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It has been known for a long time that Drosophila can learn to discriminate not only between different odorants but also between different concentrations of the same odor. Olfactory associative learning has been described as a pairing between odorant and electric shock and since then, most of the experiments conducted in this respect have largely neglected the dual properties of odors: quality and intensity. For odorant-coupled short-term memory, a biochemical model has been proposed that mainly relies on the known cAMP signaling pathway. Mushroom bodies (MB) have been shown to be necessary and sufficient for this type of memory, and the MB-model of odor learning and short-term memory was established. Yet, theoretically, based on the MB-model, flies should not be able to learn concentrations if trained to the lower of the two concentrations in the test. In this thesis, I investigate the role of concentration-dependent learning, establishment of a concentration-dependent memory and their correlation to the standard two-odor learning as described by the MB-model. In order to highlight the difference between learning of quality and learning of intensity of the same odor I have tried to characterize the nature of the stimulus that is actually learned by the flies, leading to the conclusion that during the training flies learn all possible cues that are presented at the time. The type of the following test seems to govern the usage of the information available. This revealed a distinction between what flies learned and what is actually measured. Furthermore, I have shown that learning of concentration is associative and that it is symmetrical between high and low concentrations. I have also shown how the subjective quality perception of an odor changes with changing intensity, suggesting that one odor can have more than one scent. There is no proof that flies perceive a range of concentrations of one odorant as one (odor) quality. Flies display a certain level of concentration invariance that is limited and related to the particular concentration. Learning of concentration is relevant only to a limited range of concentrations within the boundaries of concentration invariance. Moreover, under certain conditions, two chemically distinct odorants could smell sufficiently similarly such, that they can be generalized between each other like if they would be of the same quality. Therefore, the abilities of the fly to identify the difference in quality or in intensity of the stimuli need to be distinguished. The way how the stimulus is analyzed and processed speaks in favor of a concept postulating the existence of two separated memories. To follow this concept, I have proposed a new form of memory called odor intensity memory (OIM), characterized it and compared it to other olfactory memories. OIM is independent of some members of the known cAMP signaling pathway and very likely forms the rutabaga-independent component of the standard two-odor memory. The rutabaga-dependent odor memory requires qualitatively different olfactory stimuli. OIM is revealed within the limits of concentration invariance where the memory test gives only sub-optimal performance for the concentration differences but discrimination of odor quality is not possible at all. Based on the available experimental tools, OIM seems to require the mushroom bodies the same as odor-quality memory but its properties are different. Flies can memorize the quality of several odorants at a given time but a newly formed memory of one odor interferes with the OIM stored before. In addition, the OIM lasts only 1 to 3 hours - much shorter than the odor-quality memory.
Emery-Dreifuss muscular dystrophy (EDMD) is a rare genetic disorder characterised by early contractures of the elbows, Achilles tendons and spine, slowly progressive muscle wasting and cardiomyopathy associated with cardiac conduction defect. The autosomal dominant form is caused by mutations in the LMNA gene which gives rise to lamin A and lamin C proteins by alternative splicing. These A-type lamins, together with B-type lamins, form the nuclear lamina, a network of intermediate filament proteins underlining the nuclear envelope. In order to ascertain the role lamin A and C separately contribute to the molecular phenotype, we analysed ten LMNA mutations and one single nucleotide polymorphism (SNP) in transfection studies in COS7 fibroblasts and, partially, in C2C12 myoblasts. The EGFP or DsRed2 tagged lamins were exogenously expressed either individually or both A-types together and examined by light and electron microscopy. The protein mobility of lamin A mutants was determined by FRAP analysis. Additionally, a co-immunoprecipitation binding assay of in vitro synthesised A-type lamins and emerin was performed.Eight of the LMNA mutations (R50S, R133P, E358K, E358K+C<T1698, E361K, R527P, L530P, R541S and G602S) and the SNP C<T1698, when expressed in lamin A, exhibited a range of nuclear mis-localisation patterns from a wild type phenotype to the formation of nuclear aggregates. Two mutations (T150P and delQ355) led to the severe mis-localisation of the exogenous protein and additionally affected nuclear envelope reassembly and mid-body protein composition after mitosis. Exogenously expressed DsRed2 tagged wild type and mutant lamin C was only inserted into the nuclear lamina if co-expressed with the equivalent EGFP tagged lamin A construct, except for the T150P mutation which prevented either lamin from reaching the nuclear lamina. The T150P, R527P and L530P mutations reduced the ability of lamin A, but not lamin C from binding to emerin. These data indicate that mutations in the rod domain of lamin A mainly impair its function as a structural protein, whereas mutations of the globular tail domain appear to disrupt protein-protein interactions important for gene regulation and signal transduction processes. In addition, our results suggest specific functional roles for the emerin-lamin A and emerin-lamin C containing protein complexes; this is the first report to propose that the A-type lamin mutations may be differentially dysfunctional for the same LMNA mutation.
