OPUS 4 | Biologie

Development of Bacillus subtilis spores and cells for surface display of proteins (2011)

Quynh Anh Nguyen

Surface display has attracted the attention of researchers in developing efficient display systems expressing heterologous polypeptides on the surface of bioparticles such as phages, bacterial and eukaryotic cells and bacterial spores. Among these bioparticles, the endospore from B. subtilis has advantages, including feasibility of production, safety feature, the robustness of the bacterial spore allowing storage in the desiccated form, a technological platform supported by extensive tools for genetic manipulation and less size restrictions of the displayed proteins compared to cell- and phage-based systems. A strategy to engineer B. subtilis spores to display heterologous protein on their surface is to use outer spore coat proteins (CotB, CotC, CotG) or an inner-coat protein (OxdD) with the coat genes’ transcriptional and translational signals as carriers (Isticato et al., 2001; Mauriello et al., 2004; Hinc et al., 2010; Zhou et al., 2008a; Potot et al., 2010; Kim et al., 2005a; Kwon et al., 2007). This strategy guarantees the timing for fusion protein synthesis during coat formation, but the amount of produced fusion proteins cannot be controlled. Therefore, the first aim of this doctoral thesis focused on construction of more effective expression systems for spore surface protein anchoring. A novel approach of substitution of native promoter by two different IPTG-inducible promoters to the increase the production of fusion protein is presented here. CotB was used and the expression of the cotB gene was regulated by either its own promoter, the Pgrac and the PSgrac promoter in a series of plasmids which can be integrated into or replicated independently of the B. subtilis chromosomal DNA. Two reporter proteins, α-amylase Q from B. amyloliquefaciens (AmyQ) (Palva, 1982) and GFPuv – an enhanced version from the GFP protein of the jellyfish Aequorea victoria (Crameri et al., 1996), were fused downstream of the CotB protein. To assess the enhancement of GFPuv displayed on the spore surface, CotC and CotG were similarly examined. The results indicated that the Pgrac promoter is a suitable, hence recommended as a promoter of choice. Substitution of the native promoter by Pgrac promoter, the amount of proteins displayed per spore can be increased two-fold. Furthermore, the display of heterologous proteins on the spore surface when using different carriers is gene dosage dependent. And for the first time, the tendency of the three Cot proteins’ localization on the spore coat compartment is reported using the GFPuv tag. Second, a new B. subtilis spore-based system for protein expression and purification was developed. Using this system, proteins prone to form inclusion bodies can be anchored on the spore surface, separated by a mini-intein derived from the SSp DnaB, which was then used as self-cleaving tag for purification by shifting the pH and/or temperature conditions, with no addition of any proteases or thiol reagent (Mathys et al., 1999). To construct the system, the mini-intein was fused downstream of the CotB protein, followed by the reporter protein AmyQ. By changing the pH of the buffer, the mini-intein self-cleaving process was induced followed by the release of α-amylase into the supernatants. This observation suggests the use of the B. subtilis spores as an effective and low cost tool for protein purification. However, concerns related to premature of the pH-inducible mini-intein and auto-release of coat protein raise the question about the stability of the fusion coat-heterologous protein on the spore surface using the system. Hence, further investigation is needed to achieve a usable spore-based purification system. The last aim of the thesis was to apply the newly constructed B. subtilis spore display and the cell surface display systems (Nguyen and Schumann, 2006) to generate cellulose chips, in which enzymes were immobilized on the surface of microorganism cells or spores. The cellulase A (CelA) from C. thermocellum (Beguin et al., 1985) was utilized as a model enzyme. Unfortunately, the results showed an ineffective anchoring of CelA on the cell wall. This indicates the unsuccessful creation of cell-based cellulase chip when using the SrtA transpeptidase. In contrast, CelA was verified to be successfully displayed on the spore surface using CotB and CotG, but not CotC, as carriers. In general, a large volume of culture (up to one liter) must be prepared containing both cells and spores displaying CelA on the surface to assure sufficient CMC degradation. This might indicate a low activity of CelA. Further works should be done in selection of cellulase and improvement of the systems to generate the more effective cellulase chips.

