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

hythm. Furthermore, an autokinase activity of the RspKaiC could be shownby an in vitro phosporylation assay. These data suggest the existence of afunctional timing mechanism in purple photosynthetic bacteria. Futureresults may shed some light on the evolution of clock systems and circadianrhythms in bacteria.[1] Nakajima, M. (2005): Science. 308, 414-415.[2] Holtzendorff, J. (2008): Journal of Biological Rhythms, 23, 187-199.[3] Min, H. (2005): FEBS letters.579 808-812.RGP014The global regulator Hfq participates in the singletoxygen stress response of Rhodobacter sphaeroidesB. Berghoff* 1 , J. Glaeser 1 , C. Sharma 2 , M. Zobawa 3 , F. Lottspeich 3 ,J. Vogel 2 , G. Klug 11 Institute for Micro- and Molecular Biology, Justus-Liebig-University,Gießen, Germany2 InstituteMolecular Infection Biology, Julius-Maximilians-University,Würzburg, Germany3 Protein Analytics, Max Planck Institut for Biochemistry, Martinsried,GermanyRhodobacter sphaeroides is a facultative phototrophic alphaproteobacteriumwhich is intensively studied in regard to regulation ofphotosynthesis genes. Furthermore, it is an established model organism forstudying the response to singlet oxygen ( 1 O 2), a highly reactive oxygenspecies, generated by illumination of the photosynthetic apparatus underoxic conditions. The regulatory response to 1 O 2 encompasses the inductionof several alternative sigma factors, which in turn induce several smallRNAs (sRNAs). In a previous RNA-seq study based on 454pyrosequencing, we have identified five sRNAs which were either inducedor processed under 1 O 2 stress [1, 2]. Their induction depends on the RpoEand RpoH II sigma factors, which are known to be the major regulators of the1 O 2 response. Accordingly, 1 O 2 dependent regulatory networks, comprisedof sigma factors and sRNAs, exist in R. sphaeroides.The conserved RNA-chaperone Hfq is one of the key players in sRNAmediatedregulation in many bacteria and is required for the stability ofmany sRNAs as well as to facilitate the interaction between sRNAs and theirtarget mRNAs. The phenotype of the R. sphaeroides 2.4.1Δhfq straincomprises higher sensitivity towards 1 O 2, reduced pigmentation, andminicell formation.To get insights into the possible roles of Hfq in R. sphaeroides and the 1 O 2response and to identify the direct sRNA and mRNA binding partners ofHfq in this bacterium, we used a co-immunoprecipitation strategy combinedwith deep sequencing as previously described for Salmonella [3] andconfirmed the Hfq-dependency of several known and also newly identifiedsRNAs by Northern blot analysis. Strikingly, >70% of the Hfq-associatedsRNAs were 1 O 2-affected. Among Hfq-associated mRNAs we found severalmRNAs for cell division and ribosomal proteins. In addition, gel-basedproteomics revealed an influence of Hfq on RpoH II-dependent genes, aminoacid transport/metabolism, and ATP synthase.Overall, this study suggests Hfq to be a global regulator like in otherbacteria and largely explains the pleiotropic phenotype of strain 2.4.1Δhfq.The extensive work on sRNAs in R. sphaeroides will help to solve thequestion of how photosynthetic bacteria manage an effective 1 O 2 stressresponse.[1] Berghoff, B.A. et al (2009): Photooxidative stress-induced and abundant small RNAs inRhodobacter sphaeroides. Mol. Microbiol., 74(6), 1497-512.[2] Nuss, A.M. et al (2010): Overlapping alternative sigma factor regulons in the response to singletoxygen in Rhodobacter sphaeroides. J. Bacteriol., 192: 2613-2623.[3] Sittka, A. et al (2008): Deep sequencing analysis of small noncoding RNA and mRNA targets ofthe global post-transcriptional regulator, Hfq. PLoS Genet., 4(8), e1000163.RGP015Response of the three-component system NreABC ofStaphylococcus carnosus to oxygen and nitrateS. Nilkens*, M. Singenstreu, F. Reinhart, G. UndenInstitute for Microbiology and Wine Research, Johannes-Gutenberg-University, Mainz, GermanyThe NreBC two-component system is required for activation of nitraterespiration in Staphylococcus carnosus [1]. The sensor kinase NreB containsan O 2 sensitive [4Fe-4S] 2+ cluster which is converted by O 2 to a [2Fe-2S] 2+cluster followed by complete degradation and formation of FeS-lessapoNreB [2]. The accessibility of the four Cys