In this thesis, I introduce the Virtual Brain Protocol, which facilitates applications of the Standard Brain of Drosophila melanogaster. By providing reliable and extensible tools for the handling of neuroanatomical data, this protocol simplifies and organizes the recurring tasks involved in these applications. It is demonstrated that this protocol can also be used to generate average brains, i.e. to combine recordings of several brains with the same features such that the common features are emphasized. One of the most important steps of the Virtual Insect Protocol is the aligning of newly recorded data sets with the Standard Brain. After presenting methods commonly applied in a biological or medical context to align two different recordings, it is evaluated to what extent this alignment can be automated. To that end, existing Image Processing techniques are assessed. I demonstrate that these techniques do not satisfy the requirements needed to guarantee sensible alignments between two brains. Then, I analyze what needs to be taken into account in order to formulate an algorithm which satisfies the needs of the protocol. In the last chapter, I derive such an algorithm using methods from Information Theory, which bases the technique on a solid mathematical foundation. I show how Bayesian Inference can be applied to enhance the results further. It is demonstrated that this approach yields good results on very noisy images, detecting apparent boundaries between structures. The same approach can be extended to take additional knowledge into account, e.g. the relative position of the anatomical structures and their shape. It is shown how this extension can be utilized to segment a newly recorded brain automatically.
Chemical neurotransmission is a complex process of central importance for nervous system function. It is thought to be mediated by the orchestration of hundreds of proteins for its successful execution. Several synaptic proteins have been shown to be relevant for neurotransmission and many of them are highly conserved during evolution- suggesting a universal mechanism for neurotransmission. This process has checkpoints at various places like, neurotransmitter uptake into the vesicles, relocation of the vesicles to the vicinity of calcium channels in order to facilitate Ca2+ induced release thereby modulating the fusion probability, formation of a fusion pore to release the neurotransmitter and finally reuptake of the vesicles by endocytosis. Each of these checkpoints has now become a special area of study and maintains its own importance for the understanding of the overall process. Ca2+ induced release occurs at specialized membrane structures at the synapse known as the active zones. These are highly ordered electron dense grids and are composed of several proteins which assist the synaptic vesicles in relocating in the vicinity of Ca2+ channels thereby increasing their fusion probability and then bringing about the vesicular fusion itself. All the protein modules needed for these processes are thought to be held in tight arrays at the active zones, and the functions of a few have been characterized so far at the vertebrate active zones. Our group is primarily interested in characterizing the molecular architecture of the Drosophila synapse. Due to its powerful genetics and well-established behavioural assays Drosophila is an excellent system to investigate neuronal functioning. Monoclonal antibodies (MABs) from a hybridoma library against Drosophila brain are routinely used to detect novel proteins in the brain in a reverse genetic approach. Upon identification of the protein its encoding genetic locus is characterized and a detailed investigation of its function is initiated. This approach has been particularly useful to detect synaptic proteins, which may go undetected in a forward genetic approach due to lack of an observable phenotype. Proteins like CSP, Synapsin and Sap47 have been identified and characterized