Cells and organelles are enclosed by membranes that consist of a lipid bilayer harboring highly
diverse membrane proteins (MPs). These carry out vital functions, and α-helical MPs, in
particular, are of outstanding pharmacological importance, as they comprise more than half of
all drug targets. However, knowledge from MP research is limited, as MPs require membranemimetic
environments to retain their native structures and functions and, thus, are not readily
amenable to in vitro studies. To gain insight into vectorial functions, as in the case of channels
and transporters, and into topology, which describes MP conformation and orientation in the
context of a membrane, purified MPs need to be reconstituted, that is, transferred from detergent
micelles into a lipid-bilayer system.
The ultimate goal of this thesis was to elucidate the membrane topology of Mistic, which is
an essential regulator of biofilm formation in Bacillus subtilis consisting of four α-helices. The
conformational stability of Mistic has been shown to depend on the presence of a hydrophobic
environment. However, Mistic is characterized by an uncommonly hydrophilic surface, and
its helices are significantly shorter than transmembrane helices of canonical integral MPs.
Therefore, the means by which its association with the hydrophobic interior of a lipid bilayer
is accomplished is a subject of much debate. To tackle this issue, Mistic was produced and
purified, reconstituted, and subjected to topological studies.
Reconstitution of Mistic in the presence of lipids was performed by lowering the detergent
concentration to subsolubilizing concentrations via addition of cyclodextrin. To fully exploit
the advantages offered by cyclodextrin-mediated detergent removal, a quantitative model was
established that describes the supramolecular state of the reconstitution mixture and allows
for the prediction of reconstitution trajectories and their cross points with phase boundaries.
Automated titrations enabled spectroscopic monitoring of Mistic reconstitutions in real time.
On the basis of the established reconstitution protocol, the membrane topology of Mistic was
investigated with the aid of fluorescence quenching experiments and oriented circular dichroism
spectroscopy. The results of these experiments reveal that Mistic appears to be an exception
from the commonly observed transmembrane orientation of α-helical MPs, since it exhibits
a highly unusual in-plane topology, which goes in line with recent coarse-grained molecular
Membrane proteins are generally soluble only in the presence of detergent micelles or other membrane-mimetic systems, which renders the determination of the protein’s molar mass or oligomeric state difficult. Moreover, the amount of bound detergent varies drastically among different proteins and detergents. However, the type of detergent and its concentration have a great influence on the protein’s structure, stability, and functionality and the success of structural and functional investigations and crystallographic trials. Size-exclusion chromatography, which is commonly used to determine the molar mass of water-soluble proteins, is not suitable for detergent-solubilised proteins because
the protein–detergent complex has a different conformation and, thus, commonly exhibits
a different migration behaviour than globular standard proteins. Thus, calibration curves obtained with standard proteins are not useful for membrane-protein analysis. However,
the combination of size-exclusion chromatography with ultraviolet absorbance, static light scattering, and refractive index detection provides a tool to determine the molar mass of protein–detergent complexes in an absolute manner and allows for distinguishing the contributions of detergent and protein to the complex.
The goal of this thesis was to refine the standard triple-detection size-exclusion chromatography measurement and data analysis procedure for challenging membrane-protein samples, non-standard detergents, and difficult solvents such as concentrated denaturant solutions that were thought to elude routine approaches. To this end, the influence of urea on the performance of the method beyond direct influences on detergents and proteins was investigated with the help of the water-soluble bovine serum albumin. On the basis of
the obtained results, measurement and data analysis procedures were refined for different detergents and protein–detergent complexes comprising the membrane proteins OmpLA and Mistic from Escherichia coli and Bacillus subtilis, respectively.
The investigations on mass and shape of different detergent micelles and the compositions of protein–detergent complexes in aqueous buffer and concentrated urea solutions
showed that triple-detection size-exclusion chromatography provides valuable information
about micelle masses and shapes under various conditions. Moreover, it is perfectly suited for the straightforward analysis of detergent-suspended proteins in terms of composition and oligomeric state not only under native but, more importantly, also under denaturing conditions.
The heart is reported to show a net consumption of lactate. This may contribute up to 15% to the total body lactate disposal. In this work, the consumption of lactate was shown for the first
time on the single cell level with the new FRET-based lactate sensor Laconic.
