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

Psp/IM30 protein family [2, 3]. Phage-shock proteins are widely conservedin bacteria, archaea, cyanobacteria and plants. LiaH forms large oligomericring structures reminiscent of those observed for PspA (E.coli) or Vipp1(A.thaliana). Comprehensive phenotypic profiling of lia mutants onlyrevealed weak sensitivities against cell envelope and oxidative stressconditions [3]. To gain a mechanistic insight into the physiological role ofB.subtilis LiaH, we searched for potential interaction partners. Bacterial twohybrid assays revealed a complex protein-protein interaction network inwhich LiaH is embedded. Moreover, we were able to demonstrate that LiaHplays an important role in protein secretion. Our collective data indicatesthat the lia system of B.subtilis has adopted a function similar to theproteobacterial phage-shock response, despite significant regulatorydifferences.[1] Jordan et al (2008): FEMS Microbiol. Rev. 32:107-146.[2] Mascher et al (2004): Antimicrob. Agents Chemother. 48:2888-2896.[3] Wolf et al (2010): J. Bacteriol. 192: 4680-93.SRV012Characterization of the farnesol-induced stress responsein Aspergillus nidulansD. Wartenberg* 1 , M. Voedisch 1 , O. Kniemeyer 1 , R. Winkler 2 ,A.A. Brakhage 11 Department of Molecular and Applied Microbiology, Hans Knöll Institute(HKI), Jena, Germany2 Department of Biotechnology and Food Engineering, Monterrey Institute ofTechnology, MexicoFarnesol is a sesquiterpene alcohol representing the first identified quorumsensing molecule in eukaryotic organisms. It is produced by the humanpathogenic fungus Candida albicans responsibly inhibiting the yeast-tohyphaeswitch and biofilm formation. Furthermore, it represses growth offilamentous fungi by triggering apoptosis as demonstrated for the modelorganism Aspergillus nidulans. We aimed to identify the molecular targetsof farnesol an thus carried out comparative proteome analysis. Aspergillusnidulans was grown in minimal medium over night and 50 μM farnesol wasadded 3 hours before harvesting. After preparing the protein extracts wecompared farnesol-induced and non-induced conditions by 2D-DIGE. Weidentified 53 proteins showing at least 1,5 fold significant alteration inrelative spot volume. Due to farnesol treatment many proteins involved incell cycle (Cdc48), morphogenesis (HexA) and general stress response wereup-regulated (HSP and ROS-detoxifying proteins). In addition we identifieda highly up-regulated protein of unknown function with a dehydrin-likemotif. Further proteome and northern blot analysis showed its involvementin an early response to farnesol. The corresponding deletion mutantexhibited no increased sensitivity to farnesol. However, the dehydrin-likeprotein is involved in osmoadaptation and sexual development whichexemplifies an additional target of farnesol in filamentous fungi.SRV013Structural und functional insight into pilus sensing by theCpx envelope stress systemS. Hunke* 1 , P. Scheerer 2 , N. Krauß 31 Department of Biology, Humboldt University, Berlin, Germany2 Institute for Medicinal Physics and Biophysics (CC2), Charite,Berlin,Germany3 School of Biological and Chemical Sciences, Queen Mary University ofLondon, London, United KingdomTwo-component signal transduction systems (TCS) are the predominantadaption machineries of bacteria to cope with environmental changes. Inmany TCSs auxiliary proteins enable responses to additional stimuli. TheCpx-TCS is the global modulator of cell envelope-stress that integrates verydifferent signals. It consists of the kinase CpxA, the regulator CpxR and theauxiliary protein CpxP. CpxP both inhibits activation of CpxA and isindispensable for the quality control system of P pili that are crucial foruropathogenic Escherichia coli during kidney colonization. However, it isnot clear how these two essential biological functions of CpxP are linked.We have solved the crystal structure of CpxP to 1.45 Å resolution with twomonomers being interdigitated like „left hands” forming a cap-shaped dimer(1). Our combined structural and functional studies suggest that CpxPinhibits the kinase CpxA through direct protein-protein interaction.It has been proposed that Cpx pathway activation is caused by titrating CpxPaway from CpxA (2). A prerequisite of this