using this approach so far. MAB nc82 has been one of the shortlisted antibodies from the same library and is widely used as a general neuropil marker due to the relative transparency of immunohistochemical whole mount staining obtained with this antibody. A careful observation of double stainings at the larval neuromuscular junctions with MAB nc82 and other pre and post-synaptic markers strongly suggested an active zone localization of the nc82 antigen. Synaptic architecture is well characterized in Drosophila at the ultrastructural level. However, molecular details for many synaptic components and especially for the active zone are almost entirely unknown. A possible localization at the active zone for the nc82 antigen served as the motivation to initiate its biochemical characterization and the identification of the encoding gene. In the present thesis it is shown by 2-D gel analysis and mass spectrometry that the nc82 antigen is a novel active zone protein encoded by a complex genetic locus on chromosome 2R. By RT-PCR exons from three open reading frames previously annotated as separate genes are demonstrated to give rise to a transcript of at least 5.5 kb. Northern blots produce a prominent signal of 11 kb and a weak signal of 2 kb. The protein encoded by the 5.5 kb transcript is highly conserved amongst insects and has at its N-terminus significant homology to the previously described vertebrate active zone protein ELKS/ERC/CAST. Bioinformatic analysis predicts coiled-coil domains spread all over the sequence and strongly suggest a function involved in organizing or maintaining the structure of the active zone. The large C-terminal region is highly conserved amongst the insects but has no clear homologues in veretebrates. For a functional analysis of this protein transgenic flies expressing RNAi constructs under the control of the Gal4 regulated enhancer UAS were kindly provided by the collaborating group of S.Sigrist (Gِttingen). A strong pan-neuronal knockdown of the nc82 antigen by transgenic RNAi expression leads to embryonic lethality. A relatively weaker RNAi expression results in behavioural deficits in adult flies including unstable flight and impaired walking behavior. Due to this peculiar phenotype as observed in the first knockdown studies the gene was named “bruchpilot” (brp) encoding the protein “Bruchpilot (BRP)” (German for crash pilot). A pan-neuronal as well as retina specific downregulation of this protein results in loss of ON and OFF transients in ERG recordings indicating dysfunctional synapses. Retina specific downregulation also shows severely impaired optomotor behaviour. Finally, at an ultrastructural level BRP downregulation seems to impair the formation of the characteristic T-shaped synaptic ribbons at the active zones without significantly altering the overall synaptic architecture (in collaboration with E.Asan). Vertebrate active zone protein Bassoon is known to be involved in attaching the synaptic ribbons to the active zones as an adapter between active zone proteins RIBEYE and ERC/CAST. A mutation in Bassoon results in a floating synaptic ribbon phenotype. No protein homologous to Bassoon has been observed in Drosophila. BRP downregulation also results in absence of attached synaptic ribbons at the active zones. This invites the speculation of an adapter like function for BRP in Drosophila. However, while Bassoon mutant mice are viable, BRP deficit in addition to the structural phenotype also results in severe behavioural and physiological anomalies and even stronger downregulation causes embryonic lethality. This therefore suggests an additional and even more important role for BRP in development and normal functioning of synapses in Drosophila and also in other insects. However, how BRP regulates synaptic transmission and which other proteins are involved in this BRP dependant pathway remains to be investigated. Such studies certainly will attract prominent attention in the future.
