Doctoral Thesis
Refine
Year of publication
Document Type
- Doctoral Thesis (42) (remove)
Has Fulltext
- yes (42)
Is part of the Bibliography
- no (42)
Keywords
- Streptococcus pneumoniae (7)
- Saccharomyces cerevisiae (6)
- Arxula adeninivorans (5)
- Enzym (4)
- Hefe (4)
- Biochemische Analyse (3)
- Pneumokokken (3)
- Virulenz (3)
- Yeast (3)
- Bacillus subtilis (2)
- Genetik (2)
- Hantavirus (2)
- Harnsäure (2)
- Lebensmittel (2)
- Lipoproteine (2)
- Metabolismus (2)
- Pestivirus (2)
- Phospholipid biosynthesis (2)
- Phospholipidbiosynthese (2)
- Proteomanalyse (2)
- PspC (2)
- Purinderivate (2)
- RNS-Viren (2)
- Sin3 (2)
- Staphylococcus aureus (2)
- Stoffwechsel (2)
- TFIID (2)
- metabolism (2)
- 1-(S)-Phenylethanol (1)
- 1-Phenylethanol (1)
- 1-phenylethanol (1)
- Abwasser (1)
- Adenin-Deaminase (1)
- AdhA (1)
- AdhR (1)
- Adhäsine (1)
- Aktivatorprotein (1)
- Aktivität (1)
- Alcohol dehydrogenases (1)
- Alkoholdehydrogenasen (1)
- Altern (1)
- Aminosäurenstoffwechsel (1)
- Antibiotics (1)
- Aquaculture (1)
- Arginin (1)
- Arxula ade (1)
- Biochemie (1)
- Biotechnology (1)
- Blastobotrys raffinosifermentans (1)
- C. elegans (1)
- Caenorhabditis elegans (1)
- Candida albicans (1)
- Carboxylesterasen (1)
- Carboxypeptidase (1)
- ChIP (1)
- Chiral alcohols (1)
- Cholin (1)
- Choline (1)
- Coaktivator (1)
- Coenzym A (1)
- Corepressor (1)
- Deletionsmutante (1)
- Derepression (1)
- Deutschland (1)
- Diagnostic (1)
- Diagnostik (1)
- Diagonalassay (1)
- Diesterhydrolyse (1)
- Differentielle Genexpression (1)
- Domäne <Biochemie> (1)
- E(RNS) (1)
- E. coli (1)
- E0 (1)
- ELISA (1)
- ERNS (1)
- Enzymkinetik (1)
- Epidemiologie (1)
- Esterasen (1)
- Etrx (1)
- Europa (1)
- Europe (1)
- Fermentation (1)
- Fish immunology (1)
- Fish vaccination (1)
- Flaviviren (1)
- Flaviviridae (1)
- Funktionelle Polymere (1)
- GAPDH, ALDH (1)
- Gene regulation (1)
- Genregulation (1)
- Glomus intraradices (1)
- Glomus irregulare (1)
- Glutaminstoffwechsel (1)
- Glutamintransporter GlnQPH (1)
- Glykolyse (1)
- Guanin-Deaminase (1)
- Hansenula polymorpha (1)
- Hanta-Virus (1)
- Hausschwein (1)
- Heart Failure (1)
- Hefeartige Pilze (1)
- Hefezellen-Assay (1)
- Hepatitis E Virus (1)
- Herzinsuffizienz (1)
- Hormone (1)
- Host range (1)
- Hydrolyse (1)
- Identifikation (1)
- Impfstoff (1)
- In-vitro-Kultur (1)
- Infektion (1)
- Ino2 (1)
- Inositol (1)
- Intermediärstoffwechsel (1)
- Isotopolog Profiling (1)
- Kalziumsignale (1)
- Komplement (1)
- Komplementierung (1)
- Lactobacillus brevis (1)
- Langzeit-NMR (1)
- Leader Protease (1)
- Lipasen (1)
- Lymphozyt (1)
- MRSA (1)
- MSCRAMMS (1)
- MarR-type regulator (1)
- Mass spectrometry (1)
- Maul- und Klauenseuche Virus (1)
- Mausmodell (1)
- Mediator (1)
- Mediator complex (1)
- Membrane proteins (1)
- Mensch (1)
- Metabolische Deregulation (1)
- Mikrobiologie (1)
- Mitteleuropa (1)
- Monoester (1)
- MsrAB2 (1)
- Nucleocapsidprotein (1)
- Nukleokapsidprotein (1)
- Opi1 (1)
- Oxidativer Stress (1)
- Ozonung (1)
- PCV7 (1)
- Pantothenat Kinase (1)
- Pathogenität (1)
- PavB (1)
- Phospholipid-Biosynthese (1)
- Pili (1)
- Pilus Islet-1 (1)
- Pilus Islet-2 (1)
- Pneumokokkenoberflächenprotein C (1)
- Pneumonie (1)
- Polyhydroxybuttersäuren (1)
- Polyhydroxyfettsäuren (1)
- Prionprotein (1)
- Promotor <Genetik> (1)
- Proteinreparatur (1)
- Proteomics (1)
- PspC-hpIgR-vermittelten Endozytose (1)
- PsrP (1)
- Purinabbau (1)
- Ratte (1)
- Replikon (1)
- Repression <Genetik> (1)
- Repressoren (1)
- Rhodococcus ruber (1)
- SWI/SNF (1)
- Serologie (1)
- Seroprevalence (1)
- Seroprävalenz (1)
- Shotgun proteomics (1)
- Spacer (1)
- Spacergruppen (1)
- Spillover Infections (1)
- Spillover-Infektionen (1)
- Starch (1)
- Stärke (1)
- Synthese (1)
- TCC (1)
- TFIIA (1)
- Targeted proteomics (1)
- Thioredoxin (1)
- Thrombospondin (1)
- Transcriptomics (1)
- Transkriptionsaktivierung (1)
- Transkriptionsregulation (1)
- Transkriptomanalyse (1)
- Uratoxidase (1)
- VA-Mykorrhiza (1)
- Virologie (1)
- Virusdiarrhoe-Mucosal-Disease-Virus (1)
- Vitronektin (1)
- Volllängenklon (1)
- Wasserstoffperoxid (1)
- Wirtsspektrum (1)
- Xanthin Oxidoreduktase (1)
- Xplor2® Transformations-/Expressionssystem (1)
- Xplor®2 Transformations-/Expressionssystem (1)
- Xplor®2 transformation/expression system (1)
- YodB (1)
- YraA (1)
- YvaP (1)
- activator (1)
- acute pneumonia model (1)
- adenine deaminase (1)
- aging (1)
- akutes Pneumonie-Modell (1)
- aldehydes (1)
- arbuscular mycorrhizal fungi (1)
- arginine (1)
- bacterial lipocalin (1)
- bakterielle Lipocalin (1)
- cDNS (1)
- cell free conversion (1)
- complement (1)
- diagonal assay (1)
- dioxygenase (1)
- enantiomerenrein (1)
- enantiomerically pure (1)
- enzyme (1)
- fermentation (1)
- full lenght clone (1)
- fumarate reductase (1)
- genome size (1)
- glucokinase (1)
- glycolysis (1)
- goat polymorphisms (1)
- gp44/48 (1)
- hormonelle Aktivität (1)
- hydrogen peroxide (1)
- integrin (1)
- mouse model (1)
- oxidative stress (1)
- pathogenesis (1)
- phylogeny (1)
- platelets (1)
- pneumococcal surface protein C (1)
- pneumococcus (1)
- poly(hydroxyalkanoates) (1)
- protein repair (1)
- proteomics (1)
- purine degradation (1)
- quinone (1)
- receptor (1)
- recombinant Antigen (1)
- recombinant protein (1)
- rekombinantes Antigen (1)
- stress response (1)
- thiol stress (1)
- transcriptional regulation (1)
- virulence factors (1)
- virulenz (1)
- vitronectin (1)
- xanthine oxidoreductase (1)
Institute
- Institut für Mikrobiologie - Abteilung für Genetik & Biochemie (42) (remove)
Streptococcus pneumoniae (pneumococci) are lancet-shaped, Gram-positive, alpha-hemolytic, facultative anaerobic human specific commensals of the upper and lower respiratory tract. Pneumococci may convert to pathogenic bacteria and spread to the lungs and blood. In different population groups, such as children, the elderly and immunocompromised individuals, pneumococci can cause local infections such as bronchitis, rhinitis, acute sinusitis, and otitis media as well as life-threatening invasive diseases such as community-acquired pneumonia, sepsis and meningitis. Pneumococci are surrounded by a rigid and complex exoskeleton, the peptidoglycan, also referred to as murein sacculus. The peptidoglycan (PNG) protects the cells from rupture by osmotic pressure and maintains their characteristic shape. The PNG is a heteropolymer made up of glycan strands that are cross-linked by short peptides and during growth the existing murein is continuously hydrolyzed by specific lytic enzymes to enable the insertion of new peptidoglycan. Bacterial cell-wall hydrolases are essential for peptidoglycan turnover and crucial to preserve cell shape. The D,D-carboxypeptidase DacA and L,D-carboxypeptidase DacB of Streptococcus pneumoniae function in a sequential manner. This study determined the crystal structure of the surface-exposed lipoprotein DacB, which differs considerably from the DacA structure. DacB contains a Zn2+ ion in its catalytic center located in the middle of a fully exposed, large groove. Two different conformations with differently arranged active site topology were identified. In addition the critical residues for catalysis and substrate specificity were identified. Deficiency in DacA or DacB resulted in a modified peptidoglycan peptide composition and led to an altered cell shape of the dac-mutants. In contrast, lgt-mutant lacking lipoprotein diacylglyceryl transferase activity required for proper lipoprotein maturation retained L,D-carboxypeptidase activity and showed an intact murein sacculus. Furthermore, this study demonstrated the pathophysiological effects of disordered DacA or DacB activities. Real-time bioimaging of intranasally infected mice indicated a substantially attenuated virulence of dacB- and dacAdacB-mutants pneumococci, while loss of function of DacA had no significant effect. In addition, uptake of these mutants by professional phagocytes was enhanced, while their adherence to lung epithelial cells was decreased. The second part of this study focused on the functional and structure determination of the soluble dimeric pneumococcal lipoprotein PccL. Because of its calycin fold and structural homology with the lipocalin YxeF from Bacillus subtilis, PccL was introduced as the first member of the lipocalin protein family in pneumococci and named “PccL” (Pneumococcal calycin fold containing Lipoprotein). Similar to other lipocalins, the distinct beta-barrel, which is open at one end, is significantly conserved in PccL. Moreover, the application of the in vivo acute pneumonia mouse infection model and the in vitro phagocytosis as well as adherence invasion studies revealed considerable differences in colonization and invasive infection between the wild-type D39 and the pccL-mutant. In conclusion, this study characterized the crucial role of pneumococcal carboxypeptidases DacA and DacB for PGN architecture, bacterial shape and pathogenesis. By applying in vivo and in vitro approaches, a close relationship between PGN metabolism and pathophysiological effects was discovered. In addition, the high resolution structure of DacB has been solved and analyzed and a structure model with a resolution of 2.0 Å is provided. Furthermore, analysis of the PGN composition was applied to indicate the impact of an impaired lipoprotein biogenesis pathway on localization and activity of DacB. The major impact of carboxypeptidases on cell shape and virulence proposes DacB as a promising target for the development of novel drugs or due to its surface exposition also as a promising vaccine candidate. PccL is the first pneumococcal lipocalin-like protein and this study indicated its contribution to pneumococcal virulence. However, the mechanism and the mode of action of PccL are still unknown and have to be deciphered in further studies.
Microbial cell factories have been largely exploited for the controlled production of recombinant proteins, including industrial enzymes and biopharmaceuticals. The advent of high-throughput ‘-omics’ techniques have boosted the design of these production systems due to their valuable contribution to the field of systems metabolic engineering, a discipline integrating metabolic engineering with systems and synthetic biology. In order to thrive, the field of systems metabolic engineering needs absolute proteomics data to be generated, as proteins are the central players in the complex metabolic and adaptational networks. Due to advent of mass spectrometry-based proteomics, a substantial amount of absolute proteomic data became available in the past decade. However, membrane proteins remained inaccessible to these efforts.
Nonetheless, comparative studies targeting the membrane proteome have been quite successful in characterizing physiological processes. Hence, label-free proteomics was used in a study (Quesada-Ganuza et al, 2019 – Article I) to identify and optimize PrsA in Bacillus subtilis, for improved yield of amylase. Amylase is one of the most relevant enzymes in the biotechnological sector. By employing a label-free mass spectrometry approach targeting the membrane proteome of this bacterium, relative changes in heterologous and native levels of PrsA could be quantified. The results of this study evidenced that each PrsA shows different relative abundancies, but with no relevant impact in the yield of amylase.
Even though relative protein quantification can already provide a good visualization of the physiological changes occurring between different conditions, they are not sufficient to understand how resources are allocated in the cell under certain physiological conditions. Therefore, a global method for absolute membrane protein quantification remains the biggest requirement for systems metabolic engineering.
