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

the common Hfq protein. The identification of diverse sRNA candidatesindicates that riboregulation constitutes an important layer of generegulation in Helicobacter. Research in H. pylori will also help to shed lighton sRNA-mediated regulation in other ε-proteobacteria, includingwidespread and emerging pathogens such as Campylobacter.[1] Sharma, C.M. et al (2010): The primary transcriptome of the major human pathogen Helicobacterpylori. Nature, 464(7286):250-5.RGV004Regulation of Ammonium Uptake and ComplexFormation between Amt and GlnK ProteinsT. Pflüger, T. Wacker*, S.L.A. Andrade, C. Hernández, S. Maier,S. HelfmannInstitute for Organic Chemistry and Biochemistry, Albert-Ludwigs-University, Freiburg, GermanyAmmonium transport (Amt) proteins are a family of integral membraneproteins that specifically transport NH 3/NH 4 + across biological membranes.Although high resolution structures are known for E. coli AmtB [1], A.fulgidus Amt-1 [2], N. europaea Rh50 [3] and the human RhCG [4], anumber of controversies persist around several aspects of the transportmechanism [5]. In order to gain a better understanding of Amt function andregulation, we use the hyperthermophilic euryarchaeon Archaeoglobusfulgidus as a working model. Its genome organization shows three amtgenes, each directly linked to a glnK gene within one operon. GlnKs aretrimeric cytoplasmic proteins that belong to the P II family and have a keyfunction in the regulation of nitrogen assimilation in the cell. They can bind,and thus directly sense, effector molecules such as adenosine diphosphate,adenosine triphosphate and 2-oxoglutarate. The integration of these signals(energy and carbon cellular status, respectively) by GlnK proteins can resultin a complex formation with Amt in the membrane and consequent blockageof NH 3/NH 4 + uptake [6]. We have characterized all three A. fulgidus GlnKproteins in their interaction mode with effector molecules by means of X-raycrystallography and isothermal titration calorimetry [7] and investigate Amt-GlnK complex formation events. These findings provide new insights intothe regulation of ammonium uptake and nitrogen assimilation in A. fulgidusin particular, and in archaea in general.[1] Khademi, S. et al (2004): Science, 305, 1587; L. Zheng et al, PNAS 101, 17090.[2] Andrade, S. L. A. et al (2005): PNAS, 102, 14994.[3] Lupo, D. et al (2007): PNAS, 104, 19303; X. Li et al (2007): PNAS, 104, 19279.[4] Gruswitz, F. et al (2010): PNAS, 107, 9638.[5] S.L.A. Andrade, S.L.A. et al (2007): Mol. Memb. Biol. 24, 357.[6] Gruswitz, F. et al (2007): PNAS, 104, 42 ; Conroy et al (2007): PNAS 104, 1213.[7] Helfmann, S. et al (2010): JMB, 402, 165 ; C. Litz et al (2011): Acta Cryst. F, in press.RGV005The role of the cytoplasmic PAS domain of theEscherichia coli histidine kinase DcuS in signaltransductionP. Degreif-Dünnwald*, G. UndenInstitute for Microbiology and Wine Research, Johannes-Gutenberg-University, Mainz, GermanyBacteria contain membrane integral sensors for the response to changingenvironmental conditions. Many of the sensors are two-component systemsconsisting of a sensor histidine kinase and a response regulator that triggersthe cellular response [1]. DcuS, the C 4-dicarboxylate sensor of E. coli is amembrane integral histidine kinase [2]. DcuS is a multidomain proteinconsisting of a sensory periplasmic PAS P (Per-Arnt-Sim) domain, twotransmembrane helices, a cytoplasmic PAS C and the C-terminal kinasedomain.PAS domains are ubiquitous signalling modules found in all kingdoms oflife. They can detect many different stimuli including light, oxygen, redoxpotential and various small molecules and also modulate protein-proteininteractions. PAS domains are characterised by a conserved α/β-fold. ManyPAS domains with sensory function have been identified but a large numberof PAS C domains contain no apparent cofactor and their function isunknown [3].A combination of mutation, protein-protein interaction and solid-state NMRexperiments [4] were used to study the structure and function of a membraneembedded construct of DcuS and of the PAS C domain. The experimentsshow that PAS C has no sensory function and is responsible for signaltransduction from PAS P to the C-terminal histidine kinase.