VAAM-Jahrestagung 2011 Karlsruhe, 3.–6. April 2011

acteriocines, proteins involved in cell wall metabolism, cell division,pyrimidine metabolism and metabolism of amino acids. The two componentsignal transduction systems CiaRH, VicKRX, RelRP, comDE andSMU.1037/1038 were significantly affected by carolacton. While comDE isthe only positively regulated TCS, VicKRX shows the earliest response tocarolacton. Among the other differentially expressed genes many known tobe regulated by VicKRX were identified, implicating a central role of theVicKRX-system in the mode of action of carolacton.An analysis of the sensitivity of all 13 viable HK-mutants of S. mutans tocarolacton using Live/Dead Bacterial Viability staining showed that allmutants were susceptible. Recently analysed regulon of eukaryotic typeserine/threonine kinase pknB showed a strong overlap with the carolactonaffected genes. Furthermore pknB is predicted to modulate the activity of theVicKRX system and interferes with the acid resistance. A pknB mutant wasproved to be insensitive to carolacton using Live/Dead staining and Cfucounts,indicating pknB as the potential target.[1] Kunze, B. et al (2010): Damage of Streptococcus mutans biofilms by carolacton, a secondarymetabolite from the myxobacterium Sorangium cellulosum. BMC.Microbiol. 10:199.SRP033Extracellular proteolysis of A. fumigatus and eIF2αkinase signaling - is there a connection?A. Bergmann* 1 , A. Schedler 1 , C. Sasse 2 , S. Krappmann 11 Center for Infectious Diseases, University of Wuerzburg, Würzburg,Germany2 Institute for Molecular Infection Biology, Julius-Maximilians-University,Würzburg, GermanyAspergillus fumigatus is a ubiquitous mould colonizing soil anddecomposing organic matter. It produces small conidia that are distributedby the air and reach the alveoli of the lung when inhaled. Inimmunocompromised patients, this opportunistic pathogen can cause severalforms of disease, the most severe form is called Invasive Aspergillosis (IA).Several determinants contribute to pathogenicity of A. fumigatus, e.g. itsnutritional versatility and the ability to react on fast changing environmentalconditions. The prtT gene product is a global regulator of extracellularproteolytic activity in A. fumigatus and is therefore involved in degradationof polymeric proteinaceous substances of the surrounding environment. Thistranscription factor regulates expression of several secreted proteases (alp1,mep, pep1); however, it is not a virulence determinant of pulmonaryinvasive aspergillosis in leukopenic mice. Expression of PrtT appears to beregulated posttranscriptionally, also supported by a long 5’ leader region ofthe prtT transcript. Furthermore, A. fumigatus exhibits eIF2α-kinasesignaling to counteract environmental stress conditions as it expresses twofunctional kinases for this initiation factor of translation: CpcC, an integralcomponent of the so-called Cross-Pathway Control system of amino acidbiosynthesis, and IfkB (initiation factor kinase B) with as yet unknowncellular function. To further investigate any regulatory role of the prtT leaderregion, we generated 5’prtT::gfp-reporter strains in genetic deletionbackgrounds for either or both eIF2α kinases. CpcC and IfkB seem toinfluence translation of the reporter upon a shift from minimal medium toBSA as sole nitrogen source with the presence of either sensor kinase beingapparently sufficient for expression. These data imply a mechanism oftranslational regulation of PrtT expression via two redundant eIF2α kinases,which links extracellular proteolysis of A. fumigatus to this conservedregulatory cascade.A Pkc1-GFP fusion protein has been reported to relocate from the cytoplasmto mitochondria upon treatment of yeast cells with the oxidative stress agentfarnesol (Fairn et al. 2007: J. Biol. Chem., 282, 4868-4874). In order toverify these data, we also constructed a similar Pkc1-GFP fusion in ourlaboratory strain and used it in combination with a mCherry-fusion of themitochondrial marker succinate dehydrogenase. We found that Pkc1-GFPprimarily localizes to the yeast bud neck during cytokinesis and does notrelocalize to mitochondria upon treatment with farnesol, tea trea oil ordetergents like Tween20 or Tween40. However, the nonionic detergentNonidet P-40, which was used by the authors cited above as a solvent forfarnesol, led to an accumulation of Pkc1-GFP at mitochondrial structures,even in the absence of farnesol.We conclude that the reported effect ofoxidative stress on Pkc1 localization is an experimental artefact.Currently, we are investigating the involvement of Pkc1 in the regulation ofyeast cytokinesis.SRP035A proteomic signature library: Gene expression ofStaphylococcus aureus under various growth-restrictingconditionsS. Fuchs*, D. Zühlke, J. Pané-Farré, H. Kusch, C. Wolf, S. Reiß,L.T.N. Binh, M. Hecker, S. EngelmannInstitute for Microbiology, Ernst-Moritz-Arndt-University, Greifswald,GermanySince decades, gel-based proteomics has been used to get deeper insightsinto physiological processes of living cells. Now we present an interexperimentalcomparison of different protein synthesis pattern induced inStaphylococcus aureus COL by nine various stressors or growth limitingfactors as hydrogen peroxide, diamide, paraquat, nitric oxide, heat,puromycin, mupirocin, and oxygen limitation in the presence or absence ofnitrate. Based on a cytoplasmic reference map with 698 identified proteinspots (521 different proteins) common and specific features of the individualstress responses were analyzed. Whereas only the synthesis of UspA(SACOL1759) was generally induced in six out of the nine experiments,several other proteins showed stress-specific expression profiles. Forinstance, synthesis of Rex-controlled proteins (e.g. Ldh1, SrrAB) wasclearly induced only after oxygen limitation or nitrosative stress. Exposureto H 2O 2 led to a stepwise adaptation that affects mainly expression ofproteins involved in DNA repair and nucleotide metabolism. Furthermore,expression data of more than 70 so far uncharacterized proteins areavailable. This might provide initial indications of their possiblephysiological role. All data generated in this study are stored in a custommadeonline resource which might be helpful for the interpretation of new(e.g. in vivo derived) expression data in future.SRP034Yeast protein kinase C does not relocate to mitochondriaupon membrane stress: evidence for experimentalartefactsS. Meyer*Department of Genetics, University of Osnabrück, Osnabrück, GermanyProtein kinase C (Pkc1) of the Baker's yeast Saccharomyces cerevisiae is akey component of the cell wall integrity (CWI) signalling pathway, whichgoverns cell wall biosynthesis upon cell surface stress. This pathway isessential for survival under normal growth conditions (i.e. in the absence ofosmotic stabilization) and constitutes an ideal target for the development ofantifungal agents. In addition to its role in activating the central MAP kinasemodule of the CWI pathway, Pkc1 influences the dynamics of the actincytoskeleton and is involved in the secretory pathway.spektrum | Tagungsband 2011