The development of ethanol tolerance is due to changes in synaptic plasticity. Since the mechanisms mediating synaptic plasticity are probably defective in the mutant hangAE10, it was a goal of the present study to find out how HANG contributes to synaptic plasticity. In particular, it was important to clarify in which neuronal process HANG plays a role. Antibody stainings against HANG revealed that the protein is localized in all neuronal nuclei of larval and adult brains; the staining is absent in hangAE10, thus confirming that this P-element insertion stock is a protein null for HANG. Detailed analysis of the subnuclear distribution of HANG showed that HANG immunoreactivity is enriched at distinct spots in the nucleus in a speckled pattern; these speckles are found at the inside of the nuclear membrane and do not colocalize with chromatin nor with the nucleolus; thus, HANG is probably involved in the stabilization, processing or export of RNAs. As synaptic plasticity can be studied in single neurons at the larval neuromuscular junction, the morphology of the synaptic terminals of hangAE10 mutants was analyzed at muscle 6/7, segment A4. These studies revealed that hangAE10 mutants display a 40 % increase in bouton number and axonal branch length; in addition, some boutons have an abnormal hourglass-like shape, suggesting that they are arrested in a semi-separated state following the initiation of bouton division. The increase in bouton number of hang mutants is mainly due to an increase in numbers of type Ib boutons. The analysis of the distribution of several synaptic markers in hang mutants did not show abnormalities. The presynaptic expression of HANG in hang mutants rescues the increase in bouton number and axonal branch length, thus proving that the phenotypes seen in the P-element insertion hangAE10 are attributable to the lack of HANG rather than to effects of the P-element marker rosy or to a secondary hit on the same chromsome during mutagensis. This finding is further supported by the fact that postsynaptic expression of HANG does not rescue the abnormal NMJ morphology of hangAE10. Alterations in cAMP levels regulate the number of boutons; since hang mutants display an increase in bouton number, the questions was whether this morphological abnormality was due to defects in cAMP signalling. To test this hypothesis, hangAE10 NMJs were compared to those of the hypomorphic allele dnc1 that has a defective cAMP cascade. Some aspects of the NMJ phenotype (e.g. the increase in bouton number and the unaltered ratio of active zones per bouton area) are similar in hangAE10 and dnc1, other differ. Expression of a UAS-dnc transgene in hangAE10 mutants does not modify the phenotype. In summary, the results of this study indicate that nuclear protein HANG might be involved in isoform-specific splicing of genes required for synaptic plasticity at the NMJ.
Somites are repeated epithelial segments that are generated in a rhythmic manner from the presomitic mesoderm (PSM) in the embryonic tailbud. Later, they differentiate into skeletal muscle, cartilage and dermis. Somitogenesis is regulated by a complex interplay of different pathways. Notch/Delta signaling is one of the pathways well characterized in zebrafish through mutants affected in its different components. Previous work in mouse, chicken and zebrafish has shown that also additional components are required during somitogenesis, most importantly through an FGF and Retinoic acid (RA) gradient, as well as Wnt signaling. However, no zebrafish mutants with defects in these pathways showing specific somite malformations are described. This was explained by functional redundancies among related genes that have resulted from a whole genome duplication which occurred in a teleost fish ancestor 350 million years ago. As distinct duplicates exist in different teleost species, a large scale mutagenesis screen in the medaka (Oryzias latipes) has been performed successfully in Kyoto, Japan. I analyzed nine of the isolated medaka mutants that show variable aspects of somitic phenotypes. This includes a complete or partial loss of somite boundaries (e.g. bms and sne), somites with irregular sizes and shapes (e.g. krz and fsl) or partially fused and enlarged somites (e.g. dpk). Although some of these medaka mutants share characteristics with previously described zebrafish somite mutants, most of the mutants represent unique phenotypes, not obtained in the zebrafish screens. In-situ hybridization analyses with marker genes implicated in the segmentation clock (e.g. her7), establishment of anterior-posterior (A-P) polarity (e.g. mesp) and differentiation of somites (e.g. myf5, lfng) revealed that the medaka mutants can be separated into two classes. Class I shows defects in tailbud formation and PSM prepatterning, and lateron somite boundary formation was impaired in these mutants. A unique member of this class with a novel phenotype is the doppelkorn (dpk) mutant that has single fused or enlarged somites. This phenotype has not been reported till now in zebrafish somite mutants. In-situ analyses on dpk showed that stabilization of the cyclically expressed somitogenesis clock genes must be affected in this mutant. This is accompanied by a disrupted regulation of A-P