VAAM-Jahrestagung 2012 18.–21. März in Tübingen

198demonstrating its suitability as a selection marker. The introduction of afunctional promoter of M. adhaerens HP15 into the IVET vector and itssubsequent transformation intopyrB-deficient mutant allowed itscomplementation. Transformants expressing the pyrB gene and lacZ grewin absence of uracil indicating that the system wass functional. Thestandardization of the IVET screening is currently being tested. Promisinggenes obtained will be cloned, mutagenized, and characterized in terms oftheir role in diatom-bacteria interaction. Results of this study willcontribute to a better understanding of the molecular mechanisms ofdiatom-bacteria interactions.RSP015Functional analyses of small RNAs in Agrobacterium tumefaciensA. Overlöper* 1 , P. Möller 1 , B. Voss 1,2 , W. Hess 2 , C. Sharma 3 , F. Narberhaus 11 Ruhr-University, Biology of Microorganisms, Bochum, Germany 2 Institute ofBiology III, University Freiburg, Freiburg, Germany3 Institute for Molecular Infection Biology, University Würzburg, Würzburg,GermanyOver the last decade, sRNAs have been recognized as widespreadregulators of gene expression in bacteria (1). The largest and mostextensively studied set of sRNAs act through base pairing with targetRNAs, usually modulating the translation and stability of mRNAs (2).Using a comparative bioinformatic approach (3) we identified diversesRNAs in the plant pathogen Agrobacterium tumefaciens. One sRNA,called AbcR1, controls the expression of at least three ABC transportersamong them the periplasmic binding protein of the GABA transporter. It isthe first described bacterial sRNA that controls uptake of a plant-generatedsignaling molecule (4). The molecular details of the sRNA-mRNAinteraction will be presented.By using a differential RNA sequencing (dRNA-seq) technology, wediscovered many new sRNA on all four A. tumefaciens replicons, thecircular chromosome, the linear chromosome, the At-plasmid and the Tiplasmid(5). Northern blot analyses revealed that several sRNAs weredifferentially expressed in response to different growth conditions. OnesRNA from the Ti-plasmid was massively induced under virulenceconditions. Experiments to identify targets of selected sRNAs are underway.1. Narberhaus, F. and J. Vogel, Regulatory RNAs in prokaryotes: here, there and everywhere. MolMicrobiol, 2009. 74(2): p. 261-9.2. Waters, L.S. and G. Storz, Regulatory RNAs in bacteria. Cell, 2009. 136(4): p. 615-28.3. Voss, B., et al., Biocomputational prediction of non-coding RNAs in model cyanobacteria. BMCGenomics, 2009. 10: p. 123.4. Wilms, I., et al., Small RNA-mediated control of the Agrobacterium tumefaciens GABA binding protein.Mol Microbiol, 2011. 80(2): p. 492-506.5. Wilms, I., et al., Deep sequencing uncovers numerous small RNAs on all four replicons of the plantpathogen Agrobacterium tumefaciens. RNA Biology, in pressRSP016Signal transduction in the thermoacidophilic crenarchaeonSulfolobus acidocaldariusJ. Reimann*, K. Lassak, S. Khadouma, S.-V. AlbersMax Planck Institute for Terrestrial Microbiology, Molecular Biology ofArchaea, Marburg, GermanySignal transduction from extracellular stimuli to the inside and within thecell is essential for survival of microorganisms. In this process proteinkinases and phosphatases often play a key-role and are found in all threedomains of life. These enzymes catalyze one of the most importantposttranslational modifications, the reversible phosphorylation anddephosphorylation of proteins. Whereas in many Euryarchaeota both,potential histidine kinases and Ser/Thr/Tyr kinases, were found, in theCrenarchaeota just the latter are present.To date, the knowledge about signal transduction pathways, the inducingconditions and involved proteins in the Crenarchaeota is rather scarce.Therefore, we want to investigate the processes of signal transduction inSulfolobus acidocaldarius. The advantage of this important crenarchaealmodel organism is the availability of various genetic tools to performinvivoandin vitrostudies. These tools were used to examineautophosphorylation of some protein kinases and phosphorylation ofpotential interaction partners. Experimental investigations revealed aconnection between motility via the archaeal flagellum and different signaltransduction proteins in S. acidocaldarius. These results underline theimportance of protein phosphorylation in cellular processes of theArchaea.RSP017NreA, the third component of the three-component systemNreABC of Staphylococcus carnosusM. Singenstreu*, S. Nilkens, G. UndenJohannes Gutenberg University, Institute for Microbiology and WineResearch, AG Unden, Mainz, GermanyIn the facultative anaerobic Staphylococcus carnosus the NreABC threecomponent system is required for initiation of nitrate respiration [1]. NreA,NreB, and NreC are encoded within one operon (nreABC). The twocomponentsystem NreBC is involved in O 2 sensing. NreB acts as a directoxygen sensor, and the regulator NreC induces the expression of narGHJIencoding nitrate reductase under anaerobic conditions [1].Oxygen sensing by NreB is based on the conversion of the [4Fe-4S] 2+cluster to a [2Fe-2S] 2+ cluster by O 2 followed by complete degradation andformation of FeS-less apoNreB [2].The function of the third component, NreA, was analyzed. NreA is a GAFdomain protein. Deletion of NreA leads to a permanent activation ofnitrate respiration.Single-point mutants in NreA were obtained with either loss of nitrateinduction, or aerobic derepression, suggesting that NreA controls NreBCfunction in response to oxygen and nitrate availability.