Research published until today, almost exclusively reports the monocarboxylate transporter 1
(MCT1) as the transporter responsible for myocardial lactate uptake. As this membrane
transporter transports lactate together with H+ in a stoichiometry of 1:1, lactate transport is
coupled to pH regulation. Consequently, interactions of MCT1 and acid/base regulating proteins
(carbonic anhydrases (CAs and sodium bicarbonate co-transporters (NBCs)) are described in
the oocyte expression system, skeletal muscle and cancer cells.
In this work it is shown that activity of extracellular CA increases lactate uptake into mouse
cardiomyocytes by 27% and lactate induced JA/B by 42.8% to 46.2%. This effect is most likely
mediated via NBC/CA interaction because inhibition of extracellular CA reduces HCO3--
dependent acid extruding JA/B by 53.3% to 78.4%. This may link lactate uptake to cellular
respiration. When lactate was applied in medium gassed with 100% N2, lactate induced
acidification was 12.6% faster than in medium gassed with 100% O2. Thus, CO2 produced on
the pathway transferring redox energy from substrates like glucose and lactate to ADP and
phosphate via oxidative phosphorylation, may support further lactate uptake. The findings of
this work suggest an auto regulation of lactate uptake via CO2 release in ventricular mouse
Cyanobacteria are the only prokaryotes with the ability to conduct oxygenic photosynthesis,
therefore having major influence on the evolution of life on earth. Their diverse morphology
was traditionally the basis for taxonomy and classification. For example, the genus
Chroococcidiopsis has been classified within the order Pleurocapsales, based on a unique
reproduction modus by baeocytes. Recent phylogenetic results suggested a closer
relationship of this genus to the order Nostocales. However, these studies were based
mostly on the highly conserved 16S rRNA and a small selection of Chroococcidiopsis
strains. One aim of this present thesis was to investigate the evolutionary relationships of
the genus Chroococcidiopsis, the Pleurocapsales and remaining cyanobacteria using
16S rRNA, rpoC1 and gyrB gene. Including the single gene, as the multigene analyses of
97 strains clearly showed a separation of the genus Chroococcidiopsis from the
Pleurocapsales. Furthermore, a sister relationship between the genus Chroococcidiopsis
and the order Nostocales was confirmed. Consequently, the monogeneric family
Chroococcidiopsidaceae Geitler ex. Büdel, Donner & Kauff familia nova is justified. The
phylogenetic analyses also revealed the polyphyly of the remaining Pleurocapsales, due to
the fact that the strain Pleurocapsa PCC 7327 was always separated from other strains.
This is supported by differences in their metabolism, ecology and physiology.
A second aim of this study was to investigate the thylakoid arrangement of
Chroococcidiopsis and a selection of cyanobacterial strains. The investigation of 13 strains
with Low Temperature Scanning Electron Microscopy revealed two unknown thylakoidal
arrangements within Chroococcidiopsis (parietal and stacked). This result revised the
knowledge of the thylakoid arrangement in this genus. Previously, only a coiled
arrangement was known for three strains. Based on the data of 66 strains, the feature
thylakoid arrangement was tested as a potential feature for morphological identification of
cyanobacteria. The results showed a strong relationship between the group assignment of
cyanobacteria and their thylakoid arrangements. Hence, it is in general possible to
conclude from this certain phenotypic character the affiliation to a particular family, order
The third aim of this study was to investigate biogeographical patterns of the worldwide
distributed genus Chroococcidiopsis. The phylogenetic analysis suggested that the genus do not have biogeographical patterns, which is in contrast with a recent study on hypolithic
living Chroococcidiopsis strains and the majority of phylogeographic analysis of
microorganisms. Further analysis showed no separation of different life-strategies within
the genus. These results could be related to the genetic markers utilized, which may not
contain biogeographical information. Hence the present study can neither exclude nor
prove the possibility of biogeographic and life-strategy patterns in the genus
Future research should be focused on finding appropriate genetic markers investigate of
evolutionary relationships and biogeographical patterns within Chroococcidiopsis.