Hence, with this work, we successfully developed a mass spectrometry-based approach enabling the absolute quantification of membrane proteins (Antelo-Varela et al, 2019 – Article II). This study was also performed in the Gram-positive model organism Bacillus subtilis, regarded as a prolific microbial cell factory. The method developed in this work combines the comprehensiveness of shotgun proteomics with the sensitivity and accuracy of targeted mass spectrometry. Fundamental to the method is that it relies on the application of a correction and an enrichment factor to calibrate absolute membrane protein abundances derived from shotgun mass spectrometry. This has permitted, for the first time reported, the calculation of absolute membrane protein abundances in a living organism.
The newly developed approach enabled to accurately quantify ~40% of the predicted proteome of this bacterium, offering a clear visualization of the physiological rearrangements occurring upon the onset of osmotic stress. In addition, this work also provides evidence for new membrane protein stoichiometries.
Overall, this study enabled the development of a straightforward methodology long-needed in the scientific and biotechnological community and, for the first time reported, providing absolute abundances of one of the most puzzling fractions of the cell – the membrane proteome.
The next step of the work summarized here was to implement the afore described method to a biotechnological relevant strain, as absolute membrane protein abundances are essential to understand the fundamental principles of protein secretion and production stress. Hence, this work was applied in a genome-reduced B. subtilis strain, ‘midiBacillus’, expressing the major staphylococcal antigen IsaA (Antelo-Varela et al, submitted – Article III). The employed absolute membrane protein quantification methodology enabled the analysis of physiological rearrangements occurring upon the induction of heterologous protein production. This work showed that, even though IsaA was successfully secreted into the growth medium, one of the main requirements for the biotechnological sector, it was still partly accumulated in the cell membrane of this bacterium. This led to an exacerbated physiological response where membrane proteins involved in the management of secretion stress were activated. In addition, this study also showed that a rearrangement of the cell’s translocation machinery occurs upon induction of production, where a ‘game’ of in- and decrease of transporters takes place.
Anticipating the impact of genetic and environmental insults, such as the ones caused by production stress, is essential for the field of systems metabolic engineering. Thus, the highly accurate and comprehensive dataset generated during this work can be implemented in predictive mathematical models, thereby contributing in the rational design of next-generation secretion systems.
Transcriptional repression of regulated structural genes in eukaryotes often depends on pleiotropic corepressor complexes. A well-known corepressor conserved from yeast to mammalian systems is Sin3. In addition to Sin3, yeast Cyc8/Tup1 corepressor complex also regulates a diverse set of genes. Both corepressors can be recruited to target genes via interaction with specific DNA-binding proteins, leading to down-regulation of a large number of unrelated structural genes by associated histone deacetylases (HDACs). In vitro interaction studies performed in this work by GST pull-down assays showed that various repressor proteins (such as Whi5, Stb1, Gal80, Rfx1, Ure2, Rdr1, Xbp1, Yhp1, Rox1, Yox1, Dal80 and Mot3) are indeed able to bind pleiotropic corepressors Sin3 and/or Cyc8/Tup1. All repressors interacting with Sin3 contact its paired amphipathic helix domains PAH1 and/or PAH2. Mapping experiments allowed the characterization of minimum repressor domains and to derive a sequence pattern which may be important for repressor interaction with Cyc8 or Sin3. Interactions for some pathway-specific repressors such as Cti6 and Fkh1 have been studied comprehensively; minimal domains of Cti6 and Fkh1 required for interaction with Sin3 have been mapped and subsequently investigated by mutational analysis. In vitro interaction studies could show that amino acids 350-506 of Cti6 bind PAH2 of Sin3. To analyze this Cti6-Sin3 interaction domain (CSID) in more detail, selected amino acids within CSID were replaced by alanine. It turned out that hydrophobic amino acids V467, L481 and L491 L492 L493 are important for Cti6-Sin3 binding. The results of this work also suggest that repression is not executed entirely via Sin3, but rather CSID is also important for contacting pleiotropic corepressor Cyc8. In addition to PAH2 of Sin3, CSID also binds to tetratricopeptide repeats (TPR) of Cyc8. Furthermore, in vitro mapping studies revealed that Fkh1 also binds PAH2 of corepressor Sin3 via its N-terminal domain (aa 51-125). Binding studies with mutagenized Fkh1-Sin3 interaction domain (FSID) showed that Fkh151-125 variants L74A and I78A were unable to bind PAH2 of Sin3. Confirming in vitro studies, Cti6350-506 and Fkh151-125 also displayed in vivo interaction with PAH2 of Sin3 by using the “yeast two -hybrid” system. Chromatin immunoprecipitation (ChIP) analyses have demonstrated Cti6 recruitment to promoters of genes such as RNR3 and SMF3 containing iron responsive elements (IRE). Importantly, Sin3 was also recruited to these promoters but only in the presence of functional Cti6. Similarly, recruitment of Fkh1 and Sin3 to promoters of cell-cycle regulated genes CLB2 and SWI5 was shown. Recruitment of Sin3 was completely Fkh1-dependent. Additional findings of this work shed light on the fact that not only repressor proteins may contact Sin3 but also activator proteins not yet considered for interaction, e. g. specific activators such as Pho4 and Ino2. These findings indicate that Sin3 may fulfill functions beyond acting as a corepressor. In vitro studies on Sin3-Pho4 interaction showed that aa 156-208 of Pho4 are able to bind both PAH1 and PAH2 of Sin3, while an internal region of Ino2 comprising amino acids 119-212 binds to both Sin3 and Cyc8.
Streptococcus pneumoniae (pneumococci) are Gram-positive cocci and commensals of the human upper respiratory tract. Pneumococcal pathogenesis requires adherence to host cells and dissemination through cellular barriers and to evade host defense mechanisms. The Pneumococcal surface protein C (PspC) is an important virulence factor which has a crucial role in pneumococcal adhesion to host cells and immune evasion by manipulating the host complement system. To elucidate the pneumococcal adherence and uptake mechanism via factor H glycosaminoglycans (dermatan sulfate and heparin) were employed as competitive inhibitors in infection experiments with epithelial cells or human polymorphonuclear leukocytes (PMNs). Glycosaminoglycans significantly inhibited the FH mediated pneumococcal adherence and subsequent invasion to host epithelial cells. Furthermore, the short consensus repeats of FH which promotes the adhesion of pneumococci to host cells were identified by blocking experiments with domain mapped antibodies for specific regions of FH. Moreover, this study indicates that FH acts as adhesion molecule via cellular receptors recognized as integrin CR3 on human PMNs. Binding of Factor H loaded pneumococci to integrins CR3 was assessed by flow cytometry. Pneumococci coated with Factor H showed a significantly increased association with PMNs. This interaction was blocked by anti-CR3 antibodies and Pra1. This project further aims to study mechanisms of pneumococcal endocytosis by host cells, their intracellular fate, and the pathogen induced host cell signal transduction cascades including the calcium signaling upon pneumococcal infection of host cells via the PspC-hpIgR interaction. To assess now the role of protein tyrosine kinases (PTKs) during pneumococcal infection via PspC, cell culture infections were performed in presence of pharmacological inhibitors of PTKs and MAPKs or by employing genetic interference techniques. Blocking the function of Src or ER1/2 and JNK and genetic-knock down of Src and FAK reduced significantly internalization of pneumococci. These data indicated the importance of a coordinated signaling between Src PTKs, ERK1/2, and JNK during PspC-pIgR-mediated uptake of pneumococci by host epithelial cells. The impact of host cells intracellular calcium concentrations on pneumococcal PspC-hpIgR mediated internalization was studied. Intracellular calcium measurement of epithelial cells performed in the presence of pneumococci suggested a calcium influx in host epithelial cells and importantly this calcium influx was PspC- hpIgR specific as pspC-deficient pneumococci were unable to mediate calcium mobilization in host cells. The increase in intracellular calcium [Ca2+]i was dependent on phospholipase C as pretreatment of cells with a phospholipase C-specific inhibitor abolished the increase in [Ca2+]i. Furthermore, role of host intracellular calcium concentrations during pneumococcal internalization was demonstrated by employing specific pharmacological inhibitors and calcium chelators in epithelial cell culture infection assays. The results revealed that elevated host cells calcium concentrations diminished pneumococcal internalization while lower calcium concentration in host epithelial cells promoted pneumococcal uptake. This study further demonstrates that dynamin, clathrin and caveolin play a key role during pneumococcal endocytosis into host cells via PspC-hpIgR. The use of specific pharmacological inhibitors or genetic interference approaches against dynamin, clathrin and caveolin in epithelial cell culture infection assays significantly blocked pneumococcal uptake. Furthermore, confocal microscopy revealed that pneumococci co-localize with clathrin. At later stages of the infection the pathogen is sorted to early, late and recycling endosomes as indicated by co-localization of pneumococci with endosomal markers such as Rab5, Rab4, Rab 7, and Lamp1. In order to get further insights into PspC-hpIgR mediated uptake mechanisms, a chimeric PspC was constructed and expressed heterologously on the surface of Lactococcus lactis. Immunofluorescence staining, immunoblot and flow cytometric analysis of L. lactis confirmed the expression of PspC on the bacterial surface. Moreover the ability of recombinant lactococci expressing PspC to adhere to and to invade pIgR-expressing epithelial cells confirmed the functional activity of PspC when exposed on the lactococcal surface. PspC expressing lactococci confirmed the specificity of PspC-hpIgR mediated endocytosis in host epithelial cells as PspC deficient lactococci were not taken up by these host cells. Confocal microscopic analysis demonstrated that only PspC expressing lactococci were sorted to early, late and recycling endosomes, similar to the intracellular fate of S. pneumoniae.
Currently, plastic materials are an integral part of our lives, but their production mostly bases on fossil fuels or derivatives, which resources are decreasing. Extraction and processing of non-renewable resources have also negative impact on environment. One of the most promising and environmentally friendly approaches is use of microorganism. This PhD dissertation presents the non-conventional yeast Arxula adeninivorans as a host for production of bio-based and biodegradable poly(hydroxyalkanoates) plastics poly(hydroxybutyrate) and co-polymer poly(hydroxybutyrate-co-hydroxyvalerate). Additionally, the constructed yeast strain was able to secrete enantiomerically pure (R)-3-hydroxybutyric acid.
The production of PHAs requires three enzymes: β-ketothiolase, acetoacetyl-CoA reductase and PHA synthase. The strategy followed in this project was divided into two parts. While all three enzymes are responsible for intracellular production of PHA polymer, first two only lead to secretion of (R)-3-HB into culture media, which was used in a first stage of work to establish and optimize polymer production. Both, different bacterial strains and yeast A. adeninivorans were taken into account in screening of the genes encoding aforementioned enzymes. Bacterial genes were chemically synthesized using codon optimization pattern and endogenous genes were obtained using PCR and genomic DNA template from A. adeninivorans LS3 wild-type strain. Each gene was cloned into Xplor2 vector between TEF1 constitutive promoter and PHO5 terminator. Vector containing both thiolase and reductase genes was used for A. adeninivorans transformation.
The best combination of heterologous genes was overexpression of β-ketothiolase gene from Clostridium acetobutylicum and acetoacetyl-CoA reductase gene from Cupriavidus necator which led to secretion of 4.84 g L−1 (R)-3-HB, at a rate of 0.023 g L−1 h−1 over 214 h in shaking flask cultivation. Further optimization by fed-batch culturing with glucose as a carbon source did not improve (R)-3-HB secretion, but the rate of production was doubled to 0.043 g L−1 h−1 [3.78 g L−1 of (R)-3-HB at 89 h].
The product of acetoacetyl-CoA reductase is (R)-3-HB-CoA and further removing of CoA moiety is needed for acid secretion into culture media. A. adeninivorans is able to conduct this process without any additional modification but the conversion rate is unknown. Two thioesterases, cytosolic TesBp encoded by TesB gene from E. coli and mitochondrial ATes1p encoded by ATES1 gene from A. adeninivorans, were analysed to enhance secretion process. Additionally, a cytosolic version of ATES1 gene (ATES1cyt) was tested. All three genes were expressed in A. adeninivorans cells under TEF1 constitutive promoter together with thiolase and reductase genes. Despite detected enzymatic activity the yield of (R)-3-HB synthesis and secretion was not increased. Moreover, overexpressed thioesterases negatively influenced cell growth, indicating that they act on other metabolic components. The results provided two sets of information, first, the endogenous secretion system is sufficient for (R)-3-HB production; second, further screening of suitable genes needs to be performed.
Based on optimization of (R)-3-HB synthesis, thiolase gene (thl) from C. acetobutylicum and reductase gene (phaB) from C. necator were chosen to combine with PHA synthase gene (phaC) for creating the PHB-V producing strain. The PHA synthase expression module, containing TEF1 promoter and PHO5 terminator, was cloned into Xplor2 vector together with thiolase and reductase expression modules and used for A. adeninivorans transformation. The engineered strain accumulated up to 7.47% PHB of dcw. During the set of cells passaging A. adeninivorans lost the ability to accumulate polymer with maximal 23.1 % of primary accumulation level. Additionally, use of a vector including hygromycin B antibiotic resistance marker (instead of auxotrophic marker in Xplor2) did not improve polymer accumulation and stability.