polarity genes like mesp. This suggests that dpk is a mutant deficient in the wave front, which is necessary for the down-regulation of oscillating genes in the anterior PSM. Furthermore, as the initiation of oscillation of all three cyclic her genes was unaffected in dpk embryos, I could exclude that this mutant in affected in the Notch/Delta pathway. Another mutant that belongs to this class is the samidare (sam) mutant. Morphologically, sam mutants are similar to zebrafish after eight (aei). In both cases, the first 7-9 somites are formed properly, but after this somite formation ceases. Different to the situation in aei, sam mutant embryos presented an additional defect in the mid-hindbrain boundary (MHB) region. Similar MHB defects were described in the zebrafish fgf8 mutant acerebellar (ace). In ace zebrafish mutant, somites were only slightly defective, although FGF signaling has been shown to be important for somite formation in chicken, mouse and zebrafish. This was explained by functional redundancy between fgf8 and fgf24 ligands in the tailbud of zebrafish. Thus, it is interesting to suggest that the sam mutant, based on the parallel defects in somites and MHB, is a potential member of the FGF signaling pathway muatnts. It was shown that FGF plays a crucial role during MHB formation in medaka. In addition, I showed that fgf8 acts non-redundantly during tailbud formation and somitogenesis in medaka. Furthermore, I showed that FGF signaling regulates somite size also in medaka and that fgfr1 is the only FGF receptor expressed in the tailbud and somites. In class II medaka somite mutants, PSM prepatterning appears normal, whereas A-P polarity, boundary formation, epithelialization or the later differentiation of somites appears to be affected. Such mutants have not been isolated so far in zebrafish, mice or chicken. Therefore, medaka class II somite mutants seem to be a novel group of mutants that opens new perspectives to analyze A-P polarity regulation, determination and boundary formation in the presence of a normally functioning clock in the PSM. Identifying the encoding genes for all analyzed medaka somite mutants will contribute to the understanding of the molecular interactions of different signaling pathways involved during somitogenesis, and is expected to result in the identification of new components.
The genetics of species differences is an outstanding question in evolutionary biology. How do species evolve to become phenotypically distinct and how is the genetic architecture organized that underlie species differences? Phenotypic diverged traits are supposed to be frequently involved in prezygotic isolation, i.e. they prevent the formation of hybrids, whereas postzygotic isolation occurs when hybrids experience a fitness reduction. The parasitic wasp genus Nasonia represents an appropriate model system to investigate the genetics of species differences as well as the genetics of postzygotic isolation. The genus consists of three species N. vitripennis, N. longicornis and N. giraulti that differ particularly in male traits that are assumed to posses an adaptive significance: courtship behaviour and wing size differences. The courtship behaviour consists of cyclically repeated series of head nods that are separated by pauses. The stereotypic performance allowed to split up the display into distinct courtship components. Males of N. vitripennis bear vestigial forewings and are incapable of flight, whereas N. longicornis wear intermediate sized wings and N. giraulti is fully capable of flying. Nasonia species can produce interspecific hybrids after removing Wolbachia bacteria induced hybrid incompatibilities with antibiotics. Postzygotic isolation occurs to different extent and is asymmetric among reciprocal crosses, e.g. inviability is stronger in the N. vitripennis (♀) x N. longicornis (♂) cross than in the N. longicornis (♀) x N. vitripennis (♂) cross. The formation of hybrids allow to study the genetic of species differences in QTL (quantitative trait locus) analyses as well as the genetics of postzygotic isolation causing hybrid inviability. The aim of the study was to investigate the genetic architecture of differences in courtship behaviour and wing size between N. vitripennis and N. longicornis and to assess the genetics of postzygotic isolation to gain clues about the evolutionary processes underlying trait divergence and establishment of reproductive isolation between taxa. In a QTL analysis based on 94 F2-hybrid individuals of an LV cross only few QTL for wing size differences have been found with relatively large effects, although a large proportion of the phenotypic variance remained unexplained. The QTL on courtship behaviour analysis based on 94-F2 hybrid males revealed a complex genetic architecture of courtship behaviour with QTL of large phenotypic effects that explained more than 40 % of the phenotypic variance in one case. Additionally, an epistatic analysis (non-additive interlocus interaction) of courtship QTL revealed frequent genetic interchromsomal relations leading in some instances to hybrid specific effects, e.g. reversion of phenotypic effects or the transgression of phenotypes. A QTL analysis based on a threefold sample size revealed, however, an