Resurvey of a GLORIA Target Region in the Swiss National Park (2011)

Frank Breiner

There is no doubt that recent global climate change is in process and affects life on earth. Especially mountain ecosystems are supposed to be highly sensitive to climate change due to the vertical compression of life zones, rough abiotic environment and limiting ecological factors. Therefore, the European Alps is one of the best observed ecosystems where many studies figured out how climate change is affecting biodiversity. Probably the biggest and most well-known project is the GLORIA-Europe initiative established by Prof. Dr. Georg Grabherr from University of Vienna. The aim of this project is to establish a world-wide long-term monitoring network in alpine ecosystems to detect effects of climate change on the vegetation of mountain summits using standardised methods. This study is involved in the GLORIA initiative to resurvey four calcareous and four siliceous summits at Swiss National Park in summer 2009/10. The aim of this study is to answer the questions if there are changes between the first (2002/03) and second survey in plant species number, species frequency and in heterogeneity between plots. Furthermore, is altitude, cardinal direction and bedrock influencing changes or are there species groups reacting different and what are the reasons behind it? In total 226 species were found in 2009 and 2010 with almost 80% more species on the siliceous summits. Species turnover rate between the two surveys is relatively high (15-30%) and also frequency is increasing for several species. But, there are no effects of bedrock or exposition and no differences for species groups. This study shows that fluctuation of species turnover is due to fluctuation of phenological development. Furthermore, differences in plot heterogeneity can be explained by phenological fluctuation. However, there are hints for initiating effects of climate change. The occurrence of L. decidua on three lower summits and the high content of new found species with a lower distribution limit at the montane belt on PMU as well as general increase in plant frequency could be caused by climate change. Hence, these hints of climate change should be focused on in future investigations as long-term effects of climate change are expected.

Construction of an efficient secretion system for recombinant proteins in Bacillus subtilis (2011)

Kelly Cristina Leite

All proteins being translocated through the cytoplasmic membrane of bacteria cells as well as some proteins that are inserted into the cytoplasmic membrane contain a signal sequence at their N-terminus that is recognized by and targeted to the translocation machinery. Three translocation pathways have been described, so far in E. coli to allow secretion of proteins: The Sec, the Tat and the SRP (Signal Recognition Particle) pathway. While the Sec and the Tat pathway act post-translationally and accept unfolded and correctly folded polypeptides, respectively, the SRP pathway acts co-translationally. For proteins secreted through the cytoplasmic membrane via the Sec pathway, the ATP-dependent motor protein SecA is required for translocation. The translocation process of some proteins following the SRP pathway has also shown to be enhanced by the presence of SecA. The Sec and the SRP pathway share the heterotrimeric protein-conducting channel translocon complex composed of the SecYEG proteins. Based on the known characteristics of both pathways, the goal of this PhD project was to construct an efficient secretion system for recombinant proteins in Bacillus subtilis using an a-amylase as a reporter enzyme, which is secreted into the medium using the Sec pathway. Its gene amyQ was fused to an IPTG-inducible promoter. It turned out that increasing amounts of IPTG did not result in a concomitant increase of secreted a-amylase. Overproduction either formed aggregates within the cytoplasm or preproteins targeted to the translocon jammed the membrane. To release the accumulated protein within the cells two different experiments were carried out: i) a co-production and overexpression of SecA, and; ii) overexpression of an artificial secYEG operon. First, increased production of SecA showed significantly decrease in the total synthesis and secretion of a-amylase and did not reduce cytoplasmatic accumulation or membrane jamming. Second, the artificial operon enhanced expression of secY, secE and secG genes resulted in a higher amount of reporter enzyme secreted into the medium. Furthermore, two different experiments using the transposon mutagenesis strategy were carried out in order to screen for B. subtilis mutants able to increase secretion of α-amylase. Transposon mutagenesis was performed with the mariner-based transposon to inactivate gene(s) whose product might regulate directly or indirectly the secretion of α-amylase. No mutant strain presenting a higher secretion of α-amylase on indicator plates was found. In addition, I devised a modified transposon containing a xylose-expression cassette. To test the efficiency of the modified transposon, the promoter-less cat gene was used as a reporter gene and integrated into the B. subtilis chromosomal DNA. After transposon mutagenesis, mutants expressing the promoter-less cat gene were isolated. This result indicates that the modified transposon might lead to increased production of a gene in the presence of xylose and that this product might then enhance secretion of α-amylase to be detected on indicator plates. In the third part of my thesis, a terminator-test vector was constructed which should allow the identification of strong terminators acting as 5'-stabilizing element. This vector consists of an artificial bicistronic operon containing the two reporter genes bgaB and gfp allowing the insertion of the terminators between the two genes. Insertion of a terminator should lead to a reduction of the amount of GFP. The system was verified with the known sinIR transcriptional terminator. It turned out that the vector with the two reporter genes already exhibited instability in E. coli.