To counteract the effect of loss of accumulation stability, phasin gene (phaP1), originated from C. necator, was introduce together with PHA pathway genes. First screening cultivations resulted in stabilizing of polymer production reaching 9.58 % PHB of dcw and only 12.0 % loss of production ability. Further experiments increased PHB content with 19.9% PHB of dcw (3.85 g L-1) after 180 h of cultivation using rich medium. Use of another thiolase gene, the second thiolase from C. necator (bktB), which theoretically should induce production of PHBV copolymer, led to accumulation only 11.4% PHB of dcw after 139 h and no PHV fraction was detected.
Variation of the ratio between flask volume and amount of media influences the level of aeration. Importantly, decrease of aeration level significantly increased polymer synthesis. Additionally, PHB-V copolymer accumulation has been induced by use of different carbon source co-substrates. Use of rich media supplemented with ethanol allow the strain with thl thiolase to accumulate up to 42.9 % PHB of dcw without PHV fraction and with bktB thiolase to 30.5 % PHB of dcw. Nevertheless, despite of lower total amount of polymer, supplementation with 1-propanol allow both strains to accumulate PHB-V copolymer with 7.30 %mol and 22.5 %mol of PHV for thl and bktB strains, respectively.
Optimization based on genetic engineering further enhanced polymer production yield led to exceeding of 50 % PHB-V of dcw. For doubling the gene dosage, PHA synthesizing strains of A. adeninivorans were again transformed with Xplor2 vector containing PHA pathway genes. Resulting strains exhibited twice the level of enzymatic activities of thiolase and reductase compared with strains transformed once with expression vector. In a shaking flask experiment the strain transformed twice with vector containing bktB thiolase reached after 240 h 52.1% PHB-V of dcw (10.8 g L-1) with 12.3 %mol of PHV fraction which is the highest level found in yeast. As another genetic approach, a fusion strain has been created. Two different strains have been established and merged using protoplast fusion technique. Doubling of genetic material resulted in similar level of copolymer produced by Arxula as in former experiments (50.2% of dcw, 10.7 g L-1).
Culture conditions were optimized in controllable cultivation using fed-batch mode. Although optimal oxygen and pH level and continuous carbon source and nitrogen feeding were maintained, final polymer level in % of dry mass was around three times lower than for shaking flask experiment. Nevertheless, efficient growth of Arxula in fed-batch mode led to increase of total copolymer level in g L-1 (16.5 g L-1 compare to 10.8 g L-1 for shaking flasks) showing the feasibility of using Arxula strain for up-scaling production of copolymer.
Acetyl-CoA is a main precursor in synthesis of PHB-V copolymer and change of its pool was investigated. ATP citrate lyase is a cytosolic enzyme converting citrate into oxaloacetate and acetyl-CoA, supporting the biosynthesis of fatty acids. Two genes encoding Acl subunits from Aspergillus nidulans (AnAcl1 and AnAcl2) were again cloned into Xplor2 vector and transformed into A. adeninivorans PHA producing strain. Despite of higher enzymatic activity of AnAclp, accumulation of polymer was around three times higher for control without expression of lyase genes. Expectedly, the strain expressing AnAcl1/2 genes accumulated larger amount of each stearic, palmitic and oleic acid in both standard and fatty acid inducing conditions (lower nitrogen level). Thus, overexpression of AnAcl1/2 genes in A. adeninevorans cells may improve biosynthesis of fatty acids but is ineffective for PHB polymer accumulation.
The aim of the project was use of starch-based media, manufactured as by-products, for polymer production. Genetically engineered Arxula strains were cultivated using these media instead of glucose-based media. Although yeast cells were both able to secrete (R)-3-HB and to accumulate PHB, the yield was lower than for previous media. Additionally, only trace of PHV was found at the end of cultivation time when 1-propanol was supplemented. Obtained results showed that use of cheaper media is a promising approach to decrease production costs but further optimization needs to be performed especially for extended scale of production.
Determination of produced copolymer has been done based on microscopic analysis and studies of physical and chemical properties. Results revealed that Arxula accumulated PHA polymer in cytosolic granules with a similar size range compared to the ones produced by bacteria. The physicochemical study showed that produced polymer exhibited slightly different properties in comparison to bacterial polymer with similar content of PHV, i.e. very-low molecular mass, higher melting and glass transition temperature.
All above results showed that A. adeninivorans is a promising host for PHB-V production. Expression of phasin greatly increased production and stability of polymer, which led to an accumulation level never found before in yeast. Further optimization in higher production scale using cheap starch-based media may establish Arxula strain as a valuable tool for industrial production of PHB-V copolymer.
Streptococcus pneumoniae (pneumococci) and Staphylococcus aureus (S. aureus) are human-specific commensals of the upper respiratory tract. Every individual is asymptomatically colonized with both bacteria at least once in their life-time. The opportunistic pathogens can affect further organs and invade into deeper tissue. The occupation of normally sterile niches of the human body with the bacteria can lead to local infections such as sinusitis, otitis media and abscesses, or to life-threatening diseases like pneumonia, meningitis or sepsis. A strong interaction between the bacterium and the respiratory epithelial cells is a prerequisite for a successful colonization. This interaction is ensured by bacterial surface proteins, so called adhesins. The binding of the adhesins to the epithelial lineage occurs predominantly indirectly via components of the extracellular matrix (ECM), but also directly to cellular receptors. Pneumococci and S. aureus bind to various ECM glycoproteins, amongst others: fibronectin, fibrinogen, vitronectin, and collagen. Also binding of both pathogens to human thrombospondin-1 has been described. Thrombospondin-1 is mainly stored in the α-granula of thrombocytes (platelets) and released into the circulation upon activation. However, thrombospondin-1 is also produced and secreted by other cell types like endothelial cells, macrophages, and fibroblasts, which gets subsequently incorporated as component into the ECM. So far, no thrombosponin-1-binding adhesins of pneumococci were identified. PspC, Hic, and PavB are important surface-localized virulence factors, which were shown to interact with human ECM and plasma proteins. PspC and Hic bind to vitronectin and factor H, which inhibits the complement cascade of the human immune system. PavB interacts with fibronectin and plasminogen, and a pavB-deficient mutant of S. pneumoniae showed diminished capacity in colonization in a mouse model. Among the surface proteins of S. aureus, only Eap was identified as thrombospondin-1-binding adhesin. Beyond colonization, pneumococci and S. aureus can enter the blood circulation, interact with platelets, and cause their activation. The aggregation of platelets, especially initiated by S. aureus, plays an important role in the clinic, because most of the septic patients develop thrombocytopenia. Surface localized factors of
S. pneumoniae triggering platelet activation are unknown to date. In contrast, few proteins of S. aureus with potential to activate platelets, including Eap, were identified previously.
This study identified the surface proteins PavB, PspC, and Hic of S. pneumoniae as specific ligands of the human thrombospondin-1. Flow cytometric, surface plasmon resonance spectroscopic and immunological analyses revealed interactions between the pneumococcal proteins and soluble as well as immobilized thrombospondin-1. The use of specific pneumococcal deletion mutants verified the importance of the three virulence factors as binding partners of soluble thrombospondin-1. The results suggest that pneumococci are capable of acquiring soluble thrombospondin-1 from blood as well as utilizing immobilized glycoprotein of the ECM as substrate for adhesion. Furthermore, the thrombospondin-1-binding domain within the pneumococcal proteins was analyzed by use of recombinant fragments of PavB, PspC, and Hic. The binding capacity of thrombospondin-1 increased proportionally with the amount of repetitive sequences in PavB and PspC, and the length of the α-helical region within the Hic molecule. The binding behavior of thrombospondin-1 towards PavB and PspC is comparable with that of the ECM proteins vitronectin and fibronectin, but is unique towards Hic.
The localization of the binding domain of the adhesins within the thrompospondin-1 molecule occurred via use of glycosaminoglycans as competitive inhibitors for the interaction. The results suggest that the pneumococcal proteins Hic and PspC target the identical binding region within thrombospondin-1, which differs from the binding domain for PavB. However, all three virulence factors seem to bind in the N-terminal part of thrombospondin-1.
Two-dimensional gel electrophoresis, thrombospondin-1 overlay assay and subsequent mass spectrometric analysis identified AtlA of S. aureus as a surface localized interaction partner of human thrombospondin-1. Moreover, a vitronectin binding activity for AtlA was determined. Immunological and surface plasmon resonance binding studies with recombinant AtlA fragments revealed that interactions with both matrix proteins is mediated via the C-terminal located repeats R1R2 of the AtlA amidase domain. Binding of thrombospondin-1 and vitronectin occurred not simultaneously, due to a competitive inhibition.
The second part of the study focused on the activation of human platelets by recombinant pneumococcal and staphylococcal proteins. In total, 28 proteins of S. pneumoniae and 52 proteins of S. aureus were incubated with human platelets. The activation of the cells was detected by flow cytometry using the activation markers P-selectin and the dimerization of the integrin αIIbβIII. The proteins CbpL, PsaA, PavA, and SP_0899 of S. pneumoniae induced platelet activation, however, the detailed mechanism has to be deciphered in further studies. Furthermore, the secreted proteins CHIPS, FLIPr, and AtlA of S. aureus were discovered as inductors for the activation of platelets. In addition, the domains of AtlA and Eap, crucial for platelet activation, were narrowed down. Interestingly, CHIPS, FLIPr, and Eap were described as inhibitors of neutrophil recruitment. Platelets are recently recognized as immune cells, due to the expression of immune receptors. The data obtained in this study highlight a comprehensive spectrum of effects of the S. aureus proteins towards different type of immune cells. Besides the activation of platelets in suspension buffer and plasma, the aggregation of platelets in whole blood was triggered by the proteins CHIPS, AtlA, and Eap. These results suggest a contribution of the proteins during the S. aureus-induced infectious endocarditis. Secretion of the platelet activating virulence factors, which were identified within this study, might represent a pathogenic strategy during S. aureus infection in which a direct contact between S. aureus and platelets is not required or even avoided.
In conclusion, PavB, PspC, and Hic of S. pneumoniae and AtlA of S. aureus were identified as interaction partners of human thrombospondin-1. Furthermore, CHIPS, FLIPr, AtlA, and Eap were characterized as platelet activators. This study provides candidates for the development of protein-based vaccines, to prevent bacterial colonization and to neutralize secreted pathogenic factors.
Escherichia coli has been commonly used as a platform for recombinant protein production and accounts for approximately 30% of current biopharmaceuticals on the market. Nowadays, many recombinant proteins require post-translational modifications which E. coli normally cannot facilitate. Therefore, novel technological advancements are unceasingly being developed to improve the E. coli expression system. In this work, some of the most recently engineered platforms for the production of disulfide bond-containing proteins were used to study the E. coli proteome under heterologous protein production stress. The effects of protein secretion via the Sec and Tat translocation pathways were examined using a comparative LC-MS/MS analysis. The E. coli proteome responds to foreign protein production by activation of several overlapping stress responses with a high degree of interaction. In consequence, a number of important cellular processes such as cellular metabolism, protein transport, redox state of the cytoplasm and membrane structure are altered by the production stress. These changes lead to the reduction of cellular growth and recombinant product yields. Resolving the identified bottlenecks will increase the efficiency of recombinant protein expression processes in E. coli.
