Isolation and characterization of a manganese oxidizing bacterium from the Mediterranean marine sponge Suberites domuncula

In the present study of sponge-bacterial association, the presence of a marine bacterium which has not seen to be associated previously with the Mediterranean sponge Suberites domuncula was investigated. The marine sponge S. domuncula was chosen as the subject of investigation, for the identification of potential symbiotic microorganisms, since it can be kept under controlled laboratory conditions for over five years. By the use of specialized media assisting in the growth of a metal oxidizing bacterium, the manganese oxidizing bacterium was isolated from the surface of the marine sponge. The bacterium so isolated was characterized for its growth characteristics by microbiological and biochemical techniques, a detailed analysis of which showed that the bacterium followed a life cycle where the culture showed the presence of spore forming bacteria. This was correlated to the manganese oxidation activity of the bacteria and it was found that both stages are interdependent.The action of the protein responsible for carrying out the manganese (Mn) oxidation was studied by an in-gel oxidation assay, and the presence of a multi copper oxidase was confirmed by the use of copper chelators in the buffer. In parallel the effect of addition of copper was observed on the manganese oxidation by the bacteria thus supporting the observations. The manganese oxidation reaction by the bacteria was determined in the culture medium and on the surface of the cells, and it could be concluded that the oxidation was facilitated by the presence of the polysaccharides and proteins on the surface of the cells.Thus the presence of a bacterium capable of oxidizing the manganese from the surroundings was confirmed to be symbiotically associated with the marine sponge S. domuncula by monitoring its growth in axenic cultures. The reasons behind this association were studied.This bacterium displays a crucial role in the physiology/metabolism of the sponge by acting as a reversible Mn store in S. domuncula. According to this view, the presence of SubDo-03 bacteria is required as a protection against higher, toxic concentrations of Mn in the environment; manganese (II) after undergoing oxidation to manganese (IV), becomes an insoluble ion. Since only minute levels of manganese exist in the surrounding seawater a substantial accumulation of manganese has to arise, or a release by the bacterial-precipitated manganese (IV) is implicated to maintain the reversible balance. The other possible benefits provided by the bacterial association to the sponge could be in preventing cellular oxygen toxicity, help in nutrient scavenging and detoxification.

Structured antibody surfaces for bio-recognition and a label-free detection of bacteria

Antibody microarrays are of great research interest because of their potential application as biosensors for high-throughput protein and pathogen screening technologies. In this active area, there is still a need for novel structures and assemblies providing insight in binding interactions such as spherical and annulus-shaped protein structures, e.g. for the utilization of curved surfaces for the enhanced protein-protein interactions and detection of antigens. Therefore, the goal of the presented work was to establish a new technique for the label-free detection of bio-molecules and bacteria on topographically structured surfaces, suitable for antibody binding.rnIn the first part of the presented thesis, the fabrication of monolayers of inverse opals with 10 μm diameter and the immobilization of antibodies on their interior surface is described. For this purpose, several established methods for the linking of antibodies to glass, including Schiff bases, EDC/S-NHS chemistry and the biotin-streptavidin affinity system, were tested. The employed methods included immunofluorescence and image analysis by phase contrast microscopy. It could be shown that these methods were not successful in terms of antibody immobilization and adjacent bacteria binding. Hence, a method based on the application of an active-ester-silane was introduced. It showed promising results but also the need for further analysis. Especially the search for alternative antibodies addressing other antigens on the exterior of bacteria will be sought-after in the future.rnAs a consequence of the ability to control antibody-functionalized surfaces, a new technique employing colloidal templating to yield large scale (~cm2) 2D arrays of antibodies against E. coli K12, eGFP and human integrin αvβ3 on a versatile useful glass surface is presented. The antibodies were swept to reside around the templating microspheres during solution drying, and physisorbed on the glass. After removing the microspheres, the formation of annuli-shaped antibody structures was observed. The preserved antibody structure and functionality is shown by binding the specific antigens and secondary antibodies. The improved detection of specific bacteria from a crude solution compared to conventional “flat” antibody surfaces and the setting up of an integrin-binding platform for targeted recognition and surface interactions of eukaryotic cells is demonstrated. The structures were investigated by atomic force, confocal and fluorescence microscopy. Operational parameters like drying time, temperature, humidity and surfactants were optimized to obtain a stable antibody structure.