overestimation of QTL effects in the analysis based on smaller sample size pointing towards a genetic architecture of many loci with small effects governing the phenotypic differences in courtship behaviour. Furthermore, the the study comprised the analysis of postzygotic isolation in the reciprocal crosses N. vitripennis (♀) x N. longicornis (♂) versus N. longicornis (♀) x N. vitripennis (♂) located several loci distributed over different chromosomes that are involved in hybrid incompatibility. The mapping of hybrid incompatibility regions reproduced for the first time the observed asymmetries in the strength of postzygotic isolation in reciprocal crosses of between the more distant related taxa within the genus Nasonia. Stronger postzygotic incompatibilities in the VL cross are supposed to result from the superposition of nuclear-nuclear incompatibilities with nuclear-cytoplasmic incompatibilities, whereas the coincidences of these to types of incompatibilities were found to be much weaker in the reciprocal LV cross.
The genus Pogonomyrmex is predisposed for analyzing the evolution of ant colony characteristics in general and the sociogenetic structure in particular, due to the renowned biology of several species and the diversity of mating frequency and queen number. This variation in the sociogenetic structure of colonies produces a high variance in intracolonial relatedness which can be a major component driving the evolution of various colony characteristics. To exactly determine the variability of the intracolonial relatedness in the genus Pogonomyrmex both were analyzed, the number of matrilines and patrilines, in selected members of Pogonomyrmex, namely P. (sensu stricto) rugosus, P. (sensu stricto) badius and P. (Ephebomyrmex) pima using DNA fingerprint techniques. The evolution of these colony characteristics were tried to be explained within a phylogenetic framework. For that purpose we constructed a gene-tree of 39 species of the genus Pogonomyrmex. The taxon sampling covered about 83 % of the North American species and 43 % of the South American species. Effective multiple mating of queens was confirmed for P. rugosus (me=4.1) and P. badius (me=6.7). Additionally, both species are monogynous. These results corroborate behavioral observations of multiple mating for these species. Multiple mating is now known from 9 Pogonomyrmex species (behavioral evidence for 3 species – genetic evidence for 6 species). However, in P. (E.) pima all queens that were analyzed were single mated (me=1.0). Therefore, multiple mating may have either evolved early during the evolution of the genus Pogonomyrmex and has subsequently been lost in the subgenus Ephebomyrmex (plesiomorphic hypothesis), or it has first been evolved in the subgenus Pogonomyrmex sensu stricto (apomorphic hypothesis). In P. huachucanus, a species basal to the North- American sensu stricto complex, smaller effective mating number of queens compared to its sensu stricto relatives (J. Gadau and C.-P. Strehl, unpublished) probably do mirror a change from monandry to polyandry during the evolution of more advanced sensu stricto species, which would support the apomorphic hypothesis. The intracolonial relatedness in P. (E.) pima is however rather low. This is probably the result of multiple reproducing queens (polygyny). Polygyny is also documented for at least four other species of the subgenus Ephebomyrex, but so far P. (E.) pima is the only species with genetic evidence. It might be that there was an evolutionary trade-off within the subgenus Ephebomyrmex between polyandry and polygyny. Therefore, both subgenera retained a high intracolonial genetic diversity. This high genetic diversity might be one cause for the success and radiation of the genus Pogonomyrmex in arid environments. Evolution might have favored high genetic diversity of Pogonomyrmex colonies, because it helps colonies to improve their colonial organization and efficiency in performing external tasks. At least in P. badius a link between patrilines and physical polyethism was found, indicative of an improvement of colonial organization via polyandry. Furthermore, the documented extreme levels of polyandry might help P. badius females to overcome the possibility of inbreeding due to restricted dispersal. Restricted dispersal is also found in P. (E.) pima due to wingless, intermorphic queens. However, in P. (E.) pima inbreeding is probably prevented by outcrossing via males because no significant inbreeding is found. In the presented gene trees the subgenus Pogonomyrmex Ephebomyrmex was separated from the subgenus Pogonomyrmex sensu stricto. Therefore, P. Ephebomyrmex might be elevated to generic status, also due to its distinct morphological and life history characters. Nevertheless, for a precise taxonomic revision a broader complement of species has to be applied. Regularly a low number of unrelated workers was found in P. rugosus colonies, which probably stem from brood raids between mature and founding colonies. It is well known that most founding colonies are destroyed by neighboring conspecific mature colonies, but so far it was assumed that the brood of these colonies was also destroyed. This often neglected aspect might be an important fitness token for mature colonies.