Prostate cancer preferentially metastasizes to the skeleton and abundant evidence exists that osteoblasts specifically support the metastatic process, including cancer stem cell niche formation. At early stages of bone metastasis, crosstalk of prostate cancer cells and osteoblasts through soluble molecules results in a decrease of cancer cell proliferation, accompanied by altered adhesive properties and increased expression of bone-specific genes, or osteomimicry. Osteoblasts synthesize a plethora of biologically active factors, which comprise the unique bone microenvironment. By means of quantitative real-time RT-PCR it was determined that exposure to the osteoblast secretome induced gene expression changes in prostate cancer cells, including the upregulation of osteomimetic genes such as BMP2, AP, COL1A1, OPG and RANKL. IL6 and TGFbeta1 signaling pathway components also became upregulated at early time points. Moreover, osteoblast-released IL6 and TGFbeta1 contributed to the upregulation of OPG mRNA in LNCaP. Thus, the earliest response of prostate cancer cells to osteoblast-released factors, which ultimately cause metastatic cells to assume an osteomimetic phenotype, involved activation of paracrine and autocrine IL6 and TGFbeta signaling. On the other hand, a microarray analysis showed that osteoblasts exposed to the secretome of prostate cancer cells exhibited gene expression alterations suggestive of repressed proliferation, decreased matrix synthesis and inhibited immune response, which together indicate enhanced preosteocytic differentiation. TGFbeta signaling, known to inhibit osteoblast maturation, was strongly suppressed, as shown by elevated expression of negative regulators, downregulation of pathway components and of numerous target genes. Transcriptional downregulation of osteoblast inhibitory molecules such as DKK1 and FST also occurred, with concomitant upregulation of the osteoinductive molecules ADM, STC1 and BMP2, and of the transcription factors CBFA1 and HES1, which promote osteoblast differentiation. Finally, the mRNA encoding NPPB, the precursor of a molecule implicated in the inhibition of TGFbetaeffects, in bone formation and in stem cell maintenance, became upregulated after coculture both in osteoblasts and in prostate cancer cells. These results provide an insight into potential mechanisms of dysregulated bone formation in metastatic prostate cancer, as well as mechanisms by which osteoblasts might enhance the invasive, osteomimetic and stem cell-like properties of the tumor cells. In particular, the differential modulation of TGFbetasignaling in prostate cancer cells and osteoblasts appears to merit further research.
Proteins of the intermembrane space of mitochondria are generally encoded by nuclear genes that are synthesized in the cytosol. A group of small intermembrane space proteins lack classical mitochondrial targeting sequences, but these proteins are imported in an oxidation-driven reaction that relies on the activity of two components, Mia40 and Erv1. Both proteins constitute the mitochondrial disulfide relay system. Mia40 functions as an import receptor that interacts with incoming polypeptides via transient, intermolecular disulfide bonds. Erv1 is an FAD-binding sulfhydryl oxidase that activates Mia40 by re-oxidation, but the process how Erv1 itself is re-oxidized has been poorly understood. Here, I show that Erv1 interacts with cytochrome c which provides a functional link between the mitochondrial disulfide relay system and the respiratory chain. This mechanism not only increases the efficiency of mitochondrial inport by the re-oxidation of Erv1 and Mia40 but also prevents the formation of deleterious hydrogen peroxide within the intermembrane space. Thus, the miochondrial disulfide relay system is, analogous to that of the bacterial periplasm, connected to the electron transport chain of the inner membrane, which possibly allows an oxygen-dependend regulation of mitochondrial import rates. In addition, I modeled the structure of Erv1 on the basis of the Saccharomyces cerevisiae Erv2 crystal structure in order to gain insight into the molecular mechanism of Erv1. According to the high degree of sequence homologies, various characteristics found for Erv2 are also valid for Erv1. Finally, I propose a regulatory function of the disulfide relay system on the respiratory chain. The disulfide relay system senses the molecular oxygen levels in mitochondria and, thus, is able to adapt respiratory chain activity in order to prevent wastage of NADH and production of ROS.