Studies on the influence of different diets and rearing conditions on the development and growth of the two-spotted cricket Gryllus bimaculatus de Geer (2011)

Hassan Ibraik Hassan EL-Damanhouri

•The effect of rearing conditions and different diets on larvae and adults of the two-spotted cricket, Gryllus bimaculatus de Geer (Orthoptera: Gryllidae), was studied. •The percent increase in body and ovary mass of adult females reared under crowded conditions was higher than that of those reared under isolated conditions. •Higher population density increased reproductive investment in adult females, regardless of the presence or absence of adult males. •The presence or absence of adult males appeared to have no significant effect on the pronotum width, increase in body mass, DLM mass and ovary mass of adult females. •Not only the population density plays an important role in morphological, physiological and behavioural changes, but also the absolute number of animals in a population may influence the above-mentioned parameters. •A strong positive correlation was found between increase in body and ovary mass and decrease in flight muscle and fat body mass; the loss of flight muscle and fat body mass is due to a high egg production. •Five different diets were used: standard diet (ca. 20 % protein, 4.5 % lipid, 45.5 % carbohydrate), modified standard diet (ca. 22 % protein, 6 % lipid, 42 % carbohydrate), and three semi-artificial diets, all containing 30 % protein but differing amounts of lipid (A: normal, B: low, C: high). •The amount of diet consumed highly depends on the type of diet given. •Weak relationships were obtained between increase in body mass and different amounts of diets consumed. •The amount of the different storage products in the fat body of 5-day-old adult females can be arranged in the following order: lipid, protein, glycogen and free carbohydrate. •The total lipid concentration in the haemolymph was generally much higher than carbohydrate concentration. •When fed on a high calory diet, the amount of food consumed was reduced. •When crickets were fed on artificial diets from penultimate larval stage, they displayed a lower increase in body mass and some reduction in flight muscle and ovary mass compared with crickets fed on artificial diet from the day of the adult moult only. •With respect to body and ovary mass, when fed to adult females modified standard diet was heartier, followed by diet A, B, C and standard diet. •When fed to penultimate larvae onwards, however, diets B and C resulted in a significantly reduced body and ovary mass. This indicates that when diets too low or too high in lipid content are fed for a longer period, cricket cannot compensate for it. •The development and reproductive investment of Gryllus bimaculatus shows a high degree of dependence on diet availability, diet quality and quantity, and food intake. Therefore, crickets raised on the different diets did not grow at equal rates. •The results unambiguously show that population density dramatically alters cricket behavioural, morphological and physiological characteristics; as a matter of fact there was a strong correlation between crowded conditions and an increase in metabolic rate, which in turn had an influence on the effects of different diets.

Members of the Preprotein and Amino Acid Transporter Family Constitute Components of Novel Protein Import Pathways into Chloroplasts (2011)