Corynebacterium glutamicum is together with C. callunae and C. efficiens a member of the diverse group of mycolic-acid containing actinomycetes, the mycolata. These bacteria are potent producer of glutamate, lysine and other amino acids on industrial scale. The cell walls of most actinomycetes contain besides an arabinogalactan-peptidoglycan complex large amounts of mycolic acids. This three-layer envelope is called MAP (mycolyl-arabinogalactan-peptidoglycan) complex and it represents a second permeability barrier beside the cytoplasmic membrane similar to the outer membrane of Gram-negative bacteria. In analogy to the situation in the outer membrane of Gram-negative bacteria, channels are present in the mycolic acid layer of the mycobacterial cell wall for the passage of hydrophilic solutes. Molecular studies have provided far-reaching findings on the amino acid flux and its balance in C. glutamicum in general, but the L-glutamate export still remains unknown. The properties of the outer layers, typical of mycolata, seem to be of major importance in this process, and diffusion seems to play a key role for this part of the cell wall. The major aim of this thesis was to identify and study novel channel-forming proteins of the amino acid producers C. glutamicum, C. callunae and C. efficiens. Cell wall extracts of the organisms were investigated and a novel pore-forming protein, named PorH, that is homologue in all three organisms, was detected and characterized. PorHC.glut was isolated from C. glutamicum cells cultivated in minimal medium. The protein was identified in lipid bilayer experiments and purified to homogeneity by fast-protein liquid chromatography across a HiTrap-Q column. The purified protein forms cation-selective channels with a diameter of about 2.2 nm and an average single-channel conductance of about 2.5 nS in 1 M KCl in the lipid bilayer assay. Organic solvent extracts were used to study the permeability properties of the cell wall of C. callunae and C.efficiens. The cell extracts contained channel-forming activity, the corresponding proteins were purified to homogeneity by fast-protein liquid chromatography across a HiTrap-Q column and named PorHC.call and PorHC.eff. Channels formed by PorHC.call are cation-selective with a diameter of about 2.2 nm and an average single-channel conductance of 3 nS, whereas PorHC.eff forms slightly anion selective channels with an average single-channel conductance of 2.3 nS in 1 M KCl in the lipid bilayer assay. The PorH proteins were partially sequenced and the corresponding genes, which were designated as porH, were identified in the published genome sequence of C. glutamicum and C. efficiens. The chromosome of C. callunae is not sequenced, but PorHC.call shows a high homology to PorHC.eff and PorHC.glut. The proteins have no N-terminal extension, only the inducer methionine, which suggests that secretion of the proteins could be very similar to that of PorAC.glut of C. glutamicum. PorHC.glut is coded in the bacterial chromosome by a gene that is localized in the vincinity of the porAC.glut gene, within a putative operon formed by 13 genes that are encoded by the minus strand. Both porins are cotranscribed and coexist in the cell wall, which was demonstrated in RT-PCR and immunological detection experiments. The arrangement of porHC.glut and porAC.glut on the chromosome is similar to that of porBC.glut and porCC.glut and it was found that PorAC.glut, PorHC.glut, PorBC.glut and PorCC.glut coexist in the cell wall of C. glutamicum. The molecular mass of about 6 kDa of the PorH channel forming proteins is rather small and suggests that the cell wall channels are formed by oligomers. A possibly hexameric form was demonstrated for PorHC.glut in Western blot analysis with anti- PorHC.glut antibodies. Secondary structure predictions for PorHC.glut, PorHC.call and PorHC.eff predict that