Synaptic connectivity in micropatterned networks of neuronal cells

The presented thesis describes the formation of functional neuronal networks on an underlying micropattern. Small circuits of interconnected neurons defined by the geometry of the patterned substrate could be observed and were utilised as a model system of reduced complexity for the behaviour of neuronal network formation and activity. The first set of experiments was conducted to investigate aspects of the substrate preparation. Micropatterned substrates were created by microcontact printing of physiological proteins onto polystyrene culture dishes. The substrates displayed a high contrast between the repellant background and the cell attracting pattern, such that neurons seeded onto these surfaces aligned with the stamped structure. Both the patterning process and the cell culture were optimised, yielding highly compliant low-density networks of living neuronal cells. In the second step, cellular physiology of the cells grown on these substrates was investigated by patch-clamp measurements and compared to cells cultivated under control conditions. It could be shown that the growth on a patterned substrate did not result in an impairment of cellular integrity nor that it had an impact on synapse formation or synaptic efficacy. Due to the extremely low-density cell culture that was applied, cellular connectivity through chemical synapses could be observed at the single cell level. Having established that single cells were not negatively affected by the growth on patterned substrates, aspects of network formation were investigated. The formation of physical contact between two cells was analysed through microinjection studies and related to the rate at which functional synaptic contacts formed between two neighbouring cells. Surprisingly, the rate of synapse formation between physically contacting cells was shown to be unaltered in spite of the drastic reduction of potential interaction partners on the micropattern. Additional features of network formation were investigated and found consistent with results reported by other groups: A different rate of synapse formation by excitatory and inhibitory neurons could be reproduced as well as a different rate of frequency-dependent depression at excitatory and inhibitory synapses. Furthermore, regarding simple feedback loops, a significant enrichment of reciprocal connectivity between mixed pairs of excitatory and inhibitory neurons relative to uniform pairs could be demonstrated. This phenomenon has also been described by others in unpatterned cultures [Muller, 1997] and may therefore be a feature underlying neuronal network formation in general. Based on these findings, it can be assumed that inherent features of neuronal behaviour and cellular recognition mechanisms were found in the cultured networks and appear to be undisturbed by patterned growth. At the same time, it was possible to reduce the complexity of the forming networks dramatically in a cell culture on a patterned surface. Thus, features of network architecture and synaptic connectivity could be investigated on the single cell level under highly defined conditions.

optomotor-blind and the horizontal and vertical system cells of the drosophila optic lobes

The horizontal and vertical system neurons (HS and VS cells) are part of a conserved set of lobula plate giant neurons (LPGNs) in the optic lobes of the adult brain. Structure and physiology of these cells are well known, predominantly from studies in larger Dipteran flies. Our knowledge about the ontogeny of these cells is limited and stems predominantly from laser ablation studies in larvae of the house fly Musca domestica. These studies suggested that the HS and VS cells stem from a single precursor, which, at least in Musca, has not yet divided in the second larval instar. A regulatory mutation (In(1)omb[H31]) in the Drosophila gene optomotor-blind (omb) leads to the selective loss of the adult HS and VS cells. This mutation causes a transient reduction in omb expression in what appears to be the entire optic lobe anlage (OLA) late in embryogenesis. Here, I have reinitiated the laser approach with the goal of identifying the presumptive embryonic HS/VS precursor cell in Drosophila. The usefulness of the laser ablation approach which has not been applied, so far, to cells lying deep within the Drosophila embryo, was first tested on two well defined embryonic sensory structures, the olfactory antenno-maxillary complex (AMC) and the light-sensitive Bolwing´s organ (BO). In the case of the AMC, the efficiency of the ablation procedure was demonstrated with a behavioral assay. When both AMCs were ablated, the response to an attractive odour (n-butanol) was clearly reduced. Interestingly, the larvae were not completely unresponsive but had a delayed response kinetics, indicating the existence of a second odour system. BO will be a useful test system for the selectivity of laser ablation when used at higher spatial resolution. An omb-Gal4 enhancer trap line was used to visualize the embryonic OLA by GFP fluorescence. This fluorescence allowed to guide the laser beam to the relevant structure within the embryo. The success of the ablations was monitored in the adult brain via the enhancer trap insertion A122 which selectively visualizes the HS and VS cell bodies. Due to their tight clustering, individual cells could not be identified in the embryonic OLA by conventional fluorescence microscopy. Nonetheless, systematic ablation of subdomains of the OLA allowed to localize the presumptive HS/VS precursor to a small area within the OLA, encompassing around 10 cells. Future studies at higher resolution should be able to identify the precursor as (an) individual cell(s). Most known lethal omb alleles do not complement the HS/VS phenotype of the In(1)omb[H31] allele. This is the expected behaviour of null alleles. Two lethal omb alleles that had been isolated previously by non-complementation of the omb hypomorphic allele bifid, have been reported, however, to complement In(1)omb[H31]. This report was based on low resolution paraffin histology of adult heads. Four mutations from this mutagenesis were characterized here in more detail (l(1)omb[11], l(1)omb[12], l(1)omb[13], and l(1)omb[15]). Using A122 as marker for the adult HS and VS cells, I could show, that only l(1)omb[11] can partly complement the HS/VS cell phenotype of In(1)omb[H31]. In order to identify the molecular lesions in these mutants, the exons and exon/intron junctions were sequenced in PCR-amplified material from heterozygous flies. Only in two mutants could the molecular cause for loss of omb function be identified: in l(1)omb[13]), a missense mutation causes the exchange of a highly conserved residue within the DNA-binding T-domain; in l(1)omb[15]), a nonsense mutation causes a C-terminal truncation. In the other two mutants apparently regulatory regions or not yet identified alternative exons are affected. To see whether mutant OMB protein in the missense mutant l(1)omb[13] is affected in DNA binding, electrophoretic shift assays on wildtype and mutant T-domains were performed. They revealed that the mutant no longer is able to bind the consensus palindromic T-box element.