Fragmentation of habitats, especially of tropical rainforests, ranks globally among the most pervasive man-made disturbances of ecosystems. There is growing evidence for long-term effects of forest frag-mentation and the accompanying creation of artificial edges on ecosystem functioning and forest structure, which are altered in a way that generally transforms these forests into early successional systems. Edge-induced disruption of species interactions can be among the driving mechanisms governing this transformation. These species interactions can be direct (trophic interactions, competition, etc.) or indirect (modification of the resource availability for other organisms). Such indirect interactions are called ecosystem engineering. Leaf-cutting ants of the genus Atta are dominant herbivores and keystone-species in the Neotropics and have been called ecosystem engineers. In contrast to other prominent ecosystem engineers that have been substantially decimated by human activities some species of leaf-cutting ants profit from anthropogenic landscape alterations. Thus, leaf-cutting ants are a highly suitable model to investigate the potentially cascading effects caused by herbivores and ecosystem engineers in modern anthropogenic landscapes following fragmentation. The present thesis aims to describe this interplay between consequences of forest fragmentation for leaf-cutting ants and resulting impacts of leaf-cutting ants in fragmented forests. The cumulative thesis starts out with a review of 55 published articles demonstrating that herbivores, especially generalists, profoundly benefit from forest edges, often due to (1) favourable microenviron-mental conditions, (2) an edge-induced increase in food quantity/quality, and (3; less well documented) disrupted top-down regulation of herbivores (Wirth, Meyer et al. 2008; Progress in Botany 69:423-448). Field investigations in the heavily fragmented Atlantic Forest of Northeast Brazil (Coimbra forest) were subsequently carried out to evaluate patterns and hypotheses emerging from this review using leaf-cutting ants of the genus Atta as a model system. Colony densities of both Atta species occuring in the area changed similarly with distance to the edge but the magnitude of the effect was species-specific. Colony density of A. cephalotes was low in the forest interior (0.33 ± 1.11 /ha, pooling all zones >50 m into the forest) and sharply increased by a factor of about 8.5 towards the first 50 m (2.79 ± 3.3 /ha), while A. sexdens was more uniformly distributed (Wirth, Meyer et al. 2007; Journal of Tropical Ecology 23:501-505). The accumulation of Atta colonies persisted at physically stable forest edges over a four-year interval with no significant difference in densities between years despite high rates of colony turn-over (little less than 50% in 4 years). Stable hyper-abundant populations of leaf-cutting ants accord with the constantly high availability of pioneer plants (their preferred food source) as previously demonstrated at old stabilised forest edges in the region (Meyer et al. submitted; Biotropica). In addition, plants at the forest edge might be more attractive to leaf-cutting ants because of their physiological responses to the edge environment. In bioassays with laboratory colonies I demonstrated that drought-stressed plants are more attractive to leaf-cutting ants because of an increase in leaf nutrient content induced by osmoregulation (Meyer et al. 2006; Functional Ecology 20:973-981). Since plants along forest edges are more prone to experience drought stress, this mechanism might contribute to the high resource availabil-ity for leaf-cutting ants at forest edges. In light of the hyper-abundance of leaf-cutting ants within the forest edge zone (first 50 m), their po-tentially far-reaching ecological importance in anthropogenic landscapes is apparent. Based on previous colony-level estimates, we extrapolated that herbivory by A. cephalotes removes 36% of the available foliage at forest edges (compared to 6% in the forest interior). In addition, A. cephalotes acted as ecosys-tem engineers constructing large nests (on average 55 m2: 95%-CI: 22-136) that drastically altered forest structure. The ants opened gaps in the canopy and forest understory at nest sites, which allowed three times as much light to reach the nest surface as compared to the forest understory. This was accompa-nied by an increase in soil temperatures and a reduction in water availability. Modifications of microcli-mate and forest structure greatly surpassed previously published estimates. Since higher light levels were detectable up to about 4 m away from the nest edge, an area roughly four times as big as the actual nest (about 200 and 50 m2, respectively) was impacted by every colony, amounting to roughly 6% of the total area at the forest edge (Meyer et al. in preparation; Ecology). The hypothesized impacts of high cutting pressure and microclimatic alterations at nest sites on forest regeneration were directly tested using transplanted seedlings of six species of forest trees. Nests of A. cephalotes differentially impacted survival and growth of seedlings. Survival differed highly significantly between habitats and species and was generally high in the forest, yet low on nests where it correlated strongly with seed size of the species. These results indicate that the disturbance regime created by leaf-cutting ants differs from other distur-bances, since nest conditions select for plant species that profit from additional light, yet are large-seeded and have resprouting abilities, which are best suited to tolerate repeated defoliation on a nest (Meyer et al. in preparation; Journal of Tropical Ecology). On an ecosystem scale leaf-cutting ants might amplify edge-driven microclimatic alterations by very high rates of herbivory and the maintenance of canopy gaps above frequent nests. By allowing for an increased light penetration Atta may, ultimately, contribute to a dominating, self-replacing pioneer communities at forest edges, possibly creating a positive feed-back loop. Based on the persisting hyper-abundance of leaf-cutting ants at old edges of Coimbra forest and the multifarious impacts documented, we conclude that the ecological importance of leaf-cutting ants in pristine forests, where they are commonly believed to be keystone species despite very low colony densities, is greatly surpassed in anthropogenic landscapes In fragmented forests, Atta has been identified as an essential component of a disturbance regime that causes a post-fragmentation retrogressive succession. Apparently, these forests have reached a new self-replacing secondary state. I suggest additional human interference in form of thoughtful management in order to break this cycle of self-enhancing disturbance and to enable forest regeneration along the edges of threatened forest remnants. Thereby the situation of the forest as a whole can be ameliorated and the chances for a long-term retention of biodiversity in these landscapes increased.