Background: Hepatitis E virus (HEV) is the etiological agent of an acute self-limiting hepatitis in humans worldwide. The main route of infection is by ingestion of food or water contaminated with the virus. In Germany, several hundred human cases are reported each year, while preliminary studies suggest a high infestation rate of herds of domestic pig (Sus scrofa domesticus) and sounders of wild boar (Sus scrofa). Autochthonous cases are originating mainly from zoonotic transmission from domestic pig and wild boar, but other animals may also be involved. Recently, a novel strain of HEV (ratHEV) had been found in Norway rats (Rattus norvegicus) in Germany, that could contribute to human epidemiology. Therefore, the aim of this study was to assess the seroprevalence of both HEV and the novel ratHEV in human, domestic pig and rat. For each of the three mammal species, an indirect immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA) was established, that based on an Escherichia coli-expressed carboxy-terminal segment (GT3-Ctr, amino acid (aa) 326–608) of the capsid protein of the autochthonous genotype 3 (GT3), derived from a wild boar from Germany. In parallel, a segment from ratHEV homologous to GT3-Ctr was also expressed in E. coli (ratHEV-Ctr, aa315–599) and was used in the ELISA. Hence, the established tests detect antibodies directed against HEV GT3 when using GT3-Ctr as antigen and ratHEV when using ratHEV-Ctr. Results: The GT3-based in-house human IgG test was validated using a commercial assay and showed high specificity and sensitivity. The average human population (represented by a panel of blood donors from Berlin and Brandenburg) reached a seroprevalence of 12.3% (37/301) with the in-house ELISA. A panel of forestry workers from Brandenburg had an even higher seroprevalence of 21.4% (119/555). Furthermore, ratHEV-specific antibodies could be detected in several sera of forestry workers. The novel ratHEV-based rat IgG ELISA could not be compared to similar tests, however, parallel testing with GT3-Ctr and statistical inference allowed conclusion of a seroprevalence. Rats trapped from several sites in Germany had an overall seroprevalence of 24.5% (36/147). The sera were reactive exclusively with ratHEV-Ctr. As with the in-house ELISA for human sera, the porcine IgG test was validated using a commercial assay, yielding high specificity and sensitivity. A panel of domestic pigs from ten federal states of Germany showed a seroprevalence of 42.7% (383/898) when tested with the in-house ELISA. Reactivity with ratHEV was present, but seemed to be caused mostly by cross-reactivity to GT3-Ctr. Conclusion: The HEV seroprevalence observed for human sera of the average population of Germany is among the highest in Europe and has been confirmed recently by other authors. The high seroprevalence found in forestry workers suggests that they should be counted as a risk group for HEV infection. Populations of rats have been shown to be infested heavily with ratHEV, as rats from all trapping sites situated within cities had a high prevalence for ratHEV exclusively and no serum reacted exclusively with GT3-Ctr. Seroprevalence in domestic pigs was demonstrated to be distributed evenly across federal states and districts. However, a vast difference of infestation could be detected in different herds, suggesting either differences in husbandry conditions, or an external source of infection that acts locally only. The rare but exclusive reactivity of human sera with ratHEV as well as the high cross-reactivity of swine sera with ratHEV suggests that viral strains other than the ones already known may contribute to cases of hepatitis E.
Streptococcus pneumoniae, more commonly known as the pneumococcus, is a Gram-positive bacterium colonizing the human upper respiratory tract as a commensal. However, these apparently harmless bacteria have also a high virulence potential and are known as the etiologic agent of respiratory and life-threatening invasive diseases. Dissemination of pneumococci from the nasopharynx into the lungs or bloodstream leads to community-acquired pneumonia, septicaemia and meningitis. Pneumococcal diseases are treated with antibiotics and prevented with polysaccharide-based vaccines. However, due to the increase of antibiotic resistance and limitations of the current vaccines, the burden of diseases remains high. Interactions of pneumococci with soluble host proteins or cellular receptors are crucial for adherence, colonization, transmigration of host barriers and immune evasion. The pneumococcal surface-exposed proteins are the main players involved in this host-pathogen interaction. Therefore, combating pneumococcal transmission and infections has emphasized the need for a new generation of immunogenic and highly protective pneumococcal vaccines, based on surface-exposed adhesins virtually expressed by all pneumococcal strains and serotypes. The genomic analysis of S. pneumoniae strains helped to identify pneumococcal virulence factors such as pili, PsrP and PavB, which have been demonstrated to interact with human proteins playing an important role during the pathogenic process of pneumococci, and are currently considered as new potential vaccine candidates against S. pneumoniae. A subclass of pneumococcal strains produces pili that are encoded by the pathogenicity islet pilus islet-1 (rlrA islet) and/or the pilus islet-2. Both types of pili are implicated in bacterial adherence to host cells. A further pathogenicity islet encoded protein is PsrP. The presence of the psrP-secY2A2 islet correlated positively with the ability of pneumococci to cause invasive pneumococcal diseases. Recent studies indicated that PsrP is a protective adhesin interacting with keratin 10 on lung epithelial cells. In this study, the genomic loci of the pneumococcal virulence factors pili, PsrP and PavB were molecularly analyzed and used as molecular markers for molecular epidemiology studies of S. pneumoniae. The genotyping results obtained here showed the impact of the PCV7 immunization of children, started in July 2006, on the distribution of these pneumococcal virulence factors among clinical isolates in Germany. These findings gave more insights into the role of pili, PsrP and PavB in pneumococcal pathogenesis and may strongly support the idea of including these pneumococcal constituents in a broad coverage protein-based vaccine against pneumococcal infections produced by invasive serotypes in the future. The mature PavB protein contains a variable number of repetitive sequences referred to as the Streptococcal Surface Repeats (SSURE). PavB has been demonstrated to interact with fibronectin and plasminogen in a dose-dependent manner and it was identified as a surface-exposed adhesin with immunogenic properties, which contributes to pneumococcal colonization and respiratory airways infections. The complete molecular analysis performed here for PavB, allowed to know more accurately its structure and to estimate the real number of SSURE units in different pneumococcal strains. With these findings, a new primary sequence-based structural model was constructed for the PavB protein and its SSURE domain, and, at least for TIGR4, the complete pavB gene and PavB protein sequences with five SSURE units was reported in the GenBank database of the NCBI website. Due to its immediate neighborhood on the pneumococcal genome with the tcs08 genes, PavB is likely linked with this pneumococcal TCS. Here, a significant reduction of the PavB protein expression was observed in delta-tcs08-mutant strains, which may strongly suggest that the TCS08 does play a role in pneumococcal virulence and metabolisme, as further observed in growth behaviour experiments carried out with the TCS08-deficient mutants, cultured in chemically defined medium. Despite several studies suggest that the molecular mechanism underlying the bacterial signal transduction is very sophisticated, the majority of reports in prokaryotic TCS, including those for S. pneumoniae, are still focused in single cognate pairs. The pneumococcal genome encodes 14 TCSs and an orphan response regulator. It is obvious that TCS pathways are often arranged into complex circuits with extensive cross-regulation at a variety of levels, thereby endowing cells with the ability to perform sophisticated information processing tasks. This study established also the experimental and molecular bases for the construction of a comprehensive genome-wide interaction map of the complex TCS pathways for its application in the gene regulation of pneumococcal virulence factors.
Natürliche Hormone und Substanzen mit einer hormonellen Wirkung werden als organischen Spurenstoffen oder Mikroschadstoffe bezeichnet und werden über verschiedene Quellen in die Umwelt eingetragen. Dies führt insbesondere bei aquatischen Lebewesen zu Veränderungen im endokrinen System. Um die Belastung der Gewässer mit hormonell aktiven Substanzen zu verringern und einen guten chemischen und ökologischen Status nach europäischer Wasserrahmenrichtlinie zu erreichen, wird eine Reduktion des Eintrags hormonell aktiver Substanzen angestrebt. Die meisten Abwässer werden in Kläranlagen gesammelt, die somit Punktquellen für den Eintrag von hormonell aktiven Substanzen in die Umwelt darstellen. Zur Untersuchung neuer Methoden zur Abwasserreinigung sind zuverlässige und sensitive analytische Messtechniken notwendig. Da aktuelle instrumentelle Messmethoden nicht in der Lage sind hormonell aktive Substanzen im wirkungsrelevanten Konzentrationsbereich zu messen, wurden Hefezellenassays zur Detektion der östrogenen (A-YES) und androgenen (A-YAS) Aktivitäten für eine Anwendung in Oberflächengewässern und Abwässern evaluiert. Im Anschluss wurden diese Assays zur Beurteilung und Optimierung der Eliminationsleistung einer großtechnischen Ozonung auf einer kommunalen und einer Krankenhaus Kläranlage eingesetzt. Die untersuchten Abwassermatrices zeigten keine Effekte auf die Enzym Substrat Reaktion und die optische Dichte der A-YES Hefezellensuspension. Proben eines Oberflächengewässers sowie von Kläranlagen Zuläufen verursachten im A-YAS eine erhöhte optische Dichte der Zellsuspension im Vergleich zur Referenz. Eine verringerte optische Dichte der A-YAS Hefezellsuspension konnte in Extrakten von Zulaufproben bestimmt werden. Durch die Dotierung unterschiedlicher Konzentrationen der Referenzsubstanzen zu Oberflächengewässer- und Abwasserproben konnten Dosis Wirkungskurven mittels A-YES und A-YAS Assays abgebildet werden. Dabei konnte gezeigt werden, dass insbesondere in Kläranlagen-Zulaufproben sowohl eine östrogene als auch eine androgene Aktivität bereits in der undotierten Ausgangsprobe vorhanden war. Des Weiteren konnten inhibierende Effekte in den Proben detektiert werden, die auf antagonistische Substanzen hindeuten. Die Analyse von Kläranlagen Abläufen zeigte östrogene Aktivitäten zwischen 0,035 und 5,5 ng EEQ/L sowie androgene Aktivitäten zwischen < 0,31 und 6,1 ng DHTEQ/L. Während der großtechnischen Ozonung konnte die östrogene Aktivität in einer kommunalen sowie einer Krankenhaus Kläranlage um bis zu 97% bzw. 83% reduziert werden. Die Reduktion der androgenen Aktivität lag bei 80% und 66%. Für zwei Verfahren zur bedarfsabhängigen Steuerung der Ozonung basierend auf der östrogenen Aktivität und auf dem DOC Gehalt konnte die Machbarkeit gezeigt werden. Allerdings stellten sich beide Methoden zum jetzigen Zeitpunkt als nicht wirtschaftlich heraus. Antagonistische Aktivitäten konnten in einem Konzentrationsbereich von 330 - 2.700 µg OHTEQ/L (anti-östrogene Aktivität) und 550 - 730 µg FEQ/L (anti-androgene Aktivität) mittels anti A-YES und anti A-YAS detektiert werden. Während der einzelnen Reinigungsstufen konnte keine Reduktion der antagonistischen Aktivitäten nachgewiesen werden. Sowohl A-YES als auch A-YAS sind für die Analyse von Abwasserproben geeignet und ermöglichen so erstmals die Beurteilung von Verfahren zur Abwasserreinigung im wirkungsrelevanten Konzentrationsbereich.
Die Hefe Saccharomyces cerevisiae reagiert auf die sich ständig ändernden Umweltbedingungen durch eine präzise Regulation der Genexpression. Möglich wird dies durch ein komplexes Netzwerk aus spezifischen Regulatoren und pleiotropen Faktoren. Aktivatorproteine binden an Aktivierungssequenzen (UAS-Elemente) in ihren Zielpromotoren und rekrutieren basale Transkriptionsfaktoren sowie Coaktiva¬toren. Dadurch erhöhen sich Wahrscheinlichkeit und Häufigkeit der Transkriptions¬initiation und die DNA im Promotorbereich wird durch die Aktivität von Komplexen der Chromatinremodellierung und -modifizierung für die Transkriptionsmaschinerie zugänglich gemacht. Dagegen binden spezifische Repressor¬proteine an ihre Regula¬tionssequenzen (URS-Elemente) oder an Aktivatorproteine, inhibieren deren Wirkung oder rekrutieren Histondeacetylase-Komplexe wie den Sin3-Corepressor, die eine Verdichtung des Chromatins bewirken. Der Sin3-Corepressorkomplex ist an einer Vielzahl von Regulationsprozessen beteiligt. In Hefe existieren zwei Sin3-Varianten, die als Rpd3L bzw. Rpd3S bezeichnet werden und sich in ihrer Zusammensetzung unterscheiden. Neben Sin3 als zentralem Gerüst¬protein in beiden Komplexen sind im Rpd3L strukturelle Untereinheiten wie Sds3, Sap30 und Pho23 sowie die Histondeacetylase (HDAC) Rpd3 als enzymatische Komponenten enthal¬ten. Durch Funktionsanalysen von Mutanten einzelner Unterein¬heiten wurde festge¬stellt, dass zusätzlich zu Rpd3 weitere HDACs an der Repression ICRE-abhängiger Gene der Phospholipid-biosynthese Gene beteiligt sind. Interaktionsstudien zeigten, dass auch die HDACs Hda1 und Hos1 an Sin3 binden. Die Bindung erfolgt über drei sogenannte HDAC-Interaktionsdomänen (HID1-3), wobei Hda1 und Hos1 an HID2 und HID3 binden, Rpd3 dagegen an HID1 und HID3. In dieser Arbeit konnte gezeigt werden, dass die HDACs direkt an ihre jeweiligen HIDs binden. Außerdem inter¬agieren Hda1 und Hos1 auch in vivo mit Sin3. Die HID1 wurde auf die Aminosäuren 801-950 verkürzt und es wurde nachge¬wiesen, dass eine funktionsfähige katalyti¬sche Domäne von Rpd3 nicht für die Wechselwirkung mit Sin3 notwendig ist. Außerdem wurden die Interaktionsdomänen von Sds3 und Sin3 kartiert. Die erhaltenen Befunde ergänzen die Daten zu Protein-Protein-Inter¬aktionen im Sin3-Corepressorkomplex und komplettieren funktionelle Aspekte der HDAC-Rekrutierung. Eine weitere Zielstellung dieser Arbeit war die Erstellung eines Interaktionsnetzwerks zwischen spezifischen Aktivatoren und allgemeinen Faktoren der Transkription. Eukaryotische Aktivatorproteine sind modular aufgebaut und besitzen voneinander separierbare Funktionsdomänen. Die Erkennung und Bindung von UAS-Elementen in den Zielpromotoren erfolgt über die DNA-Bindedomäne (DBD), während Tran¬skriptions¬¬aktivierungs¬domänen (TADs) basale Transkriptionsfaktoren und Co¬aktiva¬toren rekrutieren und somit die aktivierende Wirkung vermitteln. Im Gegensatz zu den DBDs folgen TADs meist keinen durch Sequenzanalysen vorhersagbaren Strukturmotiven und müssen manuell eingegrenzt werden. Für die Kartierung funktioneller TADs wurden Längenvarianten von über 30 Aktiva-toren aus verschiedenen Familien DNA-bindender Proteine an die Gal4DBD fusioniert und auf ihre Fähigkeit überprüft, ein UASGAL-abhängiges Reportergen zu aktivieren. Dabei konnten 15 neue TADs eingegrenzt werden. Weiterhin wurden die bisher nicht charakterisierten Zinkcluster¬proteine Yjl206c, Yer184c, Yll054c und Ylr278c als Aktivatoren bestätigt. Dadurch stand eine Samm¬lung aus 20 bekannten und neukartierten TADs zur Verfügung, die nach Konstruktion von GST-Fusionen für in vitro-Interaktionsexperimente mit Unter¬einheiten des Mediators, des TFIID- und des SWI/SNF-Komplexes eingesetzt wurden. Es konnten direkte Wechselwirkungen von Aktivatoren (u. a. Aft2, Aro80, Mac1 und Zap1) mit den TFIID-Komponenten TBP, Taf1, Taf4 und Taf5 detektiert werden. Die Bindung an Taf1 erfolgte im Bereich von aa 1-250, der zwei Aktivator¬interaktions-domänen (AID) enthält und in vorangegangenen Experimenten auch mit Ino2 und Adr1 interagierte. Die Rap1-Bindedomäne (RBD) von Taf4 (aa 253-344) interagierte auch mit Mac1, Aft2 und Ino2. Daher wurde dieser Bereich als allgemeine AID klassifiziert. Für die Aktivatorinteraktion essentielle Aminosäuren konnten allerdings nicht identi¬fiziert werden. 17 von 20 TADs interagierten direkt mit der Mediator-Untereinheit Med15, während für Med17 10 Kontakte zu Aktivatoren detektiert wurden, was die Relevanz des Mediators für die Aktivatorfunktion unterstreicht. Die katalytische Untereinheit des SWI/SNF-Komplexes Swi2 zeigte ähnlich viele TAD-Interaktionen wie Med15. Der N-terminale Bereich von Swi2 (aa 1-450) stellte sich als ausreichend für die Bindung der Aktivatoren heraus und enthält demnach eine oder mehrere AIDs. Damit konnte das Interaktionsnetzwerk zwischen Aktivatoren und allgemeinen transkriptionalen Cofaktoren substantiell erweitert werden.