Claudia Roßig

In order to sustain their structure and metabolism, chloroplasts and other plastid types must import the majority of their proteins from the cytosol across the envelope membranes. Translocons at the outer and inner chloroplast envelope membranes, called TOC and TIC, were identified that mediate the import of proteins. N-terminal transit peptides essential for import of the protein precursors are cleaved after their entry into the stroma. It was thus far believed that all of the different cytosolic precursors would enter the chloroplast through the same, jointly acting TIC/TOC machineries. Recent evidence, however, suggests that multiple, regulated import pathways exist in plastids that involve different import machineries. Proteomics studies have revealed the presence of a large number of plastid proteins lacking predictable N-terminal transit sequences for import. The import mechanism for the majority of these proteins has not been determined yet. One example is the chloroplast envelope quinone oxidoreductase homologue, ceQORH. This protein is imported into the inner plastid envelope membrane by a TIC/TOC-independent pathway and without any proteolytic cleavage. In the present study 5 proteins were shown to interact with ceQORH during its import and were designated as ceQORH translocon components (QTC). One of these proteins, QTC24 (also called HP20), is a member of the PRAT family comprising preprotein and amino acid transporters found in chloroplasts, mitochondria and free-living bacteria. Different expression patterns and localization of PRAT proteins suggest that they are functionally diverse beyond their role in protein translocation. QTC24/HP20 is located in the outer plastid envelope membrane of chloroplasts where it establishes a hydrophilic translocation pore. Thus, chloroplasts contain besides TOC75 and OEP16-1 a third translocation channel component in their outer envelope membrane that functions in import of transit sequence-less inner envelope proteins. In vitro import into chloroplasts of corresponding isolated A. thaliana knock-out mutants revealed that the lack of HP20 could not be replaced by its close relative HP22. Athp20 plants had no phenotype when grown under standard green house conditions. However, minor defects during the very early stage of greening of etiolated seedlings were observed as the expression of mainly plastid-encoded proteins was delayed. These effects could be interpreted in terms of an impaired amino acid import at this stage of development. A second protein of the PRAT family, HP30, was further subject of this work. However, its role remains unclear at the moment. Isolated homozygous A. thaliana knock-out mutants of HP30 did not reveal any phenotype under the growth conditions analysed in this work. The preliminary investigation of stable RNA silencing mutants indicated that the function of HP30 and its close relative HP30-2 is important during the early stages of seedling development. Young leaves of respective mutant plants exhibited a chlorotic phenotype. A further member of the PRAT family is OEP16-1 that was initially identified as amino acid-selective protein channel. Other studies revealed its role as translocation pore for the PORA precursor. Analysis of the corresponding A. thaliana knock-out mutant to dissect these two mutually not exclusive functions has led to the description of different phenotypes. During a re-screen of the original seed stock, four independent OEP16-1-deficient mutant lines were isolated that exhibited different cell death properties. Two mutants contained elevated amounts of free protochlorophyllide (Pchlide) in darkness that was caused by a defect in the Pchlide-dependent import of PORA. Etiolated seedlings of these lines died after light exposure due to the production of singlet oxygen. The two other mutants did not accumulate excessive amounts of free Pchlide and greened normally. Two of the four mutant lines with seemingly no correlation between the lack of PORA and cell death were analysed in more detail in this thesis. Moreover, a complemented Atoep16-1 mutant that re-expressed functional OEP16-1 protein was shown to restore the wild-type phenotype including PORA import that prevented the accumulation of an excess of free Pchlide and singlet oxygen production upon light exposure of dark-grown seedlings.

Climate change impacts on habitats and biodiversity :  From environmental envelope modelling to nature conservation strategies (2011)

Torsten Bittner

Climate change will pose entirely new challenges for nature conservation. A literature study of 852 publications (between 2003 and 2010) illuminates this topic, examines driving research forces as well as focal points and shows recent research gaps. Here could be shown that changes in species distribution, diverse consequences for habitats, changing communities as well as biotic interactions and general aspects of diversity are the major challenges. The potential climatic modifications can alter deeply the distribution of animals and plants. Range changes due to recent climate change already exist and are traceable. In order to quantify such changes, environmental envelope modelling can be used. In addition to individual species, changes in distribution of more complex units are also conceivable. The present work mainly focuses on habitat types listed in the Annex I of the European Habitats Directive. To reveal the potential range changes of habitat types, two principally different modelling approaches have been developed. An indirect approach modelling the distribution of a habitat type using the distribution of its characteristic plant species and a direct approach, using the distribution of the habitat type itself. These two approaches were tested by modelling five grassland habitat types. Looking at the modelled results all habitats are projected to lose between 22% and 93% of their range in the ‘no dispersal’ scenario. Both approaches produce reasonable results. However, modelling an extensive set of habitat types using the indirect approach is currently not possible, because of the required but actually lacking amount of plant distribution data. Therefore, the direct approach is an appropriate instrument for modelling habitat types. Here, all 127 widespread terrestrial habitat types defined in the Annex I of the Habitats Directive were modelled and, resulting from this, a map of terrestrial habitat type diversity was calculated. Several habitat types are projected to lose many of their actually suitable areas, in particular bogs, rocky habitats, grassland and in part forests. Due to their developmental time or rather due to their special abiotic requirements, bogs and rocky habitats even lose under the assumption of a full dispersal scenario. However, most heath and grassland habitats are also projected to lose in the full dispersal scenario. Pooling all modelled results together, terrestrial habitat type diversity is shifting partly to mountain regions and the atlantic biogeographical region is projected to decrease in habitat type diversity. According to the Habitats Directive habitat types listed in Annex I are protected in ‘sites of community interest’ aiming to maintain or restore them at a favourable conservation status. Due to the projected changes a static nature conservation concept could face problems which particularly concern the coherence of the protected area network. This could lead to a loss of protective goods in spite of protected areas. To illustrate the potential problems and difficulties emerging with respect to spatial coherence of habitat types between protected areas, an analysis of spatial coherence under future conditions for a variety of habitat types in Germany was conducted. Here, a combination of environmental envelope modelling and graph theory is presented to assess the coherence of nature conservation networks. The possible incapacity of species to reach all climatically suitable areas is currently debated. Therefore, spatial scales are not only crucial for the coherence of protected areas but also for the question if future projected suitable areas could be colonized. Dispersal movements of species are only infrequently possible in our highly fragmented landscape. To answer this raising question, Odonata listed in the Habitats Directive with known dispersal distances were contemplated. The species Coenagrion ornatum, C. mercuriale and Ophiogomphus cecilia are projected to lose range when incorporating specific dispersal distances, while they are projected to extend their range in the unrestricted dispersal scenario. Furthermore, suitable climatic conditions tend to decline for Leucorrhinia albifrons and L. caudalis, whereas L. pectoralis is projected to gain distribution area assuming either species-specific or unrestricted dispersal. The nature conservation measure of translocation is an at least 100 years old methodology with pros and cons. In this thesis, the emerging problems and opportunities of this species preservation strategy are presented. Further, a new question about the ‘focal unit’ is pointed out as well as the problem of genetic variability and the aspect of pre-adopted subspecies. Moreover, a selective assisted colonisation not by moving species but ecotypes is referred.