[1] Mascher, T. et al (2006): Microbiol Mol Biol Rev 70: 910-938.[2] Zientz, E. et al (1998): J. Bacteriol 180: 5421-5425.[3] Taylor, B. L. and I. B. Zhulin (1999): Microbiol Mol Biol Rev 63: 479-506.[4] Etzkorn et al (2008): Nature Struct & Mol Biol 15:1031-1039.RGV006LaeA control of velvet family regulatory proteins forlight-dependent development and fungal cell-typespecificityÖ. Sarikaya Bayram* 1 , Ö. Bayram* 1 , O. Valerius 1 , H.S. Park 2 , S. Irniger 1 ,J. Gerke 1 , M. Ni 2 , K.-H. Han 3 , J.-H. Yu 2 , G. Braus 11 Molecular Microbiology and Genetics, Georg-August-University,Göttingen, Germany2 Departments of Bacteriology and Genetics, University of Wisconsin-Madison, Madison, USA3 Department of Pharmaceutical Engineering, Woosuk University, Wanju,Korea, Korea, Republic ofVeA is the founding member of the velvet superfamily of fungal regulatoryproteins. This protein is involved in light response and coordinates sexualreproduction and secondary metabolism in Aspergillus nidulans. In the dark,VeA bridges VelB and LaeA to form the VelB-VeA-LaeA (velvet) complex.The VeA-like protein VelB is another developmental regulator, and LaeAhas been known as global regulator of secondary metabolism. In this study,we show that VelB forms a second light-regulated developmental complextogether with VosA, another member of the velvet family, which repressesasexual development. LaeA plays a key role not only in secondarymetabolism but also in directing formation of the VelB-VosA and VelB-VeA-LaeA complexes. LaeA controls VeA modification and protein levelsand possesses additional developmental functions. The laeA null mutantresults in constitutive sexual differentiation, indicating that LaeA plays apivotal role in inhibiting sexual development in response to light. Moreover,the absence of LaeA results in the formation of significantly smaller fruitingbodies. This is due to the lack of a specific globose cell type (Hülle cells),which nurse the young fruiting body during development. This suggests thatLaeA controls Hülle cells. In summary, LaeA plays a dynamic role in fungalmorphological and chemical development, and controls expression,interactions and modification of the velvet regulators.RGP001Fluorescence-based monitoring of the nitrogen status inCorynebacterium glutamicumN. Rehm*, N. Jeßberger, S. Worsch, A. BurkovskiInstitute for Microbiology, Friedrich-Alexander-Universiy, Erlangen,GermanyAmtR, a member of the TetR protein family, is the master regulator ofnitrogen control in Corynebacterium glutamicum [1]. This repressor, whichacts as a dimer, regulates transcription of at least 38 genes when ammonium,the preferred nitrogen source of C. glutamicum, becomes limiting [2, 3].Upon ammonium starvation, AmtR-controlled genes are transcribed. Untilnow, detection of nitrogen starvation in C. glutamicum was only possible byin vitro assays such as RNA hybridization experiments and DNAmicroarrays. The aim of this study was to establish an in vivo nitrogenmonitoring system that allows a more rapid detection of nitrogen limitation.Therefore, promoter regions of AmtR-controlled genes were clonedupstream of a plasmid-encoded gfpuv gene using the pEPR1 plasmid [4].Fluorescence spectroscopy as well as fluorescence microscopy showed thatthe strictly AmtR-dependent promoters amtA P, amtB P and gltB P are wellsuited for a fluorescence-based reporter system: whereas under goodammonium supply no fluorescence was observed, cells that were starved forammonium showed high fluorescence signals. Moreover, this method alsorevealed that alternative nitrogen sources than ammonium differentiallyaffect AmtR-controlled gene expression.[1] Jakoby, M., Nolden, L., Meier-Wagner, J. Krämer, R. and Burkovski, A. (2000). Mol. Microbiol.37, 964-977.[2] Beckers, G., Strösser, J., Hildebrandt, U., Kalinowski, J., Farwick, M., Krämer, R., Burkovski, A.(2005). Mol. Microbiol. 58, 580-595.[3] Buchinger, S., Strösser, J., Rehm, N., Hänßler, E., Hans, S., Bathe, B., Schomburg, D., Krämer,R., Burkovski, A. (2009). J. Biotechnol. 140, 68-74.[4] Knoppová, M., Phensaijai, M., Veselý, M., Zemanová, M., Nesvera, J., Pátek, M. (2007). Curr.Microbiol. 55, 234-239.spektrum | Tagungsband 2011