scenario is the detection ofunfolded proteins by CpxP which might result from a chaperone-likeactivity (3). We will not only provide evidence for a chaperone-like activityof CpxP but also corroborate the functionality of an extended hydrophobiccleft on the convex surface of CpxP as a recognition site for misfolded pilussubunits. Therefore, we analyzed the capacities of the CpxP single-sitemutants to promote pili degradation in vivo.From our combined results we propose a model that elucidates bothfunctions of CpxP. Accordingly, the structural details of CpxP provide a firstinsight how a periplasmic TCS inhibitor blocks its cognate kinase and isreleased from it.[1] Zhou, Keller, Volker, Krauß, Scheerer and Hunke, in revision.[2] Isaac et al. (2005): Proc Natl Acad Sci USA 102, 17775-17779.[3] DiGiuseppe, P.A., and Silhavy, T.J. (2003): J Bacteriol 185, 2432-2440.SRV014In vivo phosphorylation patterns of key stressosomeproteins define a second feedback loop that limitsactivation of Bacillus subtilis σ BC. Eymann* 1 , S. Schulz 1 , K. Gronau 1 , D. Becher 1 , M. Hecker 1 , C.W. Price 21 Institute for Microbiology, Ernst-Moritz-Arndt-University, Greifswald,Germany2 Department of Microbiology, University of California, Davis, USAThe Bacillus subtilis stressosome is a 1.8 MDa complex that orchestratesactivation of the σ B transcription factor in response to environmental signals.It comprises members of the RsbR co-antagonist family and the RsbSantagonist, whose similar STAS domains form a core that sequesters theRsbT serine-threonine kinase. Stress-induced phosphorylation of the STASdomains by RsbT is associated with its release from core, allowing RsbT toactivate a downstream regulator. Here we investigate the in vivophosphorylation of RsbRA and its RsbRB, RsbRC and RsbRD paralogs,whose STAS domains share two conserved threonine residues. In unstressedcells these RsbR proteins are known to be phosphorylated on their more N-terminal threonine, exemplified by RsbRA T171. T171 phosphorylation isthought to be prerequisite but not the trigger for activation, which correlatesinstead with stress-induced serine phosphorylation of RsbS. We show herethat all the initial threonine modifications require RsbT kinase. Also,phosphorylation on the more C-terminal threonine, exemplified by RsbRAT205, had not been detected in vivo. We find (i) RsbRA is additionallyphosphorylated on T205 following strong stresses; (ii) this modificationdepends on RsbT; and (iii) T205A substitution greatly increases post-stressactivation of σ B . We infer that T205 phosphorylation constitutes a secondfeedback mechanism that limits σ B activation, operating in addition to theRsbX feedback phosphatase. Loss of RsbX function greatly increases thefraction of phosphorylated RsbS and doubly phosphoylated RsbRA inunstressed cells. Thus RsbX both maintains the ready state of thestressosome prior to stress, and restores it post-stress. Because similar RsbR-S-T modules are found in diverse bacteria, our results may have broadapplication.SRV015Signal perception and transduction by the transcriptionalactivator CadC of Escherichia coliI. Haneburger* 1 , S. Buchner 1 , A. Eichinger 2 , C. Koller 1 , A. Skerra 2 , K. Jung 11 Department I - Microbiology, Ludwig-Maximilians-University MunichMartinsried, Germany2 Department for Biological Chemistry, Technical University Munich,Freising-Weihenstephan, GermanyAdaptation of E. coli to acidic stress is mediated by the concerted action ofseveral proteins, among them the inducible amino acid decarboxylasesystems. One of these systems is the Cad system that is induced at lowexternal pH and concomitantly available lysine. The transcriptional activatorCadC of the Cad system belongs to the ToxR-like proteins that arecharacterized by a common topology. These proteins possess a periplasmicsensor domain, a single transmembrane helix and a cytoplasmic DNAbindingdomain. Recent data revealed that the periplasmic domain of CadCis responsible for pH sensing, while lysine signaling is mediated by aninteraction of CadC with the lysine permease LysP. We are interested inelucidating how the inner-membrane protein CadC is able to perceive andtransduce these signals across the membrane and subsequently activatestranscription of the cadBA operon.spektrum | Tagungsband 2011