Analysis of the "nurse-tree effect" of exotic shelter trees on the growth of the indigenous Podocarpus falcatus in an Ethiopian montane forest (2011)

Simone Strobl

Ethiopian forests disappear with a rate of 1.1% per year due to the high demand of firewood and timber. To protect the remaining parts of the forests, exotic tree plantations were established 60 years ago. But there are considerable concerns regarding exotic plantations: they have the reputation to cause damage to the ecosystems due to high demand of water and nutrients. Considering the environmental deterioration caused by monotonous plantations of exotic tree species, the chance for indigenous woody plants to rejuvenate naturally in those plantations appears to be very small. But there are observations of indigenous tree species regenerating under the shelter of exotic tree plantations. This enhanced growth of indigenous saplings under the canopy of exotics has been termed “nurse-tree effect”. In the Munessa-Shashamene Forest, a tropical montane forest in Ethiopia consisting of plantations of exotic tree species and remnants of natural forest, regeneration and an enhanced growth of native Podocarpus falcatus saplings under the shelter of exotic tree plantations (Pinus and Eucalyptus) was observed. The focus of this work was to examine the different growth patterns of the saplings in the sites, the effects of the on the photosynthetic performance, and to compare the water relations of the Podocarpus saplings and those of the shelter-trees. The results of the study are summarized as follows: 1. Observations over two years showed that the relative growth rates of the saplings were more than three times higher in the Pinus plantation compared to the natural forest and the Eucalyptus plantation. Relative growth rates during the dry and the rainy season were more or less identical. 2. Investigation of the sub-canopy microclimate proved PAR and VPD as major components with impact on the photosynthetic performance of the saplings. 71% of the variations in photosynthetic carbon uptake could be explained by PAR and 4% by VPD. The Pinus plantation was slightly warmer and drier compared to the other two sites. Also highest PAR values of all sites were recorded in the Pinus plantation. In the Eucalyptus plantation, PAR values were the lowest of the three sites, caused by the two-tired canopy of coppiced and uncoppiced Eucalyptus trees. 3. For an assessment of the photosynthetic efficiency of the light climate, the efficacy of the shares of the irradiation from diffuse light and light flecks were determined from light curves. The time spans and distribution of these shares of the daily accumulated radiation were recorded from the daily courses. In the Pinus plantation, the efficiency of the radiation was relatively low (70%), because of the high intensity of the sunflecks, especially during the dry season. On cloudy days the efficiency was nearly 100% and resulted in an optimum photosynthetic performance of the saplings in the Pinus plantation. In the Eucalyptus plantation, the two-tired canopy resulted in a higher proportion of diffuse radiation and less daily accumulated PAR from sunflecks (46%). Also the efficiency of the actual radiation was the lowest of all sites on cloudy (72%) and sunny (53%) days. Daily accumulated PAR under the canopy of the natural forest was in between the other forest types. Such mid-position was also true for the share of the sunflecks and the CO2 uptake. Efficacy of the radiation was 80% on sunny and 86% on cloudy days. 4. Water relations can substantially affect the photosynthetic performance of plants. Especially in the afternoons of the dry season a decrease of photosynthetic CO2 uptake by the Podocarpus saplings became apparent. Whole-tree water consumption was determined by measuring sap flow with the Granier system. In principle sap flow (and transpiration) followed VPD. Comparison of the daily courses of transpiration and stomatal conductance and sap flow showed an earlier decrease of transpiration by the leaves of the saplings than by the shelter-trees, suggesting slight water shortage especially during the dry season. This interpretation is corroborated by the higher 13C values in the leaf tissue of the saplings from the Pinus plantation. Nevertheless severe drought stress did not occur during the two years of investigation. 5. The literature on the „nurse-tree effect“ mentions in particular Eucalyptus as shelter-tree, a finding which is not in agreement with the data of this study: Neither photosynthesis nor growth was enhanced compared with the control saplings in the natural forest. The discrepancy between this work and the literature can be solved when the management of the Eucalyptus plantation is considered. As long as the Podocarpus saplings grow under the two-tired canopy of the coppiced trees, growth is as slow as in the natural forest. However, after coppicing the light climate for the saplings ameliorates considerably and growth rates increase. Thus, a shelter-tree effect could also be observed under Eucalyptus, but its dynamics is stepwise rather than continuous.

