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

CODH after overexpression in E. coli together with the other componentspossibly involved (rubredoxin, ferredoxin, reverse rubrerythrin).SRP025Cold stress in Antarctic fungi targets enzymes of theglycolytic pathway and tricarboxylic acid cycleN. Kostadinova*, M. Angelova, R. Abrashev, J. MitevaBulgarian Academy of Sciences, Mycology, Sofia, BulgariaThe ability of microorganisms to survive and thrive within hostileenvironments depends on rapid and robust stress responses. Antarctic fungihad to develop molecular mechanisms of adaptation to extreme lowtemperatures, but little is known about the effect of cold stress on theexpression of key enzymes of the basic metabolic pathways. To investigatethe role of those enzymes in cold tolerance two Antarctic fungal strains(psychrotrophic Penicillium sp. 161 and mesophilic Aspergillus glaucus363) grown at the optimal temperature (20 and 25°C, respectively) weresubjected to temperature downshift (10 and 4°C), and several enzymesinvolved in carbon metabolism, including hexokinase (HK; EC 2.7.1.1),glucose-6-phosphate dehydrogenase (G6PDH; EC 1.1.1.49),glyceraldehyde-3-phosphate dehydrogenase (GAPDH; EC 1.2.1.12), lactatedehydrogenase (LDH; EC 1.1.1.27), succinate dehydrogenase (SDH; EC1.3.99.1), isocitrate dehydrogenase (IDH; EC 1.1.1.42), and malatedehydrogenase (MDH; EC 1.1.1.37) were assessed. While the activity of theHK was decreased, the activity of G6PDH was increased at low temperatureshowing a switch from Embden-Myerhoff pathway (EMP) to pentosephosphate pathway (PPP). Enhanced GAPDH activity support thehypothesis for its crucial role in antioxidant cell response. Modulation ofLDH, a biomarker of oxidative stress, depends on temperature characteristicof the model strains. The same tendency was found about enzymes ofTricarboxylic Acid Cycle.Acknowledgements: This work was supported by the European SocialFund, Operational Programme Human Resources Development (grantBG051PO001-3.3.04/32) and National Scientific Fund of the Ministry ofEducation and Science, Bulgaria (grant VU-B-205/06).SRP026Regulation of RpoS proteolysis by multiple input signalsduring the growth phase in Escherichia coliC. Kanow-Scheel*, R. HenggeInstitute for Biology – Microbiology, Free University, Berlin, GermanyThe RpoS (σ S ) subunit of RNA polymerase is the master regulator of thegeneral stress response in Escherichia coli. Regulation of RpoS, whichresponds to many different environmental and cellular stresses, occurs at thelevels of transcription, translation, proteolysis and protein activity [3]. RpoSdegradation, which has become a paradigm of proteolytic regulation inbacteria, is initiated by binding of phosphorylated RssB, a responseregulator acting as a proteolytic targeting factor. This interaction results in astructural rearrangement that exposes the ClpX6-binding site close to theRpoS N-terminus. Using ATP hydrolysis, RpoS is then unfolded, threadedinto the proteolytic chamber formed by the ClpP14 part of the ClpXPcomplex and completely degraded, whereas RssB is released [2].In recent work, we have observed that successive RpoS stabilization andaccumulation during the post exponential and beginning stationary phase ofEscherichia coli is a complex multistep process, with terminal stabilizationin early stationary phase. Mechanistically, RpoS stabilization is based onchanges in the ratio between free RpoS and phosphorylated RssB (which islimiting for the RpoS degradation rate [4]) by titration, competition and/orsequestration of either binding partner [2]. Thus, RssB can be titrated by asudden massive increase in RpoS synthesis (e.g. in response to certainstresses). Alternatively, RpoS can be protected from proteolysis by increasedbinding to RNA polymerase, which is mutually exclusive with RssB binding[5]. Moreover, RssB can be sequestered by Ira proteins [1]. Furthermore,ClpX6 not only has to unfold RpoS, but also to strip it from tightly boundphosphorylated RssB, indicating that RpoS proteolysis has a particularlyhigh ATP requirement. Consistently, we observed that successive RpoSstabilization correlates to decreasing cellular ATP levels, suggesting thatenergy starvation seems to trigger RpoS stabilization by reducing theintracellular ATP pool below a threshold required for terminal unfolding,RssB release and degradation of RpoS.[1] Bougdour, A. et al (2008): Multiple pathways for regulation of sigmaS (RpoS) stability inEscherichia coli via the action of multiple anti-adaptors. Mol. Microbiol. 68: 298-313.