residues of NreB toalkylating agents was used to differentiate Fe-S-containing NreB and Fe-SlessapoNreB in vivo [3]. In anaerobic bacteria most of the NreB exists as[4Fe-4S] 2+ -NreB, whereas in aerobic bacteria apoNreB represents the majorand physiological relevant form. The half-life of [4Fe-4S] 2+ -NreB/apo-NreBconversion was 3 minutes after addition of air to anaerobic bacteria.NreB and NreC are encoded in one operon together with the GAF-domainprotein NreA. Deletion of NreA results in activation of nitrate respirationunder aerobic conditions. The lipase gene lip from S. hyicus was fused to thenarG promoter and used as a reporter gene to investigate mutations in NreA.NreA was required for normal function in O 2 and nitrate sensing, suggestingthe presence of an NreABC three-component system.[1] Kamps, A. et al (2004): Mol. Microbiol. 52, 713-723.[2] Müllner, M. et al: (2008): Biochem. 47, 13921-13932.[3] Reinhart, F. et al (2010): J. Bacteriol. 192(1), 86-93.RGP016Activity of the two-component regulatory system CiaRHin Streptococcus pneumoniae R6A. Schnorpfeil*, A. Halfmann, M. Müller, P. Marx, U. Günzler,R. Hakenbeck, R. BrücknerDepartment of Microbiology,University of Kaiserslautern, Kaiserslautern,GermanyThe two-component regulatory system CiaRH of Streptococcus pneumoniaeaffects a variety of processes such as competence development, autolysis,bacteriocin production, host colonization, and virulence. While the targets ofthe regulator CiaR are known, the role of phosphorylation in CiaRregulation has not been defined. To address this issue, the presumedphosphorylation site of CiaR, aspartic acid at position 51, was replaced byalanine. The mutant CiaRD51A protein was no longer able to activate CiaRdependentpromoters, strongly suggesting that the phosphorylated form ofCiaR is active in regulation. However, depending on the growth medium,inactivation of the kinase gene ciaH resulted in a subtle increase of CiaRdependentpromoter activities or in a strong reduction. Therefore, CiaH mayact as a kinase or phosphatase and CiaR is apparently able to obtain itsphosphate independently of CiaH. On the other hand, promotermeasurements in cells with an intact CiaRH system demonstrated a high,nearly constitutive, expression level of the CiaR regulon independent fromthe growth medium. Thus, in contrast to many other two-componentregulatory systems, CiaRH has apparently evolved to maintain high levels ofgene expression under a variety of conditions rather than respondingstrongly to a signal.RGP017Temporal and spatial changes in the localization and thecomposition of the RNA degrading exosome in SulfolobussolfataricusE. Evguenieva-Hackenberg*, C. Lassek, L. Hou, C. Whitharana, V. Roppelt,G. KlugInstitute for Micro- and Molecular Biology, Justus-Liebig-UniversityGießen, GermanyMany macromolecular complexes and even RNA molecules previouslythought to be distributed in the cytoplasm, were recently shown to havespecific subcellular localization in prokaryotic cells (1). Recently we haveshown that the archaeal exosome, an RNA degrading and RNA-tailingprotein complex (2), is localized at the cell periphery in thehyperthermophilic and acidophilic archaeon Sulfolobus solfataricus (3).Further studies revealed that the localization of the exosome changes indifferent growth phases: while the vast majority of the exosome is insoluble(at the cell periphery, most probably at the membrane) during theexponential growth, more than the half of the exosome is soluble (in thecytoplasm) in the stationary phase. At the cell periphery, the exosomeinteracts with the archaeal DnaG, which seems to be responsible for thelocalization. DnaG is exchanged by another protein, annotated as a premRNAsplicing protein, in the cytoplasmic form of the exosome. Data onthe analysis of the protein-protein interactions in the two forms of theexosome as well as on the impact of the composition on the function of theexosome will be shown and discussed.[1] Evguenieva-Hackenberg, E. et al (2011): Subcellular localization of RNA degrading proteins andprotein complexes in prokaryotes. RNA Biology, in press.[2] Evguenieva-Hackenberg, E. and G. Klug (2009): RNA degradation in the Archaea. Progress inMolecular Biology and Translational Science 85: 275-317.spektrum | Tagungsband 2011