Methods and strategies to identify the mode of action of phytoactive compounds

Small molecules affecting biological processes in plants are widely used in agricultural practice as herbicides or plant growth regulators and in basic plant sciences as probes to study the physiology of plants. Most of the compounds were identified in large screens by the agrochemical industry, as phytoactive natural products and more recently, novel phytoactive compounds originated from academic research by chemical screens performed to induce specific phenotypes of interest. The aim of the present PhD thesis is to evaluate different approaches used for the identification of the primary mode of action (MoA) of a phytoactive compound. Based on the methodologies used for MoA identification, three approaches are discerned: a phenotyping approach, an approach based on a genetic screen and a biochemical screening approach.rnFour scientific publications resulting from my work are presented as examples of how a phenotyping approach can successfully be applied to describe the plant MoA of different compounds in detail.rnI. A subgroup of cyanoacrylates has been discovered as plant growth inhibitors. A set of bioassays indicated a specific effect on cell division. Cytological investigations of the cell division process in plant cell cultures, studies of microtubule assembly with green fluorescent protein marker lines in vivo and cross resistant studies with Eleusine indica plants harbouring a mutation in alpha-tubulin, led to the description of alpha-tubulin as a target site of cyanoacrylates (Tresch et al., 2005).rnII. The MoA of the herbicide flamprop-m-methyl was not known so far. The studies described in Tresch et al. (2008) indicate a primary effect on cell division. Detailed studies unravelled a specific effect on mitotic microtubule figures, causing a block in cell division. In contrast to other inhibitors of microtubule rearrangement such as dinitroanilines, flamprop-m-methyl did not influence microtubule assembly in vitro. An influence of flamprop-m-methyl on a target within the cytoskeleton signalling network could be proposed (Tresch et al., 2008).rnIII. The herbicide endothall is a protein phosphatase inhibitor structurally related to the natural product cantharidin. Bioassay studies indicated a dominant effect on dark-growing cells that was unrelated to effects observed in the light. Cytological characterisation of the microtubule cytoskeleton in corn tissue and heterotrophic tobacco cells showed a specific effect of endothall on mitotic spindle formation and ultrastructure of the nucleus in combination with a decrease of the proliferation index. The observed effects are similar to those of other protein phosphatase inhibitors such as cantharidin and the structurally different okadaic acid. Additionally, the observed effects show similarities to knock-out lines of the TON1 pathway, a protein phosphatase-regulated signalling pathway. The data presented in Tresch et al. (2011) associate endothall’s known in vitro inhibition of protein phosphatases with in vivo-effects and suggest an interaction between endothall and the TON1 pathway.rnIV. Mefluidide as a plant growth regulator induces growth retardation and a specific phenotype indicating an inhibition of fatty acid biosynthesis. A test of the cuticle functionality suggested a defect in the biosynthesis of very-long-chain fatty acids (VLCFA) or waxes. Metabolic profiling studies showed similarities with different groups of VLCFA synthesis inhibitors. Detailed analyses of VLCFA composition in tissues of duckweed (Lemna paucicostata) indicated a specific inhibition of the known herbicide target 3 ketoacyl-CoA synthase (KCS). Inhibitor studies using a yeast expression system established for plant KCS proteins verified the potency of mefluidide as an inhibitor of plant KCS enzymes. It could be shown that the strength of inhibition varied for different KCS homologues. The Arabidopsis Cer6 protein, which induces a plant growth phenotype similar to mefluidide when knocked out, was one of the most sensitive KCS enzymes (Tresch et al., 2012).rnThe findings of my own work were combined with other publications reporting a successful identification of the MoA and primary target proteins of different compounds or compound classes.rnA revised three-tier approach for the MoA identification of phytoactive compounds is proposed. The approach consists of a 1st level aiming to address compound stability, uniformity of effects in different species, general cytotoxicity and the effect on common processes like transcription and translation. Based on these findings advanced studies can be defined to start the 2nd level of MoA characterisation, either with further phenotypic characterisation, starting a genetic screen or establishing a biochemical screen. At the 3rd level, enzyme assays or protein affinity studies should show the activity of the compound on the hypothesized target and should associate the in vitro effects with the in vivo profile of the compound.