[2] Hengge, R. (2009): Proteolysis of σ S (RpoS) and the general stress response in Escherichia coli.Res. Microbiol. 160: 667-676.[3] Hengge, R. (2010): The general stress response in Gram-negative bacteria. In: G. Storz and R.Hengge (ed.), Bacterial Stress Responses (2nd edition). ASM Press, Washington, D.C. pp. 251-289.[4] Pruteanu, M. and R. Hengge-Aronis (2002): The cellular level of the recognition factor RssB israte-limiting for σ S proteolysis: Implications for RssB regulation and signal transduction in σ Sturnover in Escherichia coli. Mol. Microbiol. 45:1701-1714.[5] Typas, A. et al (2007): Stationary phase reorganisation of the Escherichia coli transcriptionmachinery by Crl protein, a fine-tuner of sigmas activity and levels. EMBO. 26:1569-1578.SRP027Structural insides of the envelope stress sensor kinaseCpxA - What causes the specificity of two componentsystems?V.S. Müller* 1 , P. Scheerer 2 , T.F. Meyer 3 , S. Hunke 11 Institute for Biology, Humboldt-University, Berlin, Germany2 Institute of Biochemistry, Charité Berlin, Berlin, Germany3 Department of Molecular Biology, Max Planck Institute for InfectionBiology, Berlin, GermanyThe predominant family of signaling proteins in bacteria is the twocomponentsignal transduction system (TCS). In general it consists of asensor histidine kinase (HK) that after autophosphorylation transfers thephosphoryl group to its cognate response regulator (RR), which than effectschanges in bacterial physiology. TCSs are essential for bacteria for sensingtheir environment during infection enabling optimal virulence factorproduction and protection against the host immune response. AlthoughTCSs have remarkable similarities in sequence and structure, only smallcrucial differences seems to have a major impact, which not only results in aspecific regulatory readout but also prevents unwanted cross-talk betweennon-cognate signalling systems (1). Structural information on signaltransduction proteins are a prerequisite to identify the crucial attributes thatguarantee specificity.Here, we present a structural model of the catalytic cytosolic part of theenvelope stress HK CpxA in contact with its cognate RR CpxR based on theHK/RR co-crystal structure solved by the group of Marina (2). Thestructural model of CpxA and CpxR enabled us to identify critical aminoacids located in the interface of CpxA to CpxR that contribute specificitybetween HK and RR (1). To corroborate the functionality of these residues,we analyzed the capacities of single, double, triple or quadruple substitutionsin the interface of CpxA on the efficiency to bind CpxR. Therefore, wepreformed in vivo crosslinking with these different variants of the membraneanchored HK CpxA (Membrane-SPINE) to monitor the impact of theidentified residues on the protein-protein interaction between the HK and theRR. We could confirm the predicted effects on RR binding by thesubstitution of essential amino acids for the first time in vivo.Altogether, the structural insides of CpxA in complex with CpxR willstrikingly contribute to a better understanding of these central signaltransduction systems.[1] Capra et al (2010): PLoS Genet. e1001220.[2] Casino et al (2009): Cell 139: 325.SRP028New aspects in the regulation of the acid stress responsesystem Cad in Escherichia coliS. Ude* 1 , K. Jung 1Department of Biology I, Center for Integrated Protein Science Munich(CiPSM), Ludwig-Maximillians-Universtiy Munich, Martinsried, GermanyOn their way from the stomach to the gut enterobacteria are exposed tosubstantial change in the pH range. As the stomach can reach a pH value aslow as 1.0, neutrophilic bacteria had to evolve several strategies to survivethis extreme stress condition while maintaining their internal homeostasis.One acid response system is the lysine dependent Cad system. It consists ofthe enzyme CadA which catalyzes the decarboxylation of lysine tocadaverine while consuming a cytoplasmic proton and releasing CO 2. Thisreaction results in the increase of the internal pH. Furthermore, thelysine/cadaverine antiporter CadB, the membrane-integrated protein CadCand the lysine permease LysP are involved in the Cad system [2; 3]. Undernon-inducing conditions, the secondary lysine transporter LysP represses thetranscriptional activator CadC, whereas under low pH, anaerobiosis and inpresence of lysine CadC is released and can act as an activator oftranscription of the cadBA operon. A second repressor of the cadBA operonis the small histon-like molecule H-NS [1]. To make the picture morecomplex, several other proteins such as the lysine-2,3-aminomutase YjeK,spektrum | Tagungsband 2011