Esterases and lipases are widely used as industrial enzymes and for the synthesis of chiral drugs. Because of their rich secondary metabolism, Streptomyces species offer a relatively untapped source of interesting esterases and lipases. S. coelicolor and S. avermitilis contain 51 genes annotated as esterases and/or lipases. In this study I have cloned 14 different genes encoding for lipolytic enzymes from S. coelicolor (11 genes) and S. avermitilis (four genes). Some of these genes were over-expressed in E. coli. Three of the produced enzymes, which were produced by the genes SCO 7131, SCO6966 and SCO3644, were characterized biochemically and one of them was subjected for directed evolution. The gene estA (locus SCO 7131) was annotated as a putative lipase/esterase in the genome sequence of S. coelicolor A3(2), but does not have a homologue in the genome sequence of S. avermitilis or in other known Streptomyces sequences. estA was cloned and expressed in E. coli as a His-tagged protein. The protein was purified and could be recovered in its non-tagged form after digestion with factor Xa. The relative molecular weight was estimated to be 35.5kDa. The enzyme was only active towards acetate esters and not on larger substrates. It had a stereospecificity towards α-naphathylacetate. It was thermostable, with a half-life at 50C of 4.5 hours. Est A showed stability over pH range 5.5-10, and had optimum pH of 7.5. Its activity was drastically decreased when it was pre-incubated in 10mM PMSF, Cu+2 and Hg+2. It was not very stable in most organic solvents and had only slight enantioselectivity. Est A belongs to the HSL family whose founder member is the human hormone-sensitive lipase. I have developed a protein profile for the HSL family modifying the conserved motifs found by Arpigny and Jaeger (1999). Due to the presence of several HSL members with known 3D structure and good homology to Est A, I was able to make a homology model of Est A. Five different mutants of Est A were produced through site directed mutagenesis: W87F, V158A, W87F/V158A, M162L and S163A. The mutants M162L and S163A did not produce a significant change either in substrate specificity or enzyme kinetics. The mutants V158A and W87F/V158A could act on the larger substrates p-nitrophenylbutyrate and caproate and tributyrin. The mutant V158A had improved thermostability and its t1/2 at 50ºC increased to 24h. The affinity of V158A towards p-nitrophenyacetate increased 6-fold when compared with the wild type, whereas the affinity of W87F decreased 4-fold. Directed evolution of Est A was done through random mutagenesis and ER-PCR. A library of 6336 mutants was constructed and screened for mutants with a broader spectrum of substrate specificity. The mutant XXVF7 did show alteration in the substrate specificity of Est A. The mutant XXVF7 had 5 amino acids changes L76R, L146P, S196G, W213R and L267R. The gene locus SCO 6966 (estB gene) was cloned and expressed in E. coli as a His-tagged protein. It was not possible to remove the His-tag using factor Xa. The tagged protein had a molecular weight 31.9kDa. Est B was active against short chain fatty acid esters (C2-C6). Its optimum temperature was 30ºC and was stable for 1h at temperatures up to 37ºC. The enzyme had maximum activity at pH 8-8.5 and was stable over pH range 7.5-11 for 24h. It was highly sensitive for PMSF, Cu+2 and Hg+2. The enzymatic activity deceased in presence of organic solvents, however it was fairly stable for 1h in 20% organic solvents solutions. A third esterase was produced from the gene locus SCO 3644. This esterase was a thermosensitive one with optimum temperature of 35ºC. The three characterized enzymes included a thermophilic, mesophilic and psychrophilic ones. This indicates the high variation in the characters of Streptomyces lipolytic enzymes and highlighting Streptomyces as a source for esterases and lipases of interesting catalytic activity. This study was an initial trial to provide a strategy for a comprehensive use of genome data.