Impact of Oxygen and Pesticides on Microbial Cellulose Degradation in Aerated Agricultural Soils: A Microscaled Analysis of Processes and Prokaryotic Populations (2011)

Stefanie Schellenberger

The polysaccharide cellulose is a major component of organic matter in terrestrial ecosystems and its mineralization drives carbon fluxes in soil. The degradation of plant-derived saccharides (e.g., cellulose, cellobiose, and glucose) is catalysed by a huge diversity of aerobic and anaerobic microorganisms (including Bacteria, fungi, and protists), but there is limited information about their phylogenetic identities and their in situ relevance in soil. Soil is a heterogeneous habitat in which oxic and anoxic microzones co-occur, and in which the distribution of O2 can change rapidly. Hence, the availability of O2 is an important factor that determines the activity of the saccharide-degrading community in microzones of aerated soil. Likewise, the accumulation of potential toxic pesticides in agricultural ecosystems might influence microbial activity. It is not resolved how active cellulolytic and saccharolytic taxa respond to rapid changes in the availabilities of O2. Furthermore, it is unclear if pesticides impact on the degradation of cellulose and cellulose-linked processes, and influence the activity of active saccharide-utilizing microorganisms. Hence, this study first identified cellulolytic and saccharolytic aerobic and anaerobic Prokaryotes that catalyzed the degradation of supplemented [13C]-labeled saccharides by 16S rRNA stable isotope probing. The metabolic response of major bacterial taxa to pesticides and fluctuating availabilities of O2 was further analyzed with taxon-specific qPCR assays. Eukaryotes that contributed to soil carbon flux were identified by targeting 18S rRNA genes by the collaborative group of Dr. A. Chatzinotas at the Helmholtz Centre (UFZ) in Leipzig. Cellulose, cellobiose, and glucose were mineralized to carbon dioxide under oxic conditions, whereas different fermentation products accumulated under anoxic conditions. Fermentations occurred concomitant with the apparent reduction of nitrate and ferric iron. The degradation of supplemented saccharides was stable under oxic and anoxic conditions. Archaea were no active constituents of the cellulose-degrading community in the investigated soil. [13C]-cellulose was mainly degraded by Kineosporiaceae (Actinobacteria), the cellulolytic taxon Cluster III Clostridiaceae (Firmicutes), and the new family-level taxon 'Cellu1-3' (Bacteroidetes) under anoxic conditions. Under oxic conditions, the new family-level taxa 'Sphingo1-4' (Bacteroidetes) and 'Deha1' (Chloroflexi), and Planctomycetaceae (Planctomycetes) were involved. Active community members in [13C]-cellobiose and [13C]-glucose treatments differed from those in [13C]-cellulose treatments, and were selectively activated by O2. Twenty-eight of the 48 labeled bacterial family-level taxa did not closely affiliate with cultured species. Labeled Eukaryotes belonged to the families Bodonidae, Eustigmataceae, Mallomonadaceae, Opistonectidae, unclassified Chrysophyceae, and unclassified Stramenopiles. These families inhabit autotrophic algae and bacteriovorus flagellates. It is likely that these active Eukaryotes were labeled by incorporation of [13C]-carbon derived from grazing on active cellulolytic and saccharolytic soil bacteria. Fungi were not labeled in stable isotope probing experiments. The pesticides Bentazon, MCPA and Nonylphenol impaired cellulose-linked microbial processes only at pesticide concentrations far above the recommended rate. The impairment was most pronounced under anoxic conditions. Planctomycetaceae and the new family-level taxon 'Sphingo1-4' were sensitive to pesticide addition under oxic conditions, whereas Cluster I Clostridiaceae and the new family-level taxon 'Cellu1-3' were reduced under anoxic conditions. Nevertheless, the impact of pesticides on the degradation of saccharides at in situ-relevant concentrations seems to be minimal. These collective findings suggest that (i) a large uncultured diversity of Bacteria was involved in the degradation of cellulose, (ii) O2 selectively activates different cellulolytic and saccharolytic taxa, (iii) Cluster III Clostridiaceae, and the new family-level taxa 'Sphingo1-4' and 'Cellu1-3' represent the major cellulolytic constituents of the microbial community in the investigated agricultural soil, whereas Cluster I Clostridiaceae, Intrasporangiaceae and Micrococcaceae are saccharolytic satellite organisms that utilize cellulose-derived carbon, and (iv) the degradation of plant-derived saccharides is a community function that is stabilized by the rapid response of active bacterial taxa and independent of fluctuating availabilities of O2 and of pesticide application.