[1] Kamps et al. (2004) Mol. Microbiol. 52, 713-723[2] Müllner et al. (2008) Biochemistry 47, 13921-13932RSP018Binding properties of the transcriptional regulator AlsR ofBacillus subtilisE. Härtig*, C. Frädrich, K. HaufschildtTU Braunschweig, Microbiology, Braunschweig, GermanyThe transcriptional regulator AlsR is essential for alsSD expression inBacillus subtilis. The alsSD expression is activated in response tofermentative growth conditions, addition of acetate, low pH in the growthmedium and aerobic stationary growth. The alsSD operon encodes theacetolactate synthase and -decarboxylase catalysing the production ofacetoin from pyruvate. The AlsR regulator is a member of the LysR-typetranscriptional regulators (LTTR) composed of two domains: an N-terminal DNA binding domain with a winged HTH motif and a C-terminalregulatory domain which is involved in co-inducer binding andoligomerization.We analyzed the relevance of single amino acid residues of the DNAbindingdomain by site directed mutagenesis and in vivo functionalanalysis of produced AlsR mutant proteins in an in vivo complementationsystem. Here, mutated alsR genes were integrated into the amyE locus ofan B. subtilis alsR knock out mutant strain and expressed under the controlof the xylose-inducible xylA promoter. AlsR activity was monitored by ß-galactosidase activities derived from an AlsR-dependent alsS-lacZ reportergene fusion. Several AlsR mutants tested showed reduced alsS-lacZexpression in vivo. In addition, we produced and purified the AlsR mutantproteins as AlsR-Strep fusion proteins and analyzed their in vitro bindingability by gel retardation analyses.Using DNase I footprint analyses AlsR binding regions were identified inthe alsS promoter. A detailed analysis of the DNA sequence revealedseveral potential palindromic binding sites containing a T-N 11-A coremotif typical for LTTR proteins. To identify the DNA sequences necessaryfor AlsR binding we changed several TA bases within the proposed AlsRbinding region to GG. For this purpose a p-86alsS-lacZ reporter genefusion with 86 bp promoter sequences upstream the transcriptional startsite were used. The ß-galactosidase activities mediated by those mutantpromoters were determined and compared to the activity of B. subtiliscarrying the wild type alsS-lacZ fusion. In order to directly relate theresults of the in vivo tested mutated promoter to AlsR binding, we alsoemployed gel retardation assays.RSP019The LuxR solo PluR of Photorhabdus luminescens sensesPLAI-1, a novel endogenous signaling moleculeS. Brameyer* 1 , A.O. Brachmann 2 , Q. Zhou 2 , H. Bode 2 , R. Heermann 11 Ludwig-Maximilians-Universität München, Mikrobiologie, München, Germany2 Goethe-Universität Frankfurt, Institut für Molekulare Biowissenschaften,Frankfurt am Main, GermanyCell-to-cell communication via acyl-homoserine lactones (AHL) is wellstudied in many Gram-negative bacteria. The prototypical communicationsystem consists of a LuxI-type autoinducer synthase and a LuxR-typereceptor that detects the endogenously produced signal. The symbiotic andentomopathogenic enteric bacterium Photorhabdus luminescens harborsthe plenty of 39 LuxR-like receptors, but lacks any LuxI-type autoinducersynthase and is unable to produce AHL. Here we show that one of theseLuxR solos, Plu4562 (PluR), detects an endogenously produced signalingmolecule (PLAI-1) that is not an AHL, but a 2-pyrone derivative. Wetested different 2-pyrones for induction of plu4568-promoter activity, andshowed that a novel class of 2-pyrones named photopyrones is producedby different Photorhabdus species are the specific signal for PluR. Hence asignaling function for the chemical widespread group of pyrones wasidentified for the first time for P. luminescens. Via PluR, expression of theplu4568-plu4563 operon is activated, which encodes a putative synthesispathway correlated with cell clumping. Expression of the plu4568-plu4563operon induced cell clumping in P. luminescens by addition of PLAI-1 aswell as in E. coli when induced heterologously. PLAI-1-dependent cell-tocellcommunication and the resulting cell clumping seem to be importantfor colonization of the nematodes by P. luminescens.BIOspektrum | Tagungsband 2012