Haustoria of the rust fungus pathogen Uromyces fabae deliver RTP1 (Rust Transferred Protein1) into host plant cells. In this work, different heterologous expression systems were used to study RTP1 biological function as well as RTP1 transfer mechanism. The first part of this thesis focused on the identification of the subcellular target compartment of RTP1 in plant cells. In this respect we could identify a functional bipartite nuclear localization signal within RTP1. However, stable and transient expression studies of RTP1 in different plant species, including the host plant Vicia faba, interfered with plant cell vitality but did not result in detection of RTP1 protein. These findings led us to propose that RTP1 interferes with plant gene expression. However, the molecular basis of this interference remains unclear. By deletion studies, we could localize the active region of RTP1 within a 45 amino acid central domain. In the second part of this study, two different lines of approaches were taken to study RTP1 transfer mechanism. First, transient expression of secreted RTP1 (sRTP1) also interfered with plant cell vitality. Addition of an endoplasmic reticulum retention signal abolished sRTP1 interference with plant cell vitality, suggesting that RTP1 can reenter the plant cell from the apoplast after secretion in the absence of the pathogen. We have identified a PEST-like region within RTP1, however, contribution of this region to the stability of RTP1 is not clear. Site directed mutagenesis analysis showed that the PEST-like region is likely to play a role during the transfer of RTP1 through plant plasma membrane. In the second line of approach, we established a recombinant delivery model, using Ustilago maydis/Zea mays pathosystem, to pursue RTP1 translocation into the plant cell. Our results indicate that U. maydis is capable of secreting high amounts of recombinant RTP1, showing similar glycosylation pattern as RTP1 secreted from rust haustoria. Our data propose the use of this model system to study RTP1 domains mediating its entry into the plant cell. Haustoria of the rust fungus pathogen Uromyces fabae deliver RTP1 (Rust Transferred Protein1) into host plant cells. In this work, different heterologous expression systems were used to study RTP1 biological function as well as RTP1 transfer mechanism. The first part of this thesis focused on the identification of the subcellular target compartment of RTP1 in plant cells. In this respect we could identify a functional bipartite nuclear localization signal within RTP1. However, stable and transient expression studies of RTP1 in different plant species, including the host plant Vicia faba, interfered with plant cell vitality but did not result in detection of RTP1 protein. These findings led us to propose that RTP1 interferes with plant gene expression. However, the molecular basis of this interference remains unclear. By deletion studies, we could localize the active region of RTP1 within a 45 amino acid central domain. In the second part of this study, two different lines of approaches were taken to study RTP1 transfer mechanism. First, transient expression of secreted RTP1 (sRTP1) also interfered with plant cell vitality. Addition of an endoplasmic reticulum retention signal abolished sRTP1 interference with plant cell vitality, suggesting that RTP1 can reenter the plant cell from the apoplast after secretion in the absence of the pathogen. We have identified a PEST-like region within RTP1, however, contribution of this region to the stability of RTP1 is not clear. Site directed mutagenesis analysis showed that the PEST-like region is likely to play a role during the transfer of RTP1 through plant plasma membrane. In the second line of approach, we established a recombinant delivery model, using Ustilago maydis/Zea mays pathosystem, to pursue RTP1 translocation into the plant cell. Our results indicate that U. maydis is capable of secreting high amounts of recombinant RTP1, showing similar glycosylation pattern as RTP1 secreted from rust haustoria. Our data propose the use of this model system to study RTP1 domains mediating its entry into the plant cell.