a stretch of about 42 amino acids of PorHC.glut and 28 amino acids of PorHC.call and PorHC.eff forms amphipathic -helices with a total length of 6.3 nm and 4.2 nm respectively. This should be sufficient to cross the mycolic acid layer. Another objective of this work was to establish an heterologous expression system for corynebacterial channel-forming proteins, to investigate the channel-forming properties of the up to now only hypothetical porins PorA, PorB, PorC from C. efficiens and PorC from C. glutamicum. We could demonstrate with recombinant expression experiments in E. coli that porBC.eff and porCC.eff encode for channel-forming proteins. They are, like PorBC.glut, anion-selective with a similar single-channel conductance of 1 nS in 1 M KCl.
Rhodococcus equi is a Gram-positive intracellular pathogen which can cause severe bronchopneumonia in foals. In recent years, the role of this bacterium as human pathogen has been noted, as R.equi infections in humans have increase in frequency. This increase is associated with the rise in immunosupressed individuals, specially AIDS patients, where infection leads to symptoms and pathology similar to those seen in foals with a high mortality rate. Due to its capability to survive and multiply in murine and equine macrophages, R.equi has been classified as a facultative intracellular bacterium. R.equi is found frequently in macrophages in alveolar infiltrate from infected animals. The pathogenicity of R.equi depends on its ability to exist and multiply inside macrophages and has been associated with the presence of virulence plasmids. It has been observed that, inside foal alveolar macrophages, R.equi-containing vacuoles (RCVs) do not mature into phagolysosomes. However, most of the intracellular events during R.equi infection have not been investigated in detail. The aim of this study was to elucidate the intracellular compartmentation of R.equi and the mechanism by which the bacteria avoid destruction in host macrophages. The importance of the virulence-associated plasmids of R.equi for the establishment of RCVs was also evaluated. Furthermore, the intracellular fate of viable and non-viable R.equi was compared in order to study whether viability of R.equi influeciantes the establishment of RCVs. In this study, the RCV was characterized by using a variety of endocytic markers to follow the path of the bacteria trhough murine macropages. Transmission electron microscopy-base analysis showed that R.equi was found equally frequently in phagosomes with loosely or thightly apposed membranes, and RCV often contains numerous membranous vesicles. Laser scanning microscopy of infected macrophages showed that the majority of phagosomes containing R.equi acquired transiently the early endosomal markers Rab5, Ptlns3P, and EEA-1, suggesting initially undisturbed phagosome maturation. Although the RCV acquired some late endosomal markers, such as Rab7, LAMP-1, and Lamp-2, they did not acquired vATPase, did not interact with pre-labeled lysosomes, and failed to acidify. These data clearly suggest that the RCV is a compartment which has left vacuoles that resemble multivesicular body compartments (MVB), which are transport intermediates between early and late endosomes and display internal vesicles very similar to the ones observed within RCVs. Analyisis of several R.equi strains containing either VapA- or VapB-expressing plasmids or neither demonstrated that the possession of the virulence-associated plasmids does not affect phagosome trafficking over a two hour period of infection. The finding that non-viable R.equi was still able to inhibit phagosome maturation (although not to the same extent as viable R.equi did) suggests that heat-insensitive factors, such as cell periphery lipids, may play a major role in inhibition of phagosome maturation, although heat-sensitive factors may also be involved.