms in hostile environments like sulphide rich sediments, as was proven in several invertebrates. Among these, the bivalve family Lucinidae was reported to harbour sulphide oxidising endosymbionts to cope with the harsh conditions of their habitats and exploit the energy rich resources. Since mainly large tropical representatives of this family were examined in the past, my diploma thesis focused on the small Mediterranean species Loripes lacteus (Linnaeus, 1758) and Anodontia (Loripinus) fragilis (Philippi, 1836). The study aimed to provide a description of endosymbionts of both clam species. Therefore, in each of these bivalves collected from sulphide rich sediment layers below a Cymodocea nodosa sea-grass bed, sequences of the bacterial 16S rRNA gene were identified. Low sequence similarity based on the calculation of pairwise distances (P-distances) gave a first indication that endosymbionts of L. lacteus and A. fragilis belong to different bacterial strains. Using clone specific probes, fluorescence in situ hybridisation (FISH) proved the specificity of potential endosymbiotic sequences. Further, an even distribution of the symbionts throughout the lateral zone of gill filaments was observed using the confocal laser scanning microscope. L. lacteus and A. fragilis were shown to harbour a single, specific endosymbiont population of γ-Proteobacteria, which was stable over three months. On transmission electron micrographs, single endosymbionts enclosed by vacuoles located in large bacteriocytes along the lateral zone were detected. Additional cell-types varied slightly between the examined bivalves, probably due to different physiological adaptations of symbiont and host. Endosymbionts of L. lacteus further possess several vacuoles within the bacterial cytoplasm, most likely for sulphide storage. Phylogenetic analyses do not supported monophyly of lucinid endosymbionts. Symbiotic sequences originating from L. lacteus are related to endosymbionts of Lucina floridana and Codakia costata. 16S rRNA sequences obtained from A. fragilis form a sistergroup with A. phillipiana and Solemya terraeregina clustering within a general symbiotic clade of symbiont sequences from the lucinid Phacoides (Lucina) pectinata, Solemyidae, Thyasiridae and Vestimentifera. Thus, the phylogenetic analysis supported the existence of two different endosymbiotic species in L. lacteus and A. fragilis