In der Hefe S. cerevisiae erfolgt die Transkriptionsregulation der Strukturgene der Phospholipid-Biosynthese in Abhängigkeit der intrazellulären Konzentration der beiden Phospholipid¬vorstufen Inositol und Cholin (IC). Bei IC-Mangel kommt es zu einer Akkumulation des Signalmoleküls Phosphatidsäure, wodurch der Repressor Opi1 extranukleär am endoplasmatischen Retikulum (ER) verankert wird. Dadurch kann der heterodimere Aktivator Ino2/Ino4 an eine spezifische „upstream activation site” (UAS) in der Promotorregion, die als ICRE-Motiv („inositol/choline-responsive element“) bezeichnet wird, binden und die Initiation der Transkription vermitteln. Die aktivierende Wirkung geht dabei von zwei Transkriptions¬aktivierungsdomänen (TAD) im N-Terminus von Ino2 aus. Da bisher unbekannt war, wie die Ino2-vermittelte Genaktivierung erfolgt, bestand das Ziel dieser Arbeit in der Identifizierung der Coaktivatoren, die direkt an die TADs von Ino2 binden. Ferner sollten die für die Transkriptionsaktivierung wichtigen Wechselwirkungen innerhalb der Coaktivatoren präzise kartiert werden. Es konnte hier mit Hilfe der affinitätschromatographischen Methode des GST-„Pulldown“ gezeigt werden, dass TAD1 und TAD2 von Ino2 mit den generellen Transkriptionsfaktoren TFIID und TFIIA interagieren. Innerhalb des TFIID wurden die Untereinheiten Taf1, Taf4, Taf6, Taf10 und Taf12 in vitro als direkte Ino2-Interaktionspartner identifiziert. Dabei binden alle identifizierten Taf-Proteine an die starke TAD1, Taf10 zusätzlich an die TAD2. Frühere Untersuchungen hatten gezeigt, dass Mutationen innerhalb der TAD1 von Ino2 (D20K, F21R) zu einem vollständigen Verlust der Aktivierungsleistung führen. In dieser Arbeit wurde nachgewiesen, dass die gerichtete Mutation dieser Aminosäuren zu einem vollständigen Interaktionsverlust mit den Taf-Proteinen führt. Mit Hilfe von Interaktionsexperimenten wurden innerhalb von Taf1 zwei distinkte Aktivatorinteraktionsdomänen (AID1: AS 1-100; AID2: AS 182-250) kartiert, die die Bindung an Ino2 vermitteln. Mutationen hydrophober und basischer Aminosäure-Reste innerhalb der Taf1-AID2 hatten einen vollständigen Verlust der Interaktion mit Ino2 zur Folge. Möglicherweise sind also ionische und hydrophobe Wechselwirkungen an der Interaktion von Ino2 und Taf1 beteiligt. Mit Hilfe der Chromatin-Immunopräzipitation (ChIP) erfolgte der Nachweis, dass Taf1 in Abhängigkeit von Ino2 auch in vivo an den ICRE-haltigen Promotoren INO1 und CHO2 vorhanden ist. Im Folgenden wurden auch die Ino2-Interaktionsbereiche innerhalb der Proteine Taf6, Taf10 und Taf12 durch die Generierung sukzessiver GST-Verkürzungen eingegrenzt. Taf10 und Taf12 besitzen wie Taf1 zwei separate AIDs (Taf10: AID1 AS 1-100; AID2 AS 131-176; Taf12: AID1 AS 50-100; AID2 AS 100-178). Untersuchungen mit mutagenisierten Varianten, bei denen wie zuvor im Fall von Taf1 hydrophobe und basische Aminosäuren innerhalb der Taf12 AID2 ausgetauscht wurden, führten lediglich zu einer Verringerung der Bindungsintensität. Dies lässt vermuten, dass mehrere kleine Domänen innerhalb der AID2 existieren, die funktionell redundant sind. Mit Hilfe weiterer ChIP-Experimente konnte auch nachgewiesen werden, dass Taf6 und Taf12 abhängig von Ino2 an den untersuchten Promotoren INO1 und CHO2 vorhanden sind. Die Proteine Taf1 und Taf6 wurden exemplarisch für Genexpressionsstudien ausgewählt, um ihren Einfluss auf die Transkription des Gens INO1 unter in vivo Bedingungen nachzuweisen. Durch vergleichende Northernblot-Hybridisierungen mit temperatursensitiven (ts) taf-Mutanten wurde gezeigt, dass die INO1-Expression unter nichtpermissiven Bedingungen (37°C) auf 7% (taf1ts) bzw. 4% (taf6ts) abfällt. Diese Befunde belegen, dass INO1 zu den Taf-abhängigen Genen zählt. Der generelle Transkriptionsfaktor TFIIA wurde ebenfalls auf eine Interaktion mit Ino2 untersucht. Bekannt war bereits, dass der Aktivator Rap1, der ähnlich wie Ino2 mit mehreren TFIID-Untereinheiten interagiert, auch TFIIA kontaktiert. Durch GST-„Pulldown“-Studien konnte die Untereinheit Toa1 als direkter Ino2-Interaktionspartner identifiziert werden. Dabei zeigte sich, dass Toa1 sowohl mit der TAD1 als auch der TAD2 von Ino2 interagiert und die TAD1 Aminosäuresubstitutionen D20K und F21R zu einem vollständigen Interaktionsverlust führen. In dieser Arbeit konnte somit gezeigt werden, dass die generellen Transkriptionsfaktoren TFIID und TFIIA als Coaktivatoren des für die Transkription der Strukturgene der Phospholipid-Biosynthese essentiellen Aktivators Ino2 fungieren.
Die Transkription von Genen der Phospholipidbiosynthese in S. cerevisiae wird durch ein ICRE (inositol/choline responsive element) genanntes UAS-Element aktiviert, welches durch die Phospholipid-Vorstufen Inositol und Cholin (IC) gesteuert wird. ICRE-Motive werden durch ein Heterodimer der bHLH-Proteine Ino2 und Ino4 erkannt, wobei Ino2 über zwei Transkriptionsaktivierungsdomänen (TAD) die Expression vermittelt, während Ino4 dem Kernimport des Komplexes dient. Negativer Regulator ist Opi1, der mit Ino2 interagiert. SUA7 (TFIIB) wird durch die Interaktion mit Ino2 an den Promotor rekrutiert. Im Rahmen dieser Arbeit wurden Untersuchungen durchgeführt, um ein Heterodimer aus heterolog exprimiertem Ino2 und Ino4 über chromatographische Methoden zu reinigen. Es wurde eine affinitätschromatographische Strategie entwickelt, die es gestattet, epitopmarkiertes Ino2 und Ino4 von einem Großteil der Fremdproteine abzutrennen. Mit größeren Zellmengen könnte es künftig gelingen, ein Ino2/Ino4-Heterodimer zu reinigen und es nach Kristallisierung zusammen mit einem ICRE-Motiv einer Röntgenstrukturanalyse zugänglich zu machen. Unter Verwendung genomischer Sequenzdaten von S. cerevisiae wurden in dieser Arbeit weitere Gene identifiziert, die ICRE-Motive in ihrer Promotorregion tragen, aber keine offensichtliche Rolle bei der Phospholipidbiosynthese spielen. Untersuchungen zeigten, dass die ICRE-tragenden Gene FAR8, RSF1, YEL073C und URA8 relativ stark, die Gene ARG4, ERG20, GPD2 und VHT1 nur moderat durch IC beeinflusst werden. Für das in S. cerevisiae stark IC-abhängige INO1 Gen (50-fache Derepression bei IC-Mangel) wurde gezeigt, dass drei distinkte ICRE-Motive für diese Regulation verantwortlich sind. Die Ergebnisse dieser Arbeit wurden mit Transkriptomanalysen anderer Gruppen verglichen und die Aussagekraft von in silico-Recherchen bewertet. Candida albicans ist eine opportunistisch pathogene Hefe. Auch C. albicans ist in der Lage, Inositol, Cholin und Fettsäuren de novo zu synthetisieren. Ein Funktionshomolog zu INO1, das CaINO1, vermag eine entsprechende Nullmutation in S. cerevisiae zu komplementieren. Ebenso wurden in C. albicans die Gene CaCHO1, CaFAS1 und CaFAS2 für die Synthese von Cholin bzw. Fettsäuren identifiziert. Ferner besitzt C. albicans das dem Opi1-Protein strukturell und funktionell ähnelnde CaOpi1, welches ebenfalls in der Lage ist, eine IC-abhängige Genregulation in S. cerevisiae zu vermitteln. Die in silico Identifikation potentieller C. albicans Orthologer zu INO2 sowie zu INO4 gab Anlass zu der Annahme, dass die Regulation der Phospholipidbiosynthese in S. cerevisiae und C. albicans konserviert vorliegt. Im Rahmen dieser Arbeit wurde ein unkonventionelles Intron im mutmaßlichen CaINO4 Gen identifiziert und durch RT-PCR eine intronfreie cDNA des CaINO4 Gens erhalten. Mit den Produkten der mu tmaßlichen Gene CaINO2 und CaINO4 wurden Protein/DNA- und Protein/Protein-Interaktionen untersucht und mit der Situation in S. cerevisiae verglichen. CaIno2 und CaIno4 sind in der Lage zu heterodimerisieren und an ICRE-Motive aus S. cerevisiae zu binden, jedoch konnte keine Bindung an den CaINO1 Promotor gezeigt werden. Weiterhin ist das Heterodimer der C. albicans-Proteine in der Lage, einer S. cerevisiae ino2 ino4 Doppelmutante ein Wachstum auf IC-freiem Medium zu ermöglichen. Keines der Gene kann jedoch allein die jeweils entsprechende ino2 oder ino4 Einfachmutation komplementieren. Weder CaIno2 noch CaIno4 interagieren mit CaOpi1, hingegen interagiert CaIno2 mit Opi1, ebenso CaOpi1 mit Ino2. Ferner interagiert CaIno2 wie auch CaIno4 mit CaSua7, nicht jedoch mit Sua7. Es konnte keine Interaktion zwischen Ino2 bzw. Ino4 mit CaIno4 bzw. CaIno2 festgestellt werden, ebensowenig eine Homodimerisierung der Proteine. Ähnlich wie Ino2 enthält auch CaIno2 zwei Transkriptionsaktivi erungsdomänen an entsprechenden Positionen und vergleichbarer Aktivierungsleistung. Es gelang im Rahmen dieser Arbeit nicht, homozygote Mutationen der Gene CaINO2 und CaINO4 durch Gendisruption in die diploide Hefe C. albicans einzuführen, es konnten lediglich heteroallele Mutanten hergestellt werden. Dieser Befund ist ein Hinweis auf eine Rolle von CaIno2 und CaIno4 bei der Aktivierung essentieller Gene in C. albicans. Daher wurde mit Genaktivierungstests nach der CaIno2/CaIno4-Konsensusbindesequenz gesucht und diese dann verwendet, um potentielle Zielgene in silico zu identifizieren. Als Konsensussequenz wurde das Motiv BWTCASRTG erhalten. Dieses Motiv wurde weder vor CaINO1, CaFAS1 oder CaCHO1 gefunden, jedoch zeigte sich eine deutliche Häufung des UAS-Elements vor mitochondrialen Genen, vor Genen der Ergosterolbiosynthese und besonders vor einer Vielzahl von Genen ribosomaler Proteine. Es kann aus diesen Daten gefolgert werden, dass CaIno2 und CaIno4 für die Aktivierung anderer, vermutlich essentieller Zielgene erforderlich sind als ihre Orthologen aus S. cerevisiae, während CaINO1 durch bisher unbekannte Faktoren reguliert wird.