Regulation der Aktivität des Anti-Sigmafaktors RsiX aus Bacillus subtilis (2011)

Katharina Schäfer

Der Gram-positive Modellorganismus Bacillus subtilis besitzt 17 alternative Sigmafaktoren, von denen 7 zur Gruppe der ECF (extra-cytoplasmic function) -Sigmafaktoren gehören. Einer dieser ECF-Sigmafaktoren, SigX, und sein entsprechender Anti-Sigmafaktor, RsiX, sind Gegenstand dieser Arbeit. Dabei handelt es sich bei RsiX um ein Transmembranprotein, während SigX im Cytoplasma lokalisiert ist und von der N-terminalen RsiX-Domäne von einer Interaktion mit der RNA-Polymerase abgehalten wird. SigW, ein weiterer ECF-Sigmafaktor, und RsiW, der dazugehörende Anti-Sigmafaktor, sind bereits sehr gut untersucht. Aufgrund struktureller Ähnlichkeiten zwischen SigX/RsiX und SigW/RsiW wurde angenommen, dass die Aktivierung des SigX-Regulons in vergleichbarer Weise erfolgt, wie es für das SigW-Regulon aufgrund intensiver Analysen sehr gut untersucht ist. Die in dieser Arbeit verwendete Methode der Transposonmutagenese lieferte entscheidende Hinweise über die Regulation des sigX-rsiX-Operons. Es konnte gezeigt werden, dass das sigX-rsiX-Operon einer Regulation durch den Transkriptionsterminator Rho unterliegt. Damit wurde das erst dritte Beispiel für eine Termination der Transkription durch den Faktor Rho in B. subtilis bekannt. Als wichtiges Element für die faktorabhängige Termination der Transkription gilt das Vorhandensein einer rut-site, der Bindestelle des Faktors Rho in der naszierenden mRNA. In dieser Arbeit wurden verschiedene Analysetechniken angewandt, um mit Hilfe eines GFP-RsiX-Reporters erstmals eine rut-site in B. subtilis zu kartieren. Die hier identifizierte rut-site von rsiX ist am 5´-Ende dieses Gens lokalisiert. Bei diesen Analysen wurde außerdem deutlich, dass es im sigX-rsiX-Operon zu einer faktorabhängigen intragenischen Termination der Transkription kommt, bei welcher nicht nur die Transkription von rsiX, sondern auch die Expression des stromaufwärts liegenden sigX beeinflusst wird. Punktmutationen in der Sequenz für die rut-site von rsiX zerstörten den Einfluss des Faktors Rho auf das sigX-rsiX-Operon, so dass keine Termination der Transkription mehr stattfand und die detektierbare Menge an sigX-rsiX-Transkript deutlich anstieg. Nur so war es möglich, einen rsiX-Knockout zu komplementieren. Messungen der beta-Galaktosidase-Aktivität eines lacZ-Reporters belegten zudem die Funktion von RsiX als Anti-Sigmafaktor von SigX. Der Verlust des Einflusses von Rho auf das sigX-rsiX-Operon aufgrund einer mutierten rut site machte auch immunologische Nachweise des Anti-Sigmafaktors möglich. Infolgedessen konnte der Frage einer möglichen RsiX-Proteolyse nach dem Vorbild der RsiW-Proteolyse nachgegangen werden. Für RsiW wurde bereits gezeigt, dass ein bestimmtes Stress-Signal den Abbau des Anti-Sigmafaktors einleitet. Die Proteolyse von RsiW erfolgt dann nach dem Mechanismus der regulierten intramembranen Proteolyse (RIP) unter der Beteiligung mehrerer Proteasen. Neben der Beteiligung der Protease RasP an der RsiW-Proteolyse konnte hier auch die Beteiligung von RasP an der RsiX-Proteolyse nachgewiesen werden. Gleichzeitig wurde aber ausgeschlossen, dass die Protease PrsW, welche auch an der RsiW-Proteolyse beteiligt ist, in die RsiX-Proteolyse involviert ist. Demnach können die einzelnen Schritte der RsiW-Proteolyse nicht einfach auf die RsiX-Proteolyse übertragen werden, obwohl es sich bei beiden Transmembranproteinen um Anti-Sigmafaktoren ihrer jeweiligen ECF-Sigmafaktoren handelt.