Als Mitglieder der Ordnung Lactobacillales ist das Hauptkatabolit der Pneumokokken sowohl unter aerober wie auch microaerophiler Atmosphäre Lactat. Des Weiteren synthetisiert S. pneumoniae eine große Bandbreite an ABC-Transportersystemen, die an der Assimilation und an dem Stoffwechsel von Kohlenhydraten, löslichen Verbindungen und Aminosäuren beteiligt sind. In dieser Arbeit wurde der Kohlenstoffmetabolismus mittels 13C-Isotopologen Verteilung nach Wachstum der Pneumokokkenkultur in chemisch definiertem Medium (CDM) mit [U-13C6]Glucose, [1,2-13C2]Glucose oder [U-13C2]Glycin analysiert. GC/MS-Analysen zeigten ein Muster an schwer-markierten und unmarkierten Kohlenstoffatomen in den Aminosäuren. Die Ergebnisse ließen den Schluss zu, dass Pneumokokken sowohl einzelne Aminosäuren aufnehmen, wie auch über klassische oder nicht-klassische Biosynthesewege de novo synthetisieren können. His, Glu, Ile, Leu, Val, Pro und Gly blieben im Isotopolog Profiling unmarkiert, was ein Hinweis auf das Fehlen von Biosynthesewegen oder ihrer Regulation unter bestimmten Umweltbedingungen sein könnte. Obwohl die genetische Information für die Biosynthese der essentiellen verzweigtkettigen Aminosäuren (BAA; Ile, Leu und Val) in S. pneumoniae vorhanden ist, ergaben die 13C-Markierungsversuche keine de novo Synthese. Jedoch konnte durch Langzeit-1H-NMR (LT-NMR) Analysen eine aktive Aufnahme dieser Aminosäuren nachgewiesen werden. Darüber hinaus wird Aspartat nicht über den allgemeinen Stoffwechselweg mit Pyruvat und Acetyl-CoA synthetisiert. Die Aspartat-Synthese erfolgt im ersten Schritt durch die Umwandlung von Phosphoenolpyruvat (PEP) und CO2 zu Oxalacetat. Im zweiten Schritt wird Oxalacetat dann in Aspartat mit der Nebenreaktion Glutamat zu alpha-Ketoglutarat durch die Aspartat-Transaminase metabolisiert. GC/MS Analysen ergaben weiterhin, dass komplett markierte aromatische Aminosäuren aus Erythrose-4-Phosphat und zwei Molekülen PEP über das Intermediat Chorismat synthetisiert wurden. Es zeigte sich außerdem, dass [M+1] markiertes Serin durch die Hydroxymethylierung von unmarkiertem Glycin über 5,10-Methylentetrahydrofolat als Teil des C1-Pools hergestellt wurde. Weiterhin wurden In LT-NMR-Untersuchungen Konzentrationsänderungen der extrazellulären Metabolite quantifizert. Die homofermentative Milchsäuregärung konnte in Pneumokokken durch einen extrazellulären Anstieg der Lactatkonzentration nachgewiesen werden. Als essentielle Kandidaten wurden Glutamin und Uracil identifiziert, die das Pneumokokkenwachstum bei Mangel einschränken. Diese Ergebnisse zeigen die Vielzahl von Aminosäuren-Synthesewegen in Pneumkokken und die notwendige Rolle der Transportersysteme in Pneumokokken für die bakterielle Fitness und für die Adaption an verschiedene Wirtsnischen. Sechs mögliche Glutamin-Aufnahmesysteme konnten durch Genomanalysen von Streptococcus pneumoniae Stämmen identifiziert werden. Die Reverse Transkriptions-PCR haben gezeigt, dass die sechs gln-Operons unter in vitro Bedingungen exprimiert werden. Vier der gln-Gencluster bestehen aus den Genen glnQPH, während in zwei Regionen das Gen glnH, welches für eine lösliche Glutamin-Bindungsdomäne kodiert, fehlt. In dieser Arbeit wurde der Einfluss zwei dieser Glutamin-ABC-Transporter, mit den Operons glnQPH0411/0412 und glnQPH1098/1099, in S. pneumoniae D39 auf Virulenz und Phagozytose untersucht. Die zwei charakterisierten Transportersysteme bestehen jeweils aus der ATPase GlnQ und einem translatorischem Fusionsprotein aus der Permease GlnP und dem Bindungsprotein GlnH. Für die Untersuchungen wurden diese beiden Transporter mittels Insertations-Deletions-Mutagenese inaktiviert. CD-1 Mäuse, die intranasal mit biolumineszierenden D39delgln0411/0412 infiziert wurden, zeigten in Echtzeit eine signifikant erhöhte Überlebenszeit und eine Attenuierung bei der Ausprägung einer Pneumonie im Vergleich zu biolumineszierenden Wildtyp D39 Pneumokokken. Im murinen Sepsismodell mit der D39delgln0411/0412-Mutante zeigte sich eine gemäßigte, aber signifikante Abschwächung der Pathogenese. Im Gegensatz dazu war die D39delgln1098/1099 Mutante sowohl im murinen Pneumonie- wie auch Sepsismodell massiv attenuiert. Es war eine 100- bis 10000- fach höhere Infektionsdosis erforderlich, um mit der D39delgln1098/1099-Mutante eine vergleichbare Pathogenese der Pneumonie oder Sepsis wie beim Wildtypstamm D39 hervorzurufen. Im experimentellen Meningitismodell zeigten sich bei der D39delgln1098/1099-Mutante eine erniedrigte Anzahl an Leukozyten im Liquor und ein reduzierter Bakterientiter im Blut im Vergleich zu D39 und D39delgln0411/0412. Auch die Phagozytose-Experimente bestätigten eine signifikante verminderte Überlebensrate der beiden gln-Mutanten im Vergleich zum Wildtyp S. pneumoniae D39, was auf den Einfluss der bakteriellen Fitness auf den Schutz gegen oxidativen Stress hinweist. Diese Ergebnisse demonstrierten, dass beide Glutamin-Aufnahmesysteme für die vollständige Virulenz der Pneumokokken essentiell sind, aber verschiedene Auswirkungen auf die Pathogenese der Bakterien unter in vivo Bedingungen haben. Das Zelloberflächenprotein PavA der Pneumokokken ist ein Virulenzfaktor, der für invasive Erkrankungen wichtig ist. In dieser Arbeit wurde gezeigt, dass PavA essentiell für die in vivo Besiedlung von Streptococcus pneumoniae D39 in den oberen Atemwegen von Mäusen ist. In dem murinen Pneumoniemodell wurden pavA-Mutanten nicht aus den infizierten Mauslungen eliminiert, sondern persistierten und lösten somit eine chronische Infektion aus, während Wildtyp-Pneumokokken systemische Erkrankungen verursachten. PavA-defiziente Pneumokokken konnten unter experimentellen Bedingungen nicht aus der Lunge in die Blutbahn streuen. Diese Ergebnisse ließen den Schluss zu, dass PavA an der erfolgreichen Kolonisation der Schleimhautoberflächen und an der Translokation der Pneumokokken durch Wirtsbarrieren beteiligt ist.
Bacteria are exposed to oxidative stress as an unavoidable consequence of their aerobic lifestyle. Reactive oxygen species (ROS) are generated in the stepwise one-electron reduction of molecular oxygen during the respiration. Pathogens encounter ROS during the oxidative burst of macrophages as part of the host immune defense. Besides ROS, bacteria also have to cope with reactive chlorine, electrophilic and nitrogen species (RCS, RES, RNS). To cope with these reactive species, bacteria have evolved different defense and repair mechanisms. To maintain the reduced state of the cytoplasm, they utilize low molecular weight (LMW) thiols. LMW thiols are small thiol-containing compounds that can undergo post-translational thiolmodifications with protein thiols, termed as S-thiolations. S-thiolations function as major redox regulatory and thiol-protection mechanism under oxidative stress conditions. In eukaryotes and Gram-negative bacteria, the tripeptide glutathione (GSH) functions as major LMW thiol, which is present in millimolar concentrations. The Actinomycetes, such as Mycobacterium and Corynebacterium species do not produce GSH and utilize instead mycothiol (MSH) as their alternative LMW thiol. In Firmicutes, including Bacillus and Staphylococcus species, bacillithiol (BSH) functions as the major LMW thiol. LMW thiols protect protein thiols against the irreversible overoxidation of cystein residues to sulfinic and sulfonic acids. In addition, LMW thiols contribute to the virulence and survival of pathogens, function in metal homeostasis and serve as enzyme cofactors for detoxification of xenobiotics and antibiotics. In this doctoral thesis, we aimed to investigate the roles of MSH and BSH in redox regulation of main metabolic enzymes under oxidative stress in the pathogens Corynebacterium diphtheriae and Staphylococcus aureus. Previous redox proteomics studies identified the glyceraldehyde-3-phosphate dehydrogenase GapDH and the aldehyde dehydrogenase AldA as S-thiolated in S. aureus and C. diphtheriae. Thus, we aimed to study the redox regulation of the metabolic enzyme GapDH in C. diphtheriae in response to NaOCl and H2O2 stress by S-mycothiolation, which is described in chapter 1. Moreover, we studied the involvement of the mycoredoxin-1 (Mrx1) and thioredoxin (Trx) pathways in reactivation of S-mycothiolated GapDH in vitro. Using shotgun proteomics, 26 S-mycothiolated proteins were identified under NaOCl stress in C. diphtheriae. These are involved in energy metabolism (Ndh, GlpD) and in the biosynthesis of amino acids (ThrA, LeuB), purines (PurA) and cell wall metabolites (GlmS). The glycolytic GapDH was identified as conserved target for S-thiolation across Gram-positive bacteria. GapDH was the most abundant protein, contributing with 0.75 % to the total cystein proteome. Moreover, GapDH is a conserved target for redox regulation and S-glutathionylation in response to oxidative stress in several prokaryotic and eukaryotic organisms. Treatment of GapDH with NaOCl and H2O2 in the absence of MSH resulted in irreversible enzyme inactivation due to overoxidation. Pretreatment of GapDH with MSH prior to H2O2 or NaOCl exposure resulted in reversible inactivation due to S-mycothiolation of the active site Cys153. Since S-mycothiolation is faster compared to overoxidation, S-mycothiolation efficiently protects the GapDH active site against overoxidation. The activity of S-mycothiolated GapDH could be restored by both, the Mrx1 and Trx pathway in vitro. Interestingly, the recovery of Smycothiolated GapDH by Mrx1 was faster compared to its reduction by the Trx pathway. In previous studies, the reactivation of S-mycothiolated Mpx and MrsA by the mycoredoxin pathway occurred also faster compared to the Trx pathway, which is consistent with our results. We were further interested to analyze the redox regulation of the glyceraldehyde-3phosphate dehydrogenase Gap of S. aureus under NaOCl and H2O2 stress, which is described in chapter 2. Using the quantitative redox proteomic approach OxICAT, 58 NaOCl-sensitive cystein residues with >10% thiol oxidation under NaOCl stress were identified. Gap and AldA showed the highest oxidation increase of 29% under NaOCl stress at their active site cystein residues. Using shotgun proteomics, five S-bacillithiolated proteins were identified, including Gap, AldA, GuaB, RpmJ and PpaC. Gap contributed with 4 % as most abundant cystein protein to the total cystein proteome. Our activity assays demonstrated that Gap of S. aureus is highly sensitive to overoxidation by H2O2 and NaOCl in vitro in the absence of BSH. The active site Cys151 of Gap was oxidized to the BSH mixed disulfide under H2O2 and NaOCl stress in the presence of BSH in vitro, which resulted in the reversible Gap inactivation. Moreover, inactivation of Gap by NaOCl and H2O2 due to S-bacillithiolation was faster compared to overoxidation, indicating that S-bacillithiolation protects the Gap active site against overoxidation in vitro. We further showed that the bacilliredoxin Brx catalyzes the reduction of S-bacillithiolated Gap in vitro. Molecular docking of BSH into the Gap active site revealed that S-bacillithiolation does not require major structural changes. Apart from Gap, the aldehyde dehydrogenase AldA was identified as S-bacillithiolated at its active site Cys279 under NaOCl stress in S. aureus previously. Thus, the expression, function, redox regulation and structural changes of AldA were analysed under NaOCl and aldehyde stress in S. aureus as summarized in chapter 3. AldA was S-bacillithiolated in the presence of H2O2 and BSH as demonstrated in BSH-specific Western blots in vitro. The expression of aldA was previously shown to be regulated by the alternative sigma factor SigmaB in S. aureus. Transcription of aldA was strongly increased in a SigmaB-independent manner under formaldehyde, NaOCl and diamide stress in S. aureus. Using an aldA deletion mutant, we demonstrated that aldA is required for growth and survival under NaOCl stress in S. aureus. The purified AldA enzyme was shown to catalyze the oxidation of various aldehyde substrates, including formaldehyde, methylglyoxal, glycolaldehyde and acetaldehyde in vitro. In addition, the function of the conserved Cys279 for AldA activity was investigated in vivo and in vitro. The purified AldAC279S mutant was shown to be inactive for aldehyde oxidation in vitro. Moreover, the aldAC279S mutant was very sensitive under NaOCl stress in vivo, and this phenotype could be reversed using the aldA complemented strain. These experiments demonstrate the function of Cys279 for AldA activity both in vitro and in vivo. AldA activity assays showed that AldA is sensitive to overoxidation and irreversible inactivation by H2O2 alone in vitro. In the presence of BSH, AldA is protected against overoxidation by reversible Sbacillithiolation in vitro. Molecular docking and molecular dynamics simulations revealed that BSH occupies two different positions in the Cys279 active site, which depend on the NAD+ cofactor. In the apoenzyme, BSH forms the disulfide with Cys279 in the “resting” state position, while Cys279 is S-bacillithiolated in the “attacking” state position in the holoenzyme in the presence of the NAD+ cofactor.
Gout was described by Hippocrates in the 5th century BC as a disease of rich people and linked with excess food and alcohol. It is caused by long-lasting hyperuricemia, which is a result of an imbalance between excretion and production of uric acid. The surplus of uric acid leads to deposition of monosodium urate crystals in the joints, which can initiate a painful inflammation called a gout attack. Despite various pharmacological treatments for this disease, a low purine diet remains the basis of all gout therapies. Since food is rich in purines, the aim of this project was to develop a novel enzyme system to decrease the purine content of food, what should result in reduced serum urate concentration in patients with hyperuricemia. The system consists of five degrading enzymes (adenine deaminase, guanine deaminase, xanthine oxidoreductase, urate oxidase and purine nucleoside phosphorylase) that combined in one product are able to hydrolyse all purines to a highly soluble allantoin, which can be easily removed from the body. This approach provides the patients a possibility to reduce the symptoms and frequency of gout attacks or even doses of prescribed drugs. In order to obtain necessary system components, yeast Arxula adeninivorans LS3 was screened for enzyme activities. A. adeninivorans is known to utilise various purines and this ability is a result of activity of desired enzymes, two of which, adenine deaminase and xanthine oxidoreductase, are in focus of this thesis. The analysis of growth of A. adeninivorans on various carbon and nitrogen sources gave the first insight into the cells’ nutrient preferences indicating the presence of purine degrading enzymes, such as adenine deaminase and xanthine oxidoreductase. Purines, such as adenine and hypoxanthine, could be utilised by this yeast as sole carbon and nitrogen sources and were shown to trigger the gene expression of the purine degradation pathway. Enzyme activity tests and quantitative real-time PCR method allowed for identification of the best inducers for adenine deaminase and xanthine oxidoreductase, as well as their concentration and time of induction. The adenine deaminase (AADA) and the xanthine oxidoreductase (AXOR) genes were isolated and subjected to homologous expression in A. adeninivorans cells using Xplor®2 transformation/expression platform. The selected transgenic strains accumulated the recombinant adenine deaminase in very high concentrations. The expression of AXOR gene posed difficulties and remained a challenge. Additional expression of both proteins in alternative E. coli system was undertaken but failed for AXOR gene. The recombinant adenine deaminase and wild-type xanthine oxidoreductase were purified and characterized biochemically. The characterization included determination of optimal pH and temperature, stability in different buffers and temperatures, molecular weight, substrate spectrum, enzyme activators and inhibitors, kinetics and intracellular localisation. The determination of these parameters was necessary to ensure optimal conditions for application of these enzymes in the industry. At the final stage, the enzymes were combined in one mix with provided guanine deaminase and urate oxidase and used to degrade purines in selected food constituents. The application was successful and demonstrated the potential of this approach for the production of food with lower purine concentration.
Die Regulation der Phospholipid-Biosynthesegene in der Hefe Saccharomyces cerevisiae erfolgt über die Verfügbarkeit der Phospholipid-Vorstufen Inositol und Cholin (IC). Bei ICMangelbedingungen wird die Transkription der Strukturgene stimuliert und bei IC-Überschuss im Medium reprimiert. Im Promotorbereich dieser Gene befinden sich spezifische UAS-Elemente („inositol/choline-responsive element“, ICRE-Motive), welche von den Aktivatoren Ino2 und Ino4 gebunden werden. Bei IC-Mangel kommt es zu einer Anhäufung des Intermediats Phosphatidsäure, wodurch der Repressor Opi1 durch die Interaktion mit Scs2 außerhalb des Zellkerns am endoplasmatischen Reticulum gebunden wird. Wenn ausreichend IC im Medium vorhanden ist, kann der Repressor Opi1 in den Zellkern einwandern und den Aktivator Ino2 binden. Ferner kann Opi1 über seine Opi1-Sin3-Interaktionsdomäne (OSID) mit der PAH1 („paired amphipathic helix“) des Corepressors Sin3 interagieren. Ein Ziel dieser Arbeit war es, ausgewählte Aminosäuren in der OSID durch gerichtete Mutagenese gegen Alanin auszutauschen und die erhaltenen Opi1-Varianten auf ihre Repressorfunktion hin zu untersuchen. Die Substitution einzelner Aminosäuren innerhalb der OSID offenbarte die Notwendigkeit der Aminosäuren L56, V59 und V67 für die Opi1-Sin3 Bindung. Die Ergebnisse legten außerdem nahe, dass die Repression nicht allein über Sin3 vermittelt wird. Tatsächlich konnte gezeigt werden, dass die innerhalb der OSID von Opi1 kritischen Aminosäuren der Opi1-Sin3 Bindung (L56, V59 und V67) auch für die Interaktion von Opi1 mit Cyc8 wichtig sind. Dementsprechend rekrutiert Opi1 mit Hilfe der OSID zwei pleiotrope Corepressoren. Sin3 bindet über die PAH1 an die OSID, während die Opi1-Cyc8- Bindung über die TPR-Motive im Cyc8 vermittelt wird. Desweiteren zeigte sich, dass die sin3 cyc8 Doppelmutante synthetisch letal ist. Sin3 ist eine Untereinheit in mehreren Komplexen der Histondeacetylase (HDAC) Rpd3 und fungiert als Plattform für viele Protein-Protein Wechselwirkungen. Innerhalb des Sin3/Rpd3LKomplexes wurde der Einfluss mehrerer Untereinheiten (Pho23, Sap30, Sds3, Ume1 und Dep1) untersucht. Hier zeigte sich, dass Pho23 einen entscheidenden Einfluss auf die Regulation ICRE-abhängiger Gene hat. In den sich anschließenden Interaktionsanalysen konnte eine Bindung von Pho23, Sds3 und Sap30 an Sin3 gezeigt werden. Eine genauere Kartierung der Pho23-Sin3 Bindung zeigte, dass Pho23 über zwei voneinander unabhängige Domänen (Pho23-Sin3-Interaktionsdomäne; PSID1 und PSID2) mit Sin3 wechselwirkt, wobei die Interaktion der PSID1 und der PSID2 mit der HID (HDAC-Interaktionsdomäne) im Sin3 erfolgt. Die katalytische Aktivität innerhalb der Sin3/Rpd3-Komplexe ist durch die HDAC Rpd3 gegeben. Durch Untersuchungen der HDACs der Klasse I (Rpd3, Hos1 und Hos2) bzw. der Klasse II (Hda1 und Hos3) konnte für ICRE-abhängige Genorte gezeigt werden, dass eine rpd3 hda1 hos1 Dreifachmutante ähnlich dereguliert ist wie eine sin3 Mutante. Bei Interaktionsstudien der HDACs Rpd3, Hda1 und Hos1 mit Sin3 konnten neben der bereits bekannten HID im Sin3 (aa 801-1100) zwei neue HIDs (HID2: aa 473-600, HID3: aa 1100- 1210) identifiziert werden. Die Histondeacetylase Rpd3 bindet an die HID1 und an die HID3, während Hda1 und Hos1 jeweils an HID2 und HID3 binden. Interessanterweise stellte sich heraus, dass die Bindedomäne für die Sin3-Bindung innerhalb der Deacetylase-Domäne (DAC) aller drei HDACs liegt. Für Hos1 konnte die Sin3 Bindedomäne auf einen Aminosäurebereich von 236-400 eingegrenzt werden. Für die Hda1- Sin3 Bindung konnten zwei voneinander unabhängig interagierende Bereiche im Hda1 (aa 201-250 und aa 251-300) beschrieben werden. Neben der Deacetylierung wurde der regulative Einfluss einer weiteren kovalenten Histonmodifizierung, nämlich der der Methylierung durch Histonmethyltransferasen (HMT; Set1, Set2 und Dot1) und der Demethylierung durch Histondemethylasen (HDM; Jhd1, Jhd2, Ecm5, Gis1 und Rph1) auf die Genexpression der Phospholipid-Biosynthesegene untersucht. Hier konnte für die HMT Set2 (spezifisch für Lysin-36 im Histon H3) ein großer Einfluss auf die ICRE-abhängige Genexpression gezeigt werden. Desweiteren konnte gezeigt werden, das Set2 direkt an Ino2 bindet. Die Kartierung der Interaktionsdomäne offenbarte, dass die katalytische SET Domäne im Set2 mit der DNA-bindenden bHLHDomäne von Ino2 wechselwirkt.