Eukaryotic chromosome segregation: New aspects of separase regulation by securin, Cdk1, PP2A and auto-cleavage (2011)

Franziska Böttger

The universal triggering event of eukaryotic chromosome segregation is the proteolytic cleavage of chromosomal cohesin by separase. The activity of this essential but potentially also very dangerous protease must be tightly controlled. Prior to the onset of anaphase separase is kept inactive by association with either securin or cyclin-dependent kinase 1 (Cdk1) in conjunction with cyclin B1. Only when all chromosomes interact properly with the mitotic spindle apparatus does the anaphase promoting complex or cyclosome (APC/C), a multisubunit E3 ligase, mediate the ubiquitylation of securin and cyclin B1. Their subsequent proteasomal degradation then releases active separase. Murine embryonic stem cells, which lack securin and express a Cdk1-resistant phosphorylation site mutant separase are viable. Thus, additional regulations of sister chromatid separation by separase must exist. It was reported that human separase cleaves not only cohesin but also itself and, furthermore, that it interacts with protein phosphatase 2A (PP2A). However, the functions of separase's auto-cleavage and PP2A-interaction remain enigmatic. Moreover, securin was reported to also interact with PP2A but, strangely, with a different isoform of the phosphatase. Thus, the question needs clarification of whether separase or securin or both interact with which isoform of PP2A. In this study, further insights into the relationship between separase auto-cleavage and PP2A binding are presented. Phosphorylation of a serine residue in close proximity to the major cleavage site of separase was found to stimulate auto-cleavage of separase. Interestingly, a quantitative mass-spectrometric approach (SILAC) identified this serine residue as a substrate of separase-bound PP2A. Furthermore, a point mutation within separase was identified, which totally abolishes PP2A binding and which maps to the immediate vicinity of the auto-cleavage sites. Thus, PP2A prevents the auto-cleavage of separase both catalytically and sterically. It could further be shown that non-cleavable separase exhibits increased association with PP2A and that forced cleavage of separase displaces PP2A. Taken together, these results demonstrate that auto-cleavage and PP2A binding constitute two antagonistic mechanisms of separase regulation. Evidence is provided that the interaction of PP2A with securin is indirect and bridged by separase, and that it is the B56- and not the B55-isoform of PP2A which associates with the separase-securin complex. Moreover, free securin is shown to be degraded in early mitosis in a phosphorylation- and APC/C-dependent manner, while separase-associated securin is kept dephosphorylated and, thus, protected by PP2A. Securin levels are frequently increased in tumors. In normal cells, the early removal of excessive securin might later ensure swift separase activation and anaphase onset, thereby contributing to faithful chromosome segregation.

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