Streptococcus pneumoniae (pneumococci), a human pathobiont, express and expose several proteinaceous colonization and virulence factors on its surface to facilitate on the one hand colonization of the upper respiratory tract and on the other hand pathogenesis in the host. In this study the interaction of two of such factors referred to as pneumococcal virulence factor A (PavA) and pneumococcal virulence factor B (PavB) and acting as microbial surface components recognizing adhesive matrix molecules (MSCRAMMs), was delineated with the two host matricellular proteins fibronectin (Fn) and vitronectin (Vn). Despite similarity in nomenclature, PavA and PavB represent two diverse pneumococcal proteins with respect to their structure and association with the pneumococcal surface. PavA is a non-classical surface protein (NCSP) with an ambiguous mode of secretion and anchorage while PavB is a characteristic MSCRAMM, anchored via sortase A to pneumococcal peptidoglycan. PavB has a signature of repetitive modules termed as streptococcal surface repeats (SSURE). Pneumococci preferentially interact with immobilized human Fn. In vitro cell culture adherence assays demonstrated that cell bound Fn facilitates the adherence of pneumococci to the host cells and this particular interaction is indifferent to host cell type and is species non-specific. Flow cytometry and immunoblot analyses further indicated the ability of pneumococci to interact with the soluble form of Fn in a dose-dependent but species non-specific manner. The molecular interaction of PavA and PavB (via its SSURE domains) with Fn was delineated further in detail via several direct protein-protein interaction approaches. Ligand overlay assays, surface plasmon resonance studies and SPOT peptide arrays demonstrated that PavA and PavB target at least 13 out of the 15 type III fibronectin domains located in the C-terminal part of Fn. Strikingly, both pneumococcal fibronectin-binding proteins (FnBPs) recognize similar peptides in targeted type III repeats. Structural comparisons revealed that the targeted type III epitopes cluster on the inner strands of both β-sheets forming the fibronectin domains. Importantly, synthetic peptides of FnIII1, FnIII5 or FnIII15 bind directly to FnBPs PavA and PavB, respectively. Thus, analysis of interaction of pneumococcal FnBPs PavA and PavB revealed a probable conserved and/or common pattern of molecular interaction with human Fn. In addition to Fn, pneumococcal PavB interacts with other host matricellular proteins such as human plasminogen (Plg) and human thrombospondin-1 (hTSP-1). Pneumococcal proteins such as PspC and PspC-like Hic have earlier been demonstrated to interact with hTSP-1 as well as human Vn, thereby depicting a redundant function as MSCRAMMs. In this study the role of PavB as a pneumococcal vitronectin binding protein (VnBP) was assessed. Flow cytometric analysis suggested PavB as VnBP, because strains deficient for PavB exhibited a significantly decreased ability to acquire vitronectin compared to wild-type pneumococci. When using a double knockout, deficient in expression of PavB and the VnBP PspC, the pneumococcal interaction with vitronectin was completely abolished. The direct protein-protein interaction assays such as far western ligand overlay, ELISA, and surface plasmon resonance indicated the interaction of SSURE domains with both soluble and immobilized Vn. However, the binding activity depends on the number of SSURE domains with five SSURE showing the highest binding activity to Vn. The interaction of PavB with Vn was charge dependent and heparin sensitive as analyzed by ELISA. The importance of the heparin binding domains of Vn in this interaction was further analyzed via direct protein-protein interaction approaches. Binding studies (far western ligand overlay, ELISA, and surface plasmon resonance) with truncated recombinant Vn fragments indicated that PavB targets the C-terminal heparin-binding domain (HBD3) of vitronectin, a characteristic shared with PspC, hence, suggesting a conserved molecular interaction of pneumococci with Vn. In addition to its function as an MSCRAMM, PavB has the capability to interact directly with host epithelial cells via an unknown cellular receptor. Thus, this study aimed to identify cellular receptor(s) for PavB. In vitro cell culture adherence and invasion assays confirmed that pneumococcal PavB is involved in promoting pneumococcal adherence to respiratory epithelial cells without employing any molecular bridge. The direct interaction between PavB and host epithelial cells was further confirmed via direct binding assays when using Cy5-labeled PavB and flow cytometric analysis. Strikingly, exogenously added human vitronectin competitively inhibited binding of PavB to respiratory epithelial cells. This observation led us to hypothesize that the major vitronectin receptor αvβ3 integrin acts as a potential receptor for PavB. This hypothesis was supported by functional blocking assays with monoclonal antibodies recognizing specific integrin subunits. The results revealed reduced binding of PavB in the presence of bound antibodies recognizing αv integrin indicating that PavB employs αvβ3 integrin as its direct receptor on eukaryotic cells. This was further confirmed via a direct binding assay of PavB to mouse embryonic fibroblasts (MEFs) where cells lacking αvβ3 demonstrated a marked decrease in binding to PavB. Although functional blocking assay and direct binding assay with MEFs supported the role of αvβ3 integrin as a direct adhesin for PavB, RNA interference of αv integrin in epithelial cells did not impair the binding of PavB in αv-knocked down cells in comparison to non-transfected cells. Finally, surface plasmon resonance (SPR) analysis indicated the direct interaction between pneumococcal PavB and recombinant αvβ3 integrin. In this study we report for the first time the interaction of a Gram-positive extracellular pathogen, namely Streptococcus pneumoniae, with one of the host ICAMs, namely the αvβ3 integrin. In conclusion, the present study analysed some of the aspects of molecular interaction of pneumococcal MSCRAMMs PavA and PavB with hFn and hVn. The hot spots of interaction on C-terminal FnIII repeats were delineated for PavA and PavB. HBD3 was revealed to be pivotal for PavB-Vn interaction. In addition the redundant role of pneumococcal PavB as an MSCRAMM was demonstrated. Furthermore this study successfully identifies a direct receptor for pneumococcal PavB, namely αvβ3 integrin. The mechanism and biological rationale of this newly identified interaction is a matter of debate and awaits further scientific analyses.
Alcohol dehydrogenases as biocatalysts for the production of enantiomerically pure chiral alcohols
(2016)
Summary Enantiomerically pure chiral alcohols are key compounds in the production of certain chemicals including pharmaceuticals. Chemical synthesis allows to obtain maximal yield of 50% for one enantiomer ( >50% yield is achievable with chiral catalysts used in chemical synthesis), whereas biosynthesis leads to nearly 100% yield. Hence, expensive and time consuming resolution of racemic mixture can be avoided. Alcohol dehydrogenases are the most popular enzymes used in the chiral alcohols synthesis due to high activity with appropriate aldehydes or ketones. ADHs require a cofactor which has to be regenerated after the conversion of aldehyde/ketone to the respective alcohol. Thereby, different regeneration methods were used in the practical work to compare and choose the better one. R. erythropolis and C. hydrogenoformans alcohol dehydrogenases were chosen based on the literature screening. Each gene was cloned into Xplor2 vector and pFPMT vector. Xplor2 vector was used for the transformation of A. adeninivorans and pFPMT vector was used for the transformation of H. polymorpha. Chemically synthesized alcohol dehydrogenase sequences from R. erythropolis (ReADH) and C. hydrogenoformans (ChADH) were cloned between TEF1 promoter and PHO5 terminator which are components of Xplor2 vector or between FMD promoter and MOX terminator which are genetic elements of pFPMT vector. Moreover, ChADH and ReADH sequences with His-tag encoding sequence at the 5’ or 3’ end were constructed and the most active form of the protein was selected for further studies. ReADH-6H was used for the synthesis of 1-(S)-phenylethanol and ethyl (R)-4-chloro-3-hydroxybutanoate whereas ChADH-6H was used for the production of ethyl (R)-mandelate. ReADH-6H synthesized in A. adeninivorans and H. polymorpha was fully biochemically characterized. The enzymes from the two yeast species showed some differences in their pH and temperature optima, thermostability and activity levels. A-ReADH (A. adeninivorans) and H-ReADH (H. polymorpha) were highly active with the same substrates which were: acetophenone, 4-hydroxy-3-butanone and ethyl 4-chloroacetoacetate for reduction reaction along with 1-phenylethanol and 1,6-hexanediol for oxidation reaction. Recombinant A-ReADH-6H and H-ReADH-6H were synthesized in A. adeninivorans and H. polymorpha, respectively. Both enzymes were used for the synthesis of 1-(S)-phenylethanol and ethyl (R)-4-chloro-3-hydroxybutanoate with the use of substrate-coupled cofactor regeneration system. The enantiopurity of the products was >99%. Moreover, A. adeninivorans whole cell catalyst was also used for the synthesis of both chiral alcohols. BmGDH (Bacillus megaterium glucose dehydrogenase) was co-expressed with ReADH-6H for NADH cofactor regeneration. Comparison between isolated enzymes and permeabilized whole cell catalysts indicate that cell biocatalysts are more suitable for the production of 1-(S)-phenylethanol with 92% of acetophenone being converted in 60 min. However, cells did not show any significant advantage over isolated enzymes in the synthesis of ethyl (R)-4-chloro-3-hydroxybutanoate although the velocity of the synthesis of ethyl (R)-4-chloro-3-hydroxybutanoate was slightly improved using whole-cell catalysts, giving an 80% substrate conversion in 120 min. Recombinant C. hydrogenoformans alcohol dehydrogenase was synthesized in A. adeninivorans and biochemically characterized. Enzyme showed high activity only with one substrate, ethyl benzoylformate. The A. adeninivorans and H. polymorpha cell catalysts synthesizing ChADH and BmGDH (Bacillus megaterium glucose dehydrogenase) were constructed and used in the synthesis of ethyl (R)-mandelate (reduction product of ethyl benzoylformate) with the enantiopurity of the reaction product being >98%. H. polymorpha catalysts were more effective in the synthesis than A. adeninivorans cells. The first were able to convert 93% of ethyl benzoylformate within 180 min and the latter were converting 94% of the substrate within 360 min. Re-use of non-immobilized cells and catalysts entrapped in Lentikat® was performed and the improvement of the stability of immobilized catalysts was reported. Space time yield of 3.07 mmol l-1 h-1 and 6.07 mmol l-1 h-1 was achieved with A. adeninivorans and H. polymorpha cell catalysts, respectively. Alcohol dehydrogenase 1 from A. adeninivorans was analyzed concerning the synthesis of enantiomerically pure chiral alcohols. The enzyme did not synthesize industrially attractive products. However, based on biochemical characterization enzyme plays a role in the synthesis of 1-butanol or ethanol and thereby it is of biotechnological interest.
In der Hefe Saccharomyces cerevisiae werden die Strukturgene der Phospholipid-Biosynthese auf Transkriptionsebene in Abhängigkeit der Verfügbarkeit der Phospholipidvorstufen Inositol und Cholin (IC) über ein in der Promotorregion befindliches UAS-Element, genannt ICRE („inositol/choline-responsive element“), reguliert. Bei Mangel an IC kommt es zu einer Anhäufung des Intermediats Phosphatidsäure, wodurch der Repressor Opi1 außerhalb des Zellkerns am endoplasmatischen Reticulum verankert wird. Dadurch kann ein Heterodimer, bestehend aus den bHLH-Proteinen Ino2 und Ino4, an das ICRE-Motiv binden und die transkriptionelle Aktivierung vermitteln. Ist ausreichend IC vorhanden, gelangt der Repressor Opi1 in den Zellkern und bindet an Ino2. Dadurch ist eine Aktivierung nicht mehr möglich. Ferner kontaktiert Opi1 über seine Opi1-Sin3-Interaktionsdomäne (OSID) die Corepressor-Komplexe Sin3 und Cyc8/Tup1, die durch Rekrutierung von Histondeacetylasen (HDACs) zur Chromatinverdichtung und damit zur Genrepression führen. In einer früheren Arbeit wurde beobachtet, dass die regulierte Expression von Genen der Phospholipid-Biosynthese auch durch die Phosphatkonzentration beeinflusst wird. Es konnte festgestellt werden, dass bei Phosphatmangelbedingungen die Expression ICRE-abhängiger Gene auf 10 % reduziert ist. Eine Δopi1-Mutante zeigte dieses Expressionsmuster jedoch nicht mehr. Dieser Befund wies darauf hin, dass Opi1 seine Repressorfunktion sowohl bei IC-Überschuss als auch bei Phosphatmangel ausführt. Ein Protein, welches die Phosphatverfügbarkeit an Opi1 möglicherweise über eine Phosphorylierung vermitteln könnte, ist die cyclinabhängige Proteinkinase Pho85, für die eine in vitro Interaktion mit Opi1 gezeigt wurde. Um diese Hypothese zu überprüfen, wurden mittels gerichteter Mutagenese Aminosäurereste mutmaßlicher Pho85-Phosphorylierungsstellen im Opi1-Protein (S321, T51) gegen das nicht mehr phosphorylierbare Alanin ausgetauscht. Hefestämme, die solche Opi1-Protein-varianten (S321A, T51A) synthetisierten, zeigten jedoch weiterhin einen klaren Einfluss des Phosphatmangels auf die Expression eines ICRE-regulierten Reportergens. Dies lässt darauf schließen, dass die Repression unter Phosphatmangelbedingungen nicht über eine Phosphorylierung von Opi1 durch Pho85 zu Stande kommt. Parallel durchgeführte in vitro-Interaktionsstudien zeigten, dass die Bindung von Pho85 an Opi1 über zwei unabhängig voneinander funktionierende Interaktionsdomänen im Opi1-Protein (aa 30-70 und aa 321-350) erfolgt. Mit Hilfe des „Two-Hybrid“-Systems wurde festgestellt, dass die Opi1-Pho85 Wechselwirkung in vivo phosphatabhängig stattfindet. Die Befunde erlauben die Hypothese, dass Pho85 bei Phosphatüberschuss u. a. die OSID im Opi1 abdeckt, dadurch die Wechsel-wirkung mit Sin3/Cyc8 verhindert und eine gesteigerte Genexpression zulässt. Mittels Chromatin-Immunopräzipitation (ChIP) konnte gezeigt werden, dass Opi1, Co-Repressoren wie Sin3 und Cyc8 als auch die HDACs Hda1 und Hos1 an Promotoren ICRE-regulierter Gene Ino2-abhängig anwesend sind. Des Weiteren wurde festgestellt, dass sich Sin3 unabhängig von Opi1 an ICRE-haltigen Promotoren befindet. Dieses Ergebnis wider-sprach einer früheren Arbeitshypothese, konnte aber durch weitere Versuche, die eine direkte in vitro Interaktion von Sin3 mit dem Ino2-Aktivator zeigten, plausibel in ein neues Rekrutierungsmodell eingefügt werden. Abschließend wurden die am Beispiel von Opi1 gewonnenen Erkenntnisse durch in vitro Interaktionsanalysen diverser spezifischer Repressoren mit den pleiotropen Co-Repressoren Sin3 und Cyc8/Tup1 erweitert. Für zahlreiche Repressoren wurde gefunden, dass sie parallel mit Sin3 und Cyc8 interagieren (u. a. Rox1, Yox1, Dal80 und Mot3). Durch Kartierungsexperimente konnten minimale Repressordomänen charakterisiert werden, die die Interaktion zu Sin3 bzw. Cyc8 vermitteln, und sequenzhomologe Domänenstrukturen analysiert werden. Des Weiteren zeigte sich, dass alle Repressoren, die mit Sin3 wechselwirken, dessen Domänen PAH1 oder PAH2 („paired